A Natural Killer Cell-Centric Approach Toward New Therapeutics For

Home , Somatic hypermutation

A natural killer cell-centric approach toward new therapeutics for

autoimmune disease

A dissertation submitted to the

Graduate School of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy (Ph.D.)

in the Immunology Graduate Training Program of the College of Medicine

2019

Seth D. Reighard

B.S., University of Pittsburgh, 2010

Thesis Advisor: Stephen N. Waggoner, Ph.D.

Dissertation Committee Chair: William M. Ridgway, M.D.

Abstract

The debilitating autoimmune disease systemic lupus erythematosus (SLE) is the 5th leading cause of death for African American and Hispanic women between 15 and 24 years of age. Current therapeutics for SLE are blunt weapons that broadly suppress a patient’s immune system, and only one new treatment option has been approved in over 50 years. Standard treatments for SLE potentially lack efficacy because they do not specifically target the immune dysfunction underlying disease. SLE is characterized by the abnormal production of self-targeting autoantibodies that cause widespread organ damage. SLE autoantibodies are generated by rampant germinal center (GC) reactions: immune processes wherein memory B cells and antibody secreting cells (ASC) arise from naïve B lymphocytes following clonal expansion, somatic hypermutation, affinity maturation, and antibody class switching while receiving critical help from follicular helper T (TFH) cells. We hypothesize that inhibition of TFH and GC responses in SLE may be accomplished by harnessing the therapeutic potential of the natural killer (NK) cell. Though classically implicated in anti-tumor and antiviral immunity, NK cells also suppress activated TFH, resulting in reduced

GC reactions and decreased antibodies. Of added relevance, NK cells are diminished and defective in SLE patients. Thus, we tested whether increasing endogenous NK cell numbers or artificially enhancing their function could achieve a beneficial result in an autoimmune context like SLE. We treated mice exhibiting an SLE-like disease with drugs that expand the endogenous NK cell (and CD8 T cell) pool and, contrary to our hypothesis, observed worsened autoimmunity. Disease aggravation was attributed to the expansion of CD8 T cells, with expanded NK cells exhibiting negligible effects. Our findings suggest that current cancer treatments that expand these cell populations in vivo should be used with caution in patients with cancer and concurrent SLE. Separately, we utilized chimeric antigen receptor technology, a clinical tool currently being used to safely target tumors, to engineer an NK cell that selectively eliminates TFH cells in hopes of subduing autoreactive GCs. In vitro studies demonstrated that our CAR NK cells not only killed

TFH cells but also limited subsequent B cell-mediated generation of ASCs and the downstream production

2 of antibodies. Furthermore, we used RNA sequencing to identify differences in human SLE NK cell gene expression and uncovered several transcriptional alterations with known relevance to both SLE pathogenesis and NK cell function. Gene network analysis predicted the transcription factor PU.1 to underlie several SLE NK cell transcriptional changes, and the upregulation of PU.1 may explain an aberrant and proinflammatory phenotype often observed in disease. Future studies will aim to attenuate the expression of PU.1 in NK cells and determine whether the resulting phenotype is of benefit to disease, thereby identifying PU.1 as a potentially valuable therapeutic target in SLE. Altogether, our innovative NK cell-based therapeutic strategies have the potential to limit the autoantibody-mediated pathology of SLE in a more targeted manner, offering a new hope to thousands of people who suffer from this incurable disease.

Acknowledgments

The thesis work presented herein could not have been accomplished without the help of several people who have made a significant positive impact upon my professional and personal life. I dedicate this work to them.

First, I thank my dissertation committee members (Bill Ridgway, George Deepe, Edith Janssen, and Wenhai

Shao) for their expertise throughout graduate school. This dissertation would not be nearly as complete without their guidance. Most of them served as members of my qualifying committee as well, so I especially appreciate their willingness to endure my research presentations so many times throughout my tenure as a graduate student.

I thank all of my fellow graduate student colleagues and friends, especially Calvin and Rob, for making graduate school bearable and a lot more enjoyable. Similarly, I thank my Waggoner Lab colleagues, past and present, for their endless help and support of my projects and for their enduring friendship. I have extra gratitude for my former colleague, (Dr.) Erik, for helping me to proofread sections of this thesis in a time of dire need.

I thank my past mentors, Drs. M. Brian Traw and Thomas Cherpes, for giving me a chance to pursue scientific research early in my career, and for developing in me a strong foundation of skills, knowledge, and perseverance that is required for earning a PhD. I especially thank my PhD mentor, Dr. Stephen

Waggoner, for being the best scientist and teacher that I could have chosen for my graduate school research. Steve is the true embodiment of everything it takes to be a successful scientist: grit, optimism, creativity, sociability, salesmanship, foresight, intelligence, and determination. As I continue my journey along the research career path, I will forever use lessons learned from Steve to guide my way.

I thank the family I was born into: my mom, dad, sister and brother, for their relentless love and support of all of my academic pursuits throughout the years. I thank the family I married into: my mother, father, and siblings-in-law, for their support and frequent visits to Cincinnati that lessened the homesickness within our household. I also thank the family I chose: my closest friends from childhood to adulthood

(Justin, Mike, Matt, Sam, & Kayla) for always being there when I need them to forget about my professional life for a while.

Words cannot express the amount of love and gratitude that I feel for my wife, Brittany, who uprooted everything she knew to move with me to Cincinnati and start a new life together while I pursued an additional 8 years of school. She is truly my rock, my cornerstone, and my utmost source of motivation throughout the trials and tribulations of graduate school. I fully believe that I could not have done this without her.

Finally, I thank and dedicate this thesis to my newborn son Forrest who, at the time of finishing this thesis, is just over a month old. I never imagined I could love anything more in my life until I met him. I can’t predict what the future holds for Forrest, but I wish a few things for him. I hope that science and logic can survive and prevail in this time of “alternative facts” that he was born into. I hope that technology, reason, and unity can help humanity to heal the environmental mess he has inherited from the generations that came before him. Lastly, I hope that, no matter the passion he pursues, he will be inspired by the power of science and have a deep admiration for the phenomena that underlie the natural world.

Table of Contents

 Abstract…………………………………………………………………………………………………………………………………..……….2

 Acknowledgments……………………………………………………………………………………………………………….………..…5

 Table of Contents……………………………………………………………………………………………………………………………..7

 Abbreviations frequently used in this dissertation………………………………………………………………………….12

 Chapter 1. Introduction to critical concepts underlying this dissertation………………………………………….15

o B lymphocytes, the germinal center reaction, and follicular helper T cells…………….……..….…..16

o Systemic lupus erythematosus: disease variety and pathogenesis………………………………….…..19

o Current treatment approaches for systemic lupus erythematosus..…………………………………….25

o A brief overview of modeling systemic lupus erythematosus in mice……………………………………29

o Natural killer cell development, maturation, and function…………………………………………………..31

o Natural killer cells in systemic lupus erythematosus…………………………………………………….……..35

o The therapeutic potential of natural killer cells in human disease…………..…………………..……..38

o Summary & overall hypotheses…………………………………………………………………………………....…….42

 Chapter 2. Immunomodulatory effects of IL-2Rβ/γc-mediated expansion of natural killer cells and CD8

T cells in a mouse model of SLE-like disease………………………………………………………………………………………44

o Rationale & Hypothesis……………………………………………………………………………………………………….45

o Chapter 2 Graphical Abstract………………………………………………………………………………………………47

o Results………………………………………………………………………………………………………………………………..48

o Acknowledgements…………………………………………………………………………………………………………….52

o Figures………………………………………………………………………………………………………………………………..53

. Figure 1: Bm12-induced lupus-like disease is associated with phenotypic alteration of

NK cells, as well as a contraction in the NK cell pool that is reversed via targeted

cytokine treatment…………………………………………………………………………………..…………...53

. Figure 2: IL-2/αIL-2 (S4B6) complexes and the IL-15 superagonist N-803 selectively

expand peripheral CD8 T and NK cell populations……………………………………………………55

. Figure 3: Pretreatment with IL-2 complexes or N-803 transiently exacerbates

cGvHD……………………………………………………………………………………………………………..…….56

. Figure 4: Therapeutic application of IL-2 complexes or N-803 exacerbates lupus-like

disease…………………………………………………………………………………………………………………..58

. Figure 5: N-803 expansion of CD8 T cells, but not NK cells, contributes to enhanced

germinal center B-cell responses in bm12 model of lupus-like disease and coincides

with increased serum IFNγ……………………………………………………………………………...……..59

 Chapter 3. Elucidating the NK cell transcriptome in SLE identifies several therapeutic targets for

disease intervention…………………………………………………………………………………………………………….………….61

o Rationale & Hypothesis……………………………………………………………………………………………………….62

o Results………………………………………………………………………………………………………………………………..62

o Acknowledgements…………………………………………………………………………………………………………….66

o Figures………………………………………………………………………………………………………………………………..67

. Figure 6: Peripheral NK cell numbers trend toward a reduction in a pediatric SLE

patient cohort……….……………………………………………………………………………………………….67

. Table 1. Available clinical information and medications of the pediatric SLE patients

from which NK cells were isolated and subjected to RNA sequencing…………………….68

. Figure 7: RNA sequencing of pSLE peripheral NK cells reveals several transcriptional

differences versus peripheral NK cells from healthy controls……………………………….…69

. Table 2. Known relevance of specific genes within the top 50 most differentially-

expressed genes in pSLE peripheral NK cells vs. healthy control NK cells…………………71

. Figure 8: Gene network analysis of the pSLE NK cell transcriptome predicts the

contribution of 10 TFs to observed differences in RNA expression versus control NK

cells………………………………………………………………………………………………………………………..72

. Table 3. Known relevance of specific transcription factors predicted to be operating

based upon gene network analysis of differentially-expressed genes in pSLE

peripheral NK cells vs. healthy control NK cells………………………………………………………..73

 Chapter 4. Therapeutic targeting of follicular helper T cells with chimeric antigen receptor-expressing

natural killer cells…………………………………………………………………………………………………………………………….74

o Rationale & Hypothesis……………………………………………………………………………………………………….75

o Chapter 4 Graphical Abstract………………………………………………………………………….………………….76

o Results………………………………………………………………………………………………………………………………..77

o Acknowledgments………………………………………………………………………………………………………………81

o Figures………………………………………………………………………………………………………………………………..82

. Figure 9: PD-1 is a selective marker of human TFH cells………………………………………….82

. Figure 10: PD-L1 CAR design…………………………………………………………………………..………83

. Figure 11: Generation of CAR NK-92 via lentiviral transduction………………………………84

. Figure 12: CAR-transduced NK-92 express CAR-specific mRNA and surface PD-L1……85

. Figure 13: PD-L1 CAR NK cell responses to plate- and cell-associated PD-1…………….86

. Figure 14: Generation of target cell lines expressing human PD-1……………………………88

. Figure 15: PD-L1 CAR NK cells selectively kill TFH cells…………………………………….….…..89

. Figure 16: PD-L1 CAR NK cells kill both tonsil and peripheral TFH cells…………………...90

. Figure 17: PD-L1 CAR NK cells kill TFH cells in T:B co-cultures and suppress TFH-

dependent B-cell responses…………..…………………………………………………………………….…91

 Chapter 5. Summary, discussion, & future directions…………………………………………………………………..….93

9 o Overall summary of findings………………………………………………………………………………………….…….94 o Discussion of results and outstanding questions………………………………………………………………….95

. Is endogenous NK cell expansion a worthwhile therapeutic avenue for treating

SLE?...... …………………………………………………………………………………………………………….....95

 Figure 18: Depletion of NK cells prior to induction of SLE-like disease may

increase GC B cells and plasmablast populations……………………………………..103

. What does transcriptomic analysis reveal about NK cells in pSLE, and can this

information be utilized for therapeutic benefit?...... 103

. Is TFH targeting using CAR NK cells a reasonable therapy for autoimmune diseases such

as SLE?...... ………………………………………………………………….……………………………………...109 o Future directions………………………………………………………………………………………………………………117

. Revisiting in vivo NK cell expansion in SLE-like mice……………………………………………...117

. Attempting to “correct” NK cell function as a therapy for SLE……………………………….118

. Driving the PD-L1 CAR NK cell closer to the clinic……………………………………………………120

 Figure 19: PD-L1 CAR-expressing primary human NK cells selectively kill

autologous TFH cells in vitro………………………………………………………………….….123

 Figure 20: Functional NK cells can be differentiated from SLE patient-derived

iPSC………………………………………………………………………………………….……………..124

 Figure 21: CAR NK-92 potentially reduce PD-1-expressing human CD4 T cells

in a humanized mouse model of SLE-like disease………………………………………125

. Putting it all together: the therapeutic potential of NK cells in autoimmune

disease……………………………………………………………………………………………………………..….126 o Chapter 6. Materials & Methods……………………………………………………………………………………….127 o References…..…………………………………………………………………………………………………………………..145

10 o Appendix – Treating a mouse model of lupus nephritis using PD-L1-expressing natural killer

cells…………………………………………………………………………………………………………………….…………….171

Abbreviations frequently used in this dissertation

ANA – antinuclear antibody

ANOVA – analysis of variance

APC – antigen presenting cell

ASC – antibody-secreting cell

B6 – C57BL/6 mouse strain

BAFF – B cell activating factor

BCR – B cell receptor

CAR – chimeric antigen receptor

CD – cluster of differentiation cGvHD – chronic graft-versus-host disease

CTV – CellTrace Violet

CXCR – C-X-C motif chemokine receptor

DC – dendritic cell

ELISA – enzyme-linked immunosorbent assay

EBV – Epstein-Barr virus

FDC – follicular dendritic cell

GC – germinal center

GFP – green fluorescent protein

HSC – hematopoietic stem cells

ICOS – inducible T cell co-stimulator

IFN – interferon

Ig – immunoglobulin

IL – interleukin

IL2Rβ/γc – heterodimeric interleukin 2/interleukin 15 receptor composed of beta (CD122) and common

gamma (CD132) chain subunits iPSC – induced pluripotent stem cell

IVCCA – in vivo cytokine capture assay

LN – lupus nephritis

MFI – median fluorescence intensity

MHC-I – major histocompatibility complex 1

MHC-II – major histocompatibility complex 2

Mir – micro-ribonucleic acid

MS – multiple sclerosis

MZ – marginal zone

NK – natural killer

NSGS - NOD/LtSz-scid IL2RG–SGM3 mouse strain

OD – optical density

PD-1 – programmed cell death protein 1

PD-L1 – programmed death-ligand 1 pSLE – pediatric systemic lupus erythematosus

PU.1 – purine-rich sequence (PU-box)-binding protein 1; also known as SPI1

RA – rheumatoid arthritis

RNAi – ribonucleic acid interference

RNAseq – ribonucleic acid sequencing

RT – room temperature

SCF – stem cell factor scFv – antibody single-chain variable fragment

SEB – staphylococcus enterotoxin B

SEM – standard error of the mean

SLE – systemic lupus erythematosus

SLEDAI – systemic lupus erythematosus disease activity index

TCR – T cell receptor

TF – transcription factor

TFH – follicular helper T cell

TFR – follicular regulatory T cell

TGFβ – transforming growth factor beta

TLR – toll-like receptor

TLS – tertiary lymphoid structure

TNFα – tumor necrosis factor alpha

Treg – regulatory T cell

Chapter 1. Introduction to critical concepts underlying this

dissertation

B lymphocytes, the germinal center reaction, and follicular helper T cells

A defining aspect of the humoral immune response is the production and secretion of antibodies by B lymphocytes1. B cells arise from hematopoietic stem cells (HSC) in the fetal liver and in the bone marrow after birth. Early B cell development is characterized by the successive rearrangement of H- and L-chain immunoglobulin (Ig) loci that define the progression from pro-B, to pre-B, to the IgM-expressing immature

B cell stages2. This process is antigen-independent, highly reliant upon interleukin-7 (IL-7), and results in cells that express a B cell receptor (BCR), which is composed of Ig coupled to the signaling moiety CD79.

“Central tolerance” is the process whereby immature B cells expressing BCRs that strongly bind to self- antigen are either selected against via clonal deletion, or undergo further L-chain rearrangement3.

So-called “transitional” B cells that pass this central tolerance checkpoint leave the bone marrow, acquire cell surface IgD, and enter secondary lymphoid tissues such as the spleen and lymph nodes. Once there, these “naïve” B cells continue their maturation by forming lymphoid follicles and are then designated

“follicular” B cells4. Follicular B cells recognize antigen and present it to cognate helper T cells, then receive signals to proliferate and generate germinal center (GC) reactions5 (further discussed below). On the other hand, naïve B cells may instead undergo one of several processes outside of the follicle to achieve final maturation. Splenic “marginal zone” (MZ) B cells, found along the margins of B cell follicles, primarily recognize the antigens of encapsulated bacteria and, without input from helper T cells, subsequently differentiate into IgM-secreting plasma cells6. Other “extrafollicular” B cells that are activated by antigen directly differentiate into antibody secreting cells (ASCs) in a T cell-dependent manner. Splenic extrafollicular B cells expressing the chemokine receptor CXCR4 are activated by MZ-residing dendritic cells (DCs) and helper T cells to proliferate, undergo class-switch recombination, and directly differentiate into antibody-secreting plasmablasts7. These relatively quick routes to ASC generation (as compared to

GC reactions) are advantageous in generating a prompt response to foreign antigen, but come at a cost, as most ASC that arise from such B cells are short-lived and secrete antibodies of lower affinity8.

Some B cell subsets bypass maturation and differentiation in secondary lymphoid tissues altogether. B-1 cells, which mostly reside in pleural and peritoneal cavities, arise from the fetal liver and are characterized by their production of polyreactive natural antibodies that are critical for early responses to infection because they recognize a wide variety of antigens from common pathogens6. These antibodies are weakly autoreactive and thus play an important role in the clearance of apoptotic debris and autoantigens. Of note, B-1 cells exhibit some ability to class-switch, as shown by their differentiation into IgA-secreting plasma cells in the gut. Furthermore, in contrast to conventional B cells, they are self-renewing, long-lived, and do not require T cell-mediated help for their development, maturation, or differentiation9.

B cells function in critical ways that do not involve antibody secretion, including acting as vital antigen presenting cells (APCs) and through the secretion of a variety of important cytokines. Specifically, antigens recognized by the BCR are internalized, processed, and presented to CD4 T cells via major histocompatibility complex II (MHC-II)4. Depletion studies in mice have revealed that B cells are essential for the activation of CD4 T cells in response to both foreign antigens and autoantigens. In addition, B cells secrete cytokines such as the interleukins 2 (IL-2), 4 (IL-4), 6 (IL-6), 10 (IL-10) and interferon gamma

(IFNγ)10: all of which dramatically shape and maintain the responses of both the innate and adaptive branches of the immune system, including wound healing2. IL-10 is a critical immune-regulatory cytokine secreted by regulatory B cells (Breg), which interact with pathogenic T cells and inhibit harmful immune responses such as autoimmunity11.

A critical feature of humoral immunity is the enhancement of an antibody’s affinity for its antigen over time12. Within secondary lymphoid structures, this process of affinity maturation takes place in the specialized cellular compartment called the germinal center (GC)1. GCs are distinct anatomical regions within secondary lymphoid tissues that are composed of several key cellular players in the long-lived production of high-affinity antibodies. Following antigen exposure, GCs are formed by activated follicular

B cells and contain microanatomical regions known as the light zone (LZ) and dark zone (DZ) – areas containing activated GC B cells that appear less (LZ) or more (DZ) dense by microscopy. B cells in the DZ reside close to lymphoid tissue T cells and are highly proliferative, whereas LZ B cells are intermixed among follicular dendritic cells (FDCs) that act as an antigen reservoir to select for antigen-specific B cell clones.

During a highly complex migration back and forth between the LZ and DZ, GC B cells undergo somatic hypermutation, isotype class-switching, and affinity maturation through tightly regulated interactions with T cells, FDCs, and other cellular components5,12.

The end result of the GC reaction is critical for lifelong humoral immunity. Mature GC B cells exit the GC as plasmablasts – precursors to plasma cells that can persist for years and secrete large amounts of high- affinity antibodies. In addition to generating these ASCs, GC reactions produce memory B cells that, upon antigen re-exposure, quickly differentiate into plasma cells or re-enter a GC and undergo further somatic hypermutation and isotype class switching13. Altogether, the GC reaction is a complicated and controlled process by which naïve B cells transform into enduring, robust producers of class-switched and high- affinity antibodies.

GC reactions that give rise to ASCs are highly influenced by a CD4 T cell subset known as follicular helper

14 T (TFH) cells . These cells are phenotypically defined by the transcription factor B-cell lymphoma 6 protein

(BCL6) and by high cell-surface expression levels of C-X-C motif chemokine receptor 5 (CXCR5), inducible

15 co-stimulator (ICOS), and programmed cell death protein 1 (PD-1) . Activated and differentiated TFH cells

16 migrate into B-cell follicles and aid in GC formation, maintenance, and function . More specifically, TFH cells in GCs provide crucial signals for B cell survival and proliferation, isotype class-switching, affinity maturation, and terminal differentiation5. In fact, the amount of hypermutation a B cell clone undergoes

7 is directly proportional to the amount of TFH cell help it receives during a GC reaction . These functional contributions highlight the potent and beneficial role of TFH cells in the generation of lifelong protective humoral immunity following infection or immunization.

Systemic lupus erythematosus (SLE): disease variety and pathogenesis

The CDC estimates that 161,000 Americans, most of whom are ethnic minorities and women of child- bearing age, suffer the debilitating consequences of systemic lupus erythematosus (SLE)17,18. SLE is an autoimmune disease of unknown etiology characterized by a loss of immune tolerance and the rampant production of autoantibodies that, when complexed with antigen and deposited in the body’s tissues, drive widespread immunopathology18–20. Due to a high level of associated morbidity, the annual healthcare costs linked to SLE represents a major economic burden. As of 2016, the estimated annual direct costs for a patient with SLE was over $33,000, while an SLE patient with the kidney disease lupus nephritis (LN) carried a healthcare cost of over $71,000 annually21.

SLE is a heterogeneous disease that manifests both systemically and with organ-specific damage. While many SLE patients exhibit mild and non-life-threatening disease, some patients have severe disease that progresses rapidly with deadly consequences22. Symptoms of SLE include fever, weight loss, arthralgia, fever, and fatigue, while organ specific aspects of disease include cutaneous rash, musculoskeletal and joint inflammation, kidney damage, heart and lung complications, and neuropsychiatric manifestations23.

Anti-DNA antibodies are present in 50-70% of SLE patients, regardless of specific disease phenotype24.

Lupus nephritis (LN) develops in over half of SLE patients23 and represents a major cause of mortality within this population25. Though disease presentation in LN is diverse with varying prognoses, the renal pathology of LN is driven by IgG-dominant immune complex deposition in kidney glomeruli26.

Neuropsychiatric SLE is a significant subcategory of disease, especially as encephalopathy is a major cause of death in SLE patients25. Though the pathogenesis of neuropsychiatric SLE is unclear, it is considered to result from microvascular inflammation and is linked to specific autoantibodies, including anti-NMDA receptor, anti-ribosomal P, and anti-phospholipid antibody27. Similarly, antibody complex deposition in the skin leads to cutaneous lesions that are common to SLE, and are highly associated with specific autoantibodies such as anti-ribosomal P, anti-Ro52, and anti-galectin-322. Therefore, such heterogeneity in SLE has led to the development of a multifaceted, point-based classification system that utilizes 22 disease criteria clustered into 7 clinical domains and 3 immunologic domains28.

The etiology of SLE is multifactorial, with both genetic and environmental factors playing a significant role.

The posited underlying pathogenesis of SLE encompasses several mechanisms involving many different aspects of the immune system29. A prevailing theory is that SLE arises due to accelerated cell apoptosis and defects in the body’s ability to clear cellular debris. The pathologic anti-nuclear antibodies (ANA) that characterize SLE are thought to originate when nucleic acids (e.g. DNA), proteins that bind nucleic acids

(e.g. histones, ribosomes), and other intracellular elements (e.g. mitochondrial proteins) are released from apoptotic cells and (in genetically predisposed individuals) become the autoantigens toward which

ANA and other autoantibodies are targeted22. Neutrophils are implicated in this process due to their ability to secrete large amounts of nucleic acids as a component of extracellular traps – thus further adding to autoantigen pools in SLE30. Although these nuclear materials would normally be cleared and inaccessible to immune responses, evidence in SLE patients and relevant mouse models demonstrates the enhanced activity of cell death pathways, impaired ability to clear apoptotic debris, and specific post-translational

20 modification of apoptosis-related proteins that favor autoantibody generation31. For example, mutations leading to lowered expression of DNase1, an enzyme critical for the elimination of DNA debris, are associated with mouse models of SLE-like disease and SLE patients31. Furthermore, serum from SLE patients and SLE-prone mice contain autoantibodies specific for acetylated, methylated, and ubiquitinated DNA histones, which are post-translationally modified versions of histones that are more immunogenic than their unmodified counterparts31.

The direct response of the immune system to apoptosis-related self-antigens (regardless of their origin) is a central component of SLE pathogenesis. Nucleic acid sensors play a critical role in SLE, as accumulated nuclear debris triggers several Toll-like receptors (TLRs) that are expressed on and within a variety of leukocytes, resulting in cellular over-activation22. In fact, sustained activation of TLR7 and TLR8 alone is enough to induce an SLE-like disease in specific mice32,33. Nucleic acid receptors such as TLRs are involved in mounting an immune reaction against viral and intracellular bacterial pathogens, and the overt activation of these receptors in SLE generates a massive type I interferon (IFN) response22. Overabundance of type I IFN in SLE primarily arises from TLR-activated plasmacytoid DCs (pDC), although several cell types contribute to IFN elevation29. Type I IFN signatures are highly characteristic of both human SLE and several relevant mouse models34, with the latter revealing mechanisms by which elevated IFNα modulates the immune system and directly exacerbate SLE35. These effects include, but are not limited to, a decrease in regulatory T cells (Treg), increased secretion of (aforementioned) nucleic acids from neutrophils, and enhancement of B cell activating factor (BAFF) production22. Furthermore, the strong elicitation of type I

IFN following infection with Epstein-Barr virus (EBV) offers one explanation of why this virus is highly linked to SLE36, although molecular mimicry between specific EBV proteins and SLE autoantigens is another prevailing theory of viral involvement37,38.

Abnormalities in immune-associated secreted factors (other than type I IFN) contribute to SLE pathogenesis. For example, SLE is associated with elevated levels of tumor necrosis factor alpha (TNFα) and interleukin-17 (IL-17) that both recruit and activate myeloid cells (such as neutrophils and monocytes) to be proinflammatory and destructive, particularly at sites of antibody-complex deposition in tissues34.

Increased levels of interleukin 6 (IL-6) and BAFF in SLE are critical mediators of enhanced B cell proliferation and survival, as well as B cell loss of tolerance to self-antigen39. Levels of the interleukin-2

(IL-2) are decreased in SLE and some SLE-prone mice, causing loss of T cell homeostasis and reduced Treg populations22,40. This represents just a few of the ways that T and B cells are disrupted in SLE.

Aside from the many possible mechanisms that may underlie SLE, the downstream production of autoantibodies against self-antigens is both fundamental and universal to disease pathology. As described above, the generation of antibodies, and thus autoantibodies, hinges upon the activation and differentiation of B lymphocytes, often (but not always) with the help of T lymphocytes1. B cells are so central to disease that autoantibodies sometimes arise in SLE patients years before other manifestations of disease or immune dysfunction are detected24, while adoptive transfer of T cells alone triggers SLE-like disease in some mouse models41. In fact, the MRL/lpr SLE-prone mouse exhibits defects in the Fas receptor-mediated apoptotic pathway that causes uncontrolled proliferation of both T and B cells 31,42.

The way in which these lymphocytes contribute to SLE, however, goes beyond autoantibody production and originates with a loss of self-tolerance.

There is strong evidence supporting that defects in B cell central tolerance may be genetically determined in SLE, including mutations that lead to dysregulated BCR editing of ANA-reactive B cells3,43. Furthermore, genetic defects in BCR signaling are also linked to autoimmunity, as diminished signaling to self-antigen allows autoreactive B cells to evade negative selection in the bone marrow and escape to the periphery

22 for eventual autoantibody production24. Other evidence suggests that a loss of peripheral B cell tolerance is an effect of the SLE disease process. In fact, the activation of B cells through TLRs or via BAFF signaling, both of which are increased in SLE, bypasses B cell tolerance checkpoints22,24. B cells from control human tonsil samples reactive to apoptosis-related self-antigens are typically excluded from entering GC reactions (through a mechanism currently unknown), whereas these tonsil-residing B cells from SLE patients enter GC reactions and emerge as IgG-secreting, autoreactive ASCs24. Thus, GC exclusion is another checkpoint of peripheral B cell tolerance that may underlie SLE pathogenesis. In addition, Bregs from SLE patients have an impaired secretion of IL-10, potentially leading to further reductions in peripheral tolerance44.

As previously mentioned, B cells also secrete cytokines and act as critical APCs via expression of MHC-II.

Even when B cells lack the ability to produce antibody, their role as APCs and cytokine-producers results in some (albeit attenuated) disease manifestations in SLE mouse models24. B cells can secrete IL-6 and

IFNγ, both of which exacerbate inflammation in SLE and relevant mouse models24,45,46. Furthermore, B cells secrete lymphotoxin-α, which is critical for formation of tertiary lymphoid structures (TLS) – atypical pockets of lymphoid architecture observed in non-lymphoid tissues, such as the kidney in SLE24,47.

Nearly every type of B cell is implicated in the pathogenesis of SLE, including subsets that do not participate in GC reactions. For example, SLE-prone MRL/lpr mice generate autoreactive B cells and ASCs in non-follicular regions of lymphoid tissues48, including zones devoid of FDCs7. The generation of anti-

DNA autoantibodies originates outside of GCs in several autoimmune mouse models, while ties to aberrant TLR signaling and the generation of autoreactive extrafollicular B cells have also been observed in these models7. MZ B cell populations are expanded in some SLE-like mouse models and may contribute to serum autoantibodies, although the definition and impact of this B cell subset in humans remains

23 unclear8,24. B-1 cells are elevated in both SLE patients and SLE-prone mice, potentially due to underlying genetic mutations or induced via high levels of IFNα. In these mouse models, B-1 cells may contribute to autoimmune disease by the inherent autoreactivity of the natural antibodies they produce, as well as their enhanced antigen presentation to autoreactive T cells. Nevertheless, manipulations of other SLE-like mouse models have shown that the B-1 cell compartment is neither necessary or sufficient for autoimmune disease.9 Altogether, the autoantibodies that arise from extrafollicular B cell differentiation into ASC are notable in SLE, albeit of lower affinity, with less somatic hypermutation, and infrequently class-switched7,8,24.

Pathogenic high-affinity autoantibodies in SLE are generated by long-lived ASCs that arise from GCs49,50.

Indeed, all aspects of the GC reaction are aberrant and highly implicated in both SLE and animal models of SLE-like disease50–55. In regard to peripheral tolerance, B cell autoreactivity results from somatic hypermutation in the GC, as defects in BCR editing during this process generate autoreactive B cells directly from non-self-reactive naïve precursors24,56. Indeed, the presence of autoantibodies that are highly-specific to particular self-antigens suggests their derivation from B cells that have undergone somatic hypermutation24. Class-switching is associated with SLE, as IgG anti-dsDNA autoantibodies are more pathogenic than IgM anti-dsDNA24. In fact, certain autoreactive IgM may actually be protective in autoimmune disease, possibly due to clearance of both apoptotic debris and antibody complexes without aberrant immune activation57. Moreover, approximately half of SLE patients harbor anti-dsDNA autoantibodies that have class-switched to IgE22. Affinity maturation of B cells is also connected to SLE.

TLR signaling, which is elevated in SLE, directly enhances affinity maturation in GCs56. Thus, data in SLE patients and mouse models supports the role of increased pathogenicity of autoantibodies that highly bind autoantigen and originate from GC reactions49,51. Furthermore, studies examining autoreactive GC- generated ASCs in human SLE and relevant mouse models have found an increased numerical output of

24 plasma cells with enhanced survival, potentially due to hyperactive B cell states that precede them and elevated survival factors (e.g. BAFF, IL-6, etc.) that accompany the autoimmune disease state8.

A growing body of evidence supports that TFH cells play essential roles in the generation and maintenance of systemic autoimmunity. SLE patients harbor increased TFH cell numbers, particularly during disease

58,59 flares . The number of circulating TFH positively correlates with autoantibody titers in patients with SLE.

TFH cells are both necessary and sufficient to trigger SLE-like disease in select mutant mouse models (e.g.

Sanroque mice)51,60, while other SLE-like mouse models also demonstrate similar aberrant and pathogenic

61 TFH responses . Aberrant TFH responses are also associated with the development and persistence of pathogenic ASCs in a variety of other autoimmune diseases, including Sjögren’s syndrome, juvenile dermatomyositis, scleroderma, multiple sclerosis (MS), type-1 diabetes mellitus (T1DM), and rheumatoid arthritis (RA).

Apart from autoimmunity, exaggerated or dysregulated TFH functions contribute to disease in several

62 realms . TFH cells are implicated in immunoglobulin E (IgE) production and potentially contribute to allergic reactions63. Certain subtypes of lymphoma, including angioimmunoblastic T-cell lymphoma,

64 demonstrate a TFH cell phenotype . Collectively, the established roles of TFH cells in disease justifies exploring novel therapeutic strategies that selectively eliminate this cellular pool altogether.

Current treatment approaches for systemic lupus erythematosus

Current standard of care for SLE relies heavily upon the use of immunosuppressive drugs, such as corticosteroids, methotrexate, mycophenolic acid, leflunomide, and several others. Amongst a wide array of side effects, these treatments broadly dampen immune responses, causing impaired wound healing and an increased susceptibility to dangerous infections19,20,65. In fact, infection is currently the leading

25 cause of death in patients with SLE and is highly linked to immunosuppressant drug regimens25. The risk of deadly infection is further elevated by genetic factors and intrinsic immunologic abnormalities that predispose SLE patients toward having simultaneous immunodeficiencies66. The antimalarial drug hydroxychloroquine is often used in SLE to modify TLR signaling and does reduce mortality65, but must be taken with caution to avoid an adverse risk of retinopathy67. The gold standard for treating severe LN, cyclophosphamide, is a chemotherapeutic agent with carcinogenic and teratogenic potential68,69, making it very unsafe for women with SLE who are, or wish to become, pregnant. Despite receiving the standard- of-care via the conventional drugs above, many SLE patients do not respond or respond poorly to such treatments65, prompting the search for new and more targeted therapeutics in recent years.

In recent decades, attempts were made to both suppress and eliminate B cells as a more targeted approach to SLE treatment. This effort has led to the FDA approval of only one drug for SLE, belimumab, in more than a half-century17. Belimumab is a monoclonal human antibody that binds and neutralizes

BAFF, and has shown modest efficacy in patients with SLE (especially with cutaneous and articular manifestations). The assessment of its impact in patients with LN is currently ongoing39. Other treatments that target B cells, however, have not been as successful.

The mouse/human chimeric anti-CD20 monoclonal antibody rituximab has established benefits in cancer due to its ability to deplete B cells70. As CD20 is absent from naïve B cell progenitors and ASCs like long- lived plasma cells, rituximab treatment tends to deplete B cells between these stages while allowing for replenishment of the mature B cell pool (in 6 to 12 months) and minimizing negative effects on plasma cell secretion of protective antibodies39. When assessed in two large double-blind clinical trials of SLE patients, however, rituximab failed to achieve its primary endpoints, despite minor beneficial effects in certain ethnic demographics and disease subtypes39,70. Why rituximab failed clinical trials in SLE is still a

26 matter of debate, including the theory that trials were both conducted incorrectly and confounded by the use of corticosteroids39,65, the ability of SLE patients to develop antibodies against the murine portion of rituximab39, the fact that the drug doesn’t target pools of long-lived autoreactive ASCs71, and even the opinion that rituximab may exacerbate disease in some SLE patients via increased presence of BAFF72.

Nevertheless, the disappointing results of rituximab were echoed by other attempts at targeting B cells in

SLE, including the anti-CD22 B cell-depleting antibody epratuzumab and the anti-BAFF antibody tabalumab, both of which failed clinical testing39,71.

Because effective treatments are still lacking, a variety of efforts are currently underway to develop novel therapeutics options for SLE patients. As of 2019, ongoing clinical trials are currently testing treatment strategies for SLE that target a wide range of pathogenic disease mechanisms39. Two current trials are aiming to block type I IFN signaling via IFNAR blockade or by stimulating B cell production of neutralizing anti-IFNα antibodies. Several trials are underway to examine the effect of inhibiting T cell activation in

SLE, including Janus kinase (JAK) inhibitors and monoclonal antibodies that neutralize the cytokines IL-12 and IL-23. Fully-humanized anti-CD20 antibodies are being tested, as well as agents that block BCR signaling. Moreover, the potential benefits of combined rituximab and belimumab treatment are being tested for the additive effect of B cell depletion and BAFF blockade.

The broadly immunosuppressive agents that currently embody the standard-of-care in SLE, in addition to more targeted approaches like rituximab, may fail to stop disease because they don’t target the GC B cells,

TFH cells, and ASCs that are highly involved in SLE pathogenesis and maintenance. In regard to ASCs, most of the attempts to deplete this population in SLE have focused on testing the chemotherapeutic drug bortezomib, which inhibits the cellular proteasome and results in apoptosis of highly protein-synthesizing

27 cells like ASCs. Though bortezomib exhibited promising effects in both mouse models of SLE73 and early clinical trials in a small patient cohort74, larger investigations of its clinical effects were disappointing and failure was attributed to rapid reconstitution of the plasma cell pool from B cell precursors (aided by T cells) that were untouched by treatment75. Though efforts are currently underway to test the effect of combining bortezomib with agents such as rituximab76 and belimumab77, the future of therapeutically targeting plasma cells in SLE remains uncertain. Nevertheless, it is encouraging that part of the efficacy of belimumab in SLE is believed to occur due to reduced plasma cell survival via BAFF blockade71.

78 In more recent years, thought has shifted toward targeting TFH cells as a potential strategy to combat SLE .

Stimulation of the P2X7 receptor restricts the expansion of aberrant TFH cells and limits the generation of autoantibodies in an SLE-like mouse model, as well as inhibits the proliferation of human SLE TFH cells in vitro79. In addition, a recent study revealed that inhibiting glucose metabolism in SLE-prone mice reduced

80 the TFH cell compartment, leading to reduced measures of autoimmune disease . These two investigations both made the observation that, despite the inhibition of aberrant TFH cells driving autoimmunity, antigen-specific T cell-mediated effects upon vaccination and in the context of viral

79,80 infection remained intact . Such findings suggest that pathogenic TFH cell effects may be specifically targeted without concomitantly eliminating the beneficial impact of non-pathogenic TFH cells. The therapeutic effect of the CTLA-4-Ig fusion protein abatacept in autoimmune contexts such as RA is partially

81 attributed to its ability to repress TFH cell activity . More specifically, this drug blocks CD80 and CD86 interaction with the CD28 and CTLA-4 receptors, thereby inhibiting T cell activation. For reasons still

81 71 unclear, abatacept was shown to selectively diminish TFH cell populations in patients with MS and RA .

Although trials of abatacept in SLE were largely disappointing71, some mild benefits were observed in the context of LN82. In addition, clinical trials are currently underway to test the effect of separately blocking

CD40L and ICOSL, thus interfering in critical TFH cell interactions with autoreactive B cells. Furthermore,

83 BAFF is known to cause TFH cell accumulation in an autoimmune disease context , suggesting that the beneficial effects of belimumab may be due to more than just its impact on the B cell compartment.

A brief overview of modeling systemic lupus erythematosus in mice

Mouse models of SLE-like disease exist that manifest from either spontaneous (i.e. genetic) or induced mechanisms41,84. One of the most widely-used spontaneous models arises in F1 generations of New

Zealand White mice crossed to the New Zealand Black strain (NZB/W). This model exhibits a female bias and results in lymphadenopathy, splenomegaly, anti-dsDNA IgG production, glomerulonephritis, and early death. NZB/W mice show a type I IFN signature and carry genetic alterations (Sle1, Sle2, and Sle3) that map to genes governing tolerance to nuclear antigens, B cell hyperactivity, B-1 cell accumulation and polyreactive IgM production, and CD4 T cell apoptosis. Transfer of these genetic mutations to B6 mice results in a similar spontaneous SLE-like model (B6.NZMSle1/Sle2/Sle3) that is conveniently on the (fully genetically-characterized) B6 background85. The BXSB/Yaa mouse strain exhibits SLE-like spontaneous lymphoproliferation, lymphoid tissue hyperplasia, ANA production, and reduced mortality due to glomerulonephritis. Disease is worse in male BXSB/Yaa mice due to a Y chromosome-linked enhancement in TLR-signaling. MRL/lpr mice represent another longstanding genetic model of SLE-like disease that do not exhibit a sex bias and arise from aberrant underlying lymphoproliferation due to genetic defects in

Fas-signaling (an uncommon defect in human SLE). These mice develop autoantibodies against RNA and

DNA, antibody complex-mediated glomerulonephritis and dermatitis, and lymphadenopathy characterized by an atypical accumulation of double-negative (CD4- CD8-) T cells. Although a clear role for type I IFN is lacking in MRL/lpr mice, an essential role of IFNγ is well-documented46. Furthermore, the

BXD2 mouse strain, derived through extensive intercrossing of B6 and DBA/2J mice, displays a spontaneous gender-neutral SLE-like phenotype that includes autoantibody-mediated glomerulonephritis and arthritis86. Similar to the aforementioned Sanroque strain, disease in BXD2 mice is highly dependent

53 upon aberrant GC reactions, expanded TFH cells, and IFNγ production . Altogether, spontaneous models mirror different clinically-relevant facets of SLE and shed light on important underlying genetics of disease.

However, these models can be inconvenient to use because disease is not always fully-penetrant and develops relatively slowly over the course of several months84.

By comparison, some inducible mouse models of SLE-like disease develop quickly (i.e. weeks instead of months), are often more penetrant, and better elucidate environmental triggers of disease.

Intraperitoneal injection of the hydrocarbon pristane (a major component of mineral oil) triggers a robust

SLE-like inflammatory response in mice that manifests with ANA production, arthritis, anemia, and deadly glomerulonephritis87. It is convenient that pristane induces SLE-like disease in most mouse strains, including humanized mice41,88. Mirroring human SLE, pristane-induced disease is more severe in females and exhibits significant roles for TLR-signaling and type I IFN84. Inducing chronic graft-versus-host disease

(cGvHD) in laboratory mice often elicits an SLE-like phenotype41,84. Specifically, the injection of DBA mouse splenocytes into the F1 offspring of DBA/B6 crossed mice results in an MHC-I and MHC-II mismatch that triggers cGvHD89. Recipient mice exhibit massive and rapid polyclonal expansion of their B cells that results in ANA production and glomerulonephritis. This model has a female bias and is dependent upon activated populations of both CD4 and CD8 T cells. Similarly, injection of splenocytes from the Bm12 strain into B6 mice (or vice-versa) establishes an MHC-II restricted cGvHD that is not gender-biased, exhibits a type I IFN signature, and depends upon the allo-activation of donor CD4 T cells. In fact, engrafted T cells quickly differentiate into TFH cells and generate rampant GC reactions with host B cells, resulting in splenomegaly,

ANA production, glomerulonephritis, and fatal kidney disease54. Signs of disease are observed in as little as two weeks, and (theoretically) any mutant mouse on a B6 genetic background can be injected with

Bm12 splenocytes to examine specific genetic alterations in an SLE-like context84.

Natural killer cell development, maturation, and function

Natural killer (NK) cells are critical innate lymphocytes well-known for their inherent ability to fight cancer and eradicate virus-infected cells90–92. Among several other mechanisms of killing, NK cells most often eliminate target cells via synapse formation and the release of the cytolytic granules perforin and granzyme91.

In mice and humans, NK cells arise from common lymphoid progenitors in the bone marrow that are partially identified by their expression of IL-7 receptor, CD27, Flt3, and c-kit93. A critical early step toward the NK cell lineage requires the expression of the IL-2/IL-15 receptor beta chain (CD122), followed closely by its companion, the common gamma chain (CD132). In vitro evidence suggests that these receptor chains may be upregulated in response to progenitor cell-sensing of stem cell factor (SCF), which binds c- kit, as well as Flt3 ligand binding to Flt394. Together, CD122 and CD132 form the intermediate-affinity IL-

2/IL-15 receptor that, when activated by IL-15 trans-presented (via the IL-15 receptor alpha subunit) on the surface of bone marrow stromal cells, generates crucial signals for NK cell precursors to continue their differentiation. In parallel, NK cell precursors in the bone marrow downregulate the IL-7 receptor, thus shifting their dependence from IL-7 (which is critical for T and B cell development) toward IL-15. Essential transcription factors come into play during this stage in development, including Stat5, Nfil3, Notch, Tcf-1,

Runx3, Cbfβ, and PU.193. For example, evidence supports that PU.1 may regulate the NK cell response to growth factors such as Flt3 ligand and cytokines such as IL-15. Fittingly, PU.1-deficient mice show reduced populations of both NK cell progenitors and the cells that follow: immature NK cells95.

In mouse bone marrow, differentiation of NK cell progenitors to immature NK cells involves the successive gain in expression of the surface receptors CD161 (NK1.1), NKG2A, NKp46 and TRAIL93,94. In humans, a very similar progression occurs in the bone marrow where progenitor cells increase their surface

31 expression of CD161, CD56, NKG2A, and NKp4694. NK cells in this stage are still dependent upon signaling from trans-presented IL-15, and are highly governed by the transcription factors Ets-1 and E4pb493,96.

Importantly, immature NK cells are partly defined by their relative inability to secrete effector cytokines

(e.g. IFNγ) or initiate perforin-mediated cytotoxicity97.

The maturation of mouse bone marrow NK cells is characterized by the initial expression of the surface marker DX5 (CD49b), Slamf4, CD16 (a critical receptor for antibody-dependent cell-mediated cytotoxicity, or ADCC), as well as Ly49 – the major receptor family for NK cell recognition of MHC-I93. Human NK cells exhibit a similar maturation as they begin to express surface receptors such as NKG2D, CD16, and KIR receptors (the human homolog of mouse Ly49 receptors)97. The NK cell maturation process is influenced by several transcription factors including Id2, T-bet, Eomes, Mef, and Foxo193,95. At this point in development, NK cells are able to exit the bone marrow for the blood and peripheral tissues, even though their full differentiation is not yet complete.

Immature NK cells must become educated, or “licensed”, to kill. Licensing is a process by which NK cells learn to discriminate their own healthy cells from malignant and virus-infected cells98. In brief, NK cells express receptors such as NKG2A, KIR (in humans), and Ly49 (in mice) that recognize MHC-I on cells and generally elicit inhibitory signals that prevent NK cell from a killing response91,99. When MHC-I is downregulated on tumor or virus-infected cells, NK cells receive less inhibitory signaling and become activated, killing these pathogenic cells via a so-called “missing self” mechanism93. In addition, NK cells express activating receptors such as NKG2D that recognize stress-associated ligands (commonly upregulated on virally-infected cells) and trigger cytotoxicity toward these distressed targets. Through complex mechanisms still debated and not fully understood, the licensing process results in NK cells that

32 balance the input from inhibitory and activating receptors, often by altering their expression, and thus gain the ability to discriminate between autologous cells that are harmless or pathogenic98.

Apart from licensing, NK cells in the periphery exhibit a phenotypic transition from an immature and less- cytotoxic state to a more mature and fully-differentiated form. In mice, this process is generally characterized by the loss of CD27 expression, downregulation of NKG2A and TRAIL, and increased expression of CD11b93. In humans, this is recognized by a downregulation of NKG2A and transition from more immature NK cells that highly-express CD56 (“CD56bright”) to cells expressing CD56 at a lower level

(“CD56dim”)100. Maturation outside of the bone marrow is still influenced by IL-15, but is also affected by a plethora of other cytokines, including IL-2, IL-4, IL-10, IL-12, IL-18, IL-21, type I and II IFNs, and TGFβ97.

IL-2 and IL-21 have profound effects on NK cell survival and proliferation. IL-12 and IL-18 mature NK cells and activate them to produce effector cytokines such as IFNγ. Furthermore, IFNα typically primes NK cells for antiviral defense by upregulating perforin expression and enhancing cytotoxicity93.

There is evidence to support that “immature” NK cells outside of the bone marrow may not just be precursors to a mature and functional cell state; rather they may represent a subset of their own with specific immune functions. In healthy adults, NK cells typically make up 5-15% of peripheral blood lymphocytes97, and CD56bright NK cells constitute up to 10% of this cellular pool101. Conversely, in non- inflamed human lymph nodes where NK cells represent 5% of total mononuclear cells102, 75-95% are

CD56bright and are predominantly found in parafollicular T cell zones101. Peripheral CD56bright NK cells express chemokine receptors (e.g. CXCR3) that influence their migration to secondary lymphoid tissues103,104. Functionally speaking, CD56bright NK cells secrete more cytokines than their CD56dim counterparts, including higher amounts of IL-10105,106. Although mouse NK cells do not express CD56, evidence supports that a CD27hi NK cell population in mice very closely correlates with the CD56bright NK

33 cell population in humans103,104,107. More specifically, CD27hi mouse NK cells express CXCR3100,104, are found in much greater proportions in secondary lymphoid tissues than peripheral blood103, and secrete large amounts of cytokines like IL-10108–110. When viewed as a whole, these NK cells could represent a regulatory subset that primarily resides in lymphoid tissues and exerts immune-suppressive effects upon nearby leukocytes. However, the identity and impact of a true regulatory NK cell subset remains unclear and debated111.

In addition to their aptitude for tumor killing and viral clearance, work emanating from our lab and others has demonstrated that NK cells, in fact, exhibit regulatory effects upon the adaptive immune system.

More specifically, NK cells are known to kill virus-specific T cells and thereby limit pathology associated

112,113 with chronic viral infection . Through perforin-dependent killing of TFH cells, NK cells indirectly reduce

GC reactions, ASC generation, and resultant immunoglobulin (Ig) titers in a context of immunization and

114 acute viral infection . Moreover, the effect of NK cell cytotoxicity toward TFH cells is further supported by data showing enhanced somatic hypermutation and increased affinity maturation in the GC B cells of vaccinated mice lacking NK cells115.

This evidence, in combination with other demonstrations of NK cell-mediated immunoregulatory effects in both mice and man116, has sparked discussion about the purpose of NK cell regulatory functions beyond preventing overt antiviral immunopathology. Fittingly, a body of evidence supports the possibility that NK cells may act to prevent or quell autoimmune responses via regulation of the adaptive immune system117,118.

Natural killer cells in systemic lupus erythematosus

Immunologic defects in SLE go beyond the adaptive and humoral branches and include components of the innate immune system as well119. More specifically, NK cells are up to 75% less frequent in the peripheral blood of SLE patients, especially during disease flares120,121. Mouse models bolster these findings, as spontaneous emergence of SLE-like disease in Fas-mutant lpr mice correlates with a diminishment of (CD3- NK1.1+) NK cells122. Moreover, antibody-mediated depletion of NK cells in these mice accelerated disease, while adoptive transfer of NK cells delayed disease onset122. Although direct genetic factors may contribute to the observed NK cell deficiency123,124, it has also been speculated that an elevated systemic level of type I interferon (IFN), which is characteristic of SLE, leads to phenotypic changes in the NK cell pool that contribute to disease risk and progression125. A more direct effect of autoreactive T cells on NK cells could be occurring via secretion of interleukin 21 (IL-21)126, especially as

NK cell death upon IL-21-sensing is observed in the context of autoimmunity127. Other possibilities include the production of autoantibodies in SLE that specifically target and deplete certain NK cell subsets128.

Regardless of the underlying cause, these data suggest that an inherent or acquired NK-cell functional deficiency may contribute to the pathogenesis of SLE.

Besides reduced numbers, NK cells are also known to have functional defects in SLE and autoimmune disease in general129. Though tempting to speculate that active inflammation or disease treatment drives such deficits, several studies reveal that a more primary flaw in the NK cell compartment is associated with diseases such as SLE117. For example, a study in 1980 showed reduced NK cell cytotoxicity against tumor cell lines in SLE patients versus healthy controls, an effect that was observed several times thereafter and is not explained by concomitant steroid therapy130. More recent studies have revealed that

NK cells from SLE patients differ in a myriad of ways from the NK cells of healthy patients.

SLE NK cells have impaired differentiation and maturation. Specifically, the differentiation of NK cells from

SLE hematopoietic stem cells is markedly impaired and suggests a state of arrested development 121. NK cells are often labeled as less mature in SLE patients, mostly due to an atypical abundance of the CD56bright subset that is observed in both adult cohorts125 and pediatric SLE patients131. Furthermore, human genomic studies have uncovered specific polymorphisms associated with SLE risk that map to a gene

(CD266) with relevance to NK cell licensing132. Investigations of the mouse genome linked a mutation that elicits an SLE-like phenotype in NZB/W mice to a gene that is important for proper NK cell development and maturation (i.e. SLAMF4)133. SLAMF4 was later implicated in human SLE genomics as well134.

SLE NK cells have altered functionality. Several studies have observed an impaired cytolytic ability in SLE

NK cells121,130,135. Studies have linked this finding in SLE to observations that include increased expression of the inhibitory receptors NKG2A136 and CD161137, reduced expression of CD16 and activating KIR receptors120, reduced expression of the activating receptor NKG2D138, reduced expression of the intracellular activating domain CD3ζ139, and reduced NK cell production of the critical cytolytic mediators perforin and granzyme B121,140 (although these may be increased during SLE flares141). Moreover, the production of classic NK cell cytokines is altered in SLE, as several studies have shown the increased NK cell secretion of IFNγ and TNFα120,140,142 in disease. The aforementioned downregulation of CD3ζ was specifically linked to increased expression of IFNγ and a general proinflammatory NK cell phenotype in SLE patients139 – a phenotype that is echoed in other SLE contexts in both humans and mice120,142,143.

Although clear defects in the NK cell compartment are related to SLE and other autoimmune diseases, some evidence exists that these dysfunctional NK cells represent a pathogenic population that exacerbates disease. Despite being less cytotoxic121, SLE NK cells are reported to exhibit increased cytokine production, especially IFNγ and particularly during enhanced disease activity120. Of relevance, excess IFNγ

36 is detrimental to autoimmunity and SLE45,46,144,145, including through the generation of autoreactive GCs.

NK cells are also known to produce BAFF146, the effects of which could be detrimental in an SLE context.

One study using an SLE-like mouse model showed that, in the absence of T cell help, NK cells contribute to T-independent production of anti-nuclear antibodies (ANA)147. Highly activated NK cells are associated with increased LN disease in both human patients143 and mouse models of SLE148,149, suggesting that they may play a detrimental role in end-organ tissue damage through interactions such as autoantibody complex-binding of CD16 on the NK cell surface150. Furthermore, a specific and atypical subset of NK cells is present in various SLE-like mouse models that shares characteristics with DCs (e.g. CD11c and MHC-II expression, and the ability to present antigen) as well as characteristics of immature NK cells (e.g. expression of the transcription factor E4BP4), and secretes excessive type I and II IFN upon activation142,151.

Adoptive transfer of a relatively small number of these NK cells from TLR7-transgenic, SLE-prone mice was sufficient to induce autoinflammation in wildtype recipients152, suggesting an inherent pathogenic phenotype.

Alternatively, the possibility exists that a regulatory subset of NK cells may fail to prevent a loss of self- tolerance, leading to autoimmune diseases such as SLE. As previously mentioned, the verification and relevance of CD56bright NK cells in humans (or CD27hi NK cells in mice) as a “regulatory” subset is still a topic of ongoing investigation111. Nevertheless, despite an overall decrease of NK cells in SLE patients, the frequency of peripheral CD56bright NK cells is increased125. It remains undetermined whether and how this expansion of CD56bright NK cells relates to the pathogenesis of SLE. What is known about CD56bright NK cells and their function, however, highly suggests a protective role for them in SLE and other autoimmune diseases. For example, the therapeutic effect of daclizumab (anti-CD25) in MS patients is largely attributed to its ability to both expand and enhance the CD56bright population153, leading to its ongoing evaluation in clinical trials for other autoimmune diseases154. In a trial of patients with autoimmune uveitis, daclizumab

37 treatment expanded CD56bright NK cells that secrete high amounts of IL-10106, suggesting one potential mechanism by which these NK cells exhibit regulatory effects and suppress autoimmunity. Moreover,

CD56bright NK cells from RA patients have the ability to produce adenosine through a CD38-mediated pathway and suppress the proliferation of autologous CD4 T cell in vitro155. Thus, a subset of NK cells may exist that exhibits regulatory features and whose perturbation, genetically or environmentally, may underlie or exacerbate autoimmune diseases such as SLE.

Despite the phenotypic incongruities between healthy and SLE NK cells, no investigation to-date has examined RNA transcriptional differences between them. Elucidation of such discrepancies could help to explain the observed phenotypic abnormalities of NK cells in SLE, or may further characterize the genetic underpinnings of what we call a “regulatory” NK cell. More importantly, transcriptomics could reveal genetic defects in SLE NK cells that, if corrected, could have beneficial effects upon the course of disease.

The therapeutic potential of natural killer cells in human disease

NK cell defects and insufficiencies are repeatedly observed in the context of autoimmunity, yet the question remains: how can an inadequate NK cell compartment be rectified to potentially achieve a therapeutic effect? NK cell-based tools currently being used and developed in the realm of cancer treatment156 may provide avenues toward new treatments for autoimmunity as well. Because of the trail blazed by cancer researchers, several realistic options exist to potentially reverse, transform, or circumvent the shortfalls of ineffective NK cells, thereby providing possible new treatment options for combatting autoimmune diseases such as SLE.

Of relevance to reduced NK cell numbers in SLE, several methods currently exist for selectively expanding endogenous NK cell populations in vivo. Most of these treatments target and exploit the

38 signaling effects of the dimeric, intermediate-affinity interleukin 2 receptor (IL-2Rβ/γc). This IL-2 receptor, composed only of the beta and common gamma chain IL-2 receptor subunits, is primarily expressed on the surface of both NK cells and CD8 T cells and is responsible for the activation and proliferation of these cell types. In contrast, the trimeric, high-affinity IL-2 receptor (IL-2Rα/β/γc), which includes the alpha subunit (a.k.a. CD25), is highly expressed on regulatory T cells (Tregs) and elicits their expansion157.

158,159 Selective targeting of IL-2Rβ/γc has demonstrated success as a cancer treatment , but also gained traction in treating patients with the autoimmune disease multiple sclerosis (MS) – a condition that is also associated with reduced NK cell frequencies117. Treatment of MS with a recombinant anti-CD25 antibody, daclizumab, has shown efficacy in the clinic160. Though initially developed to target and expand Tregs, the therapeutic power of daclizumab is largely attributed to its ability to both expand and improve the functionality of CD56bright NK cells153. This may be due to inadvertent blockade of the CD25- containing high-affinity IL-2 receptor and subsequent IL-2 signaling that is shunted toward the intermediate-affinity receptor that is primarily expressed on CD8 T and NK cells161. This finding has led to the ongoing evaluation of daclizumab in clinical trials for other autoimmune diseases, including uveitis and juvenile arthritis154. Unfortunately, though phase 3 trials of daclizumab in MS revealed better efficacy over the standard of care162, serious adverse events were associated with treatment that led to its recent suspension and recall163.

Another method of expanding NK cells in vivo utilizes IL-2 bound to an IL-2 antibody in a soluble complex. When bound to the anti-IL-2 antibody clone S4B6, the half-life of IL-2 is extended and its signaling is specifically targeted toward IL-2Rβ/γc (and away from IL-2Rα/β/γc), resulting in selective expansion of CD8 T and NK cell populations157,164. Treatment with this IL-2/anti-IL-2 complex was even

39 more effective than daclizumab at expanding NK cells from MS patients165. In the context of SLE mouse models, one study demonstrated a beneficial effect of treating MRL/lpr mice with these IL-2 complexes, although the mechanism by which this occurred was underexplored40. In a separate study, when B6 ×

DBA/2 F1 mice were pre-treated with IL-2/anti-IL-2 (S4B6) complexes and injected with DBA/2 T cells (an inducible model of SLE-like disease that arises from MHC class I and II mismatch), disease was worsened, possibly due to expanded TFH cell populations. When mice were treated after disease initiation, however, SLE-like pathology was decreased, speculatively through expanded donor CD8 T cell allo- elimination of recipient B cells166. Clearly, deeper insight into whether IL-2/anti-IL-2 (S4B6) complexes are beneficial or harmful in the context of SLE is needed.

An additional, highly-effective way to selectively expand NK cells in vivo is through IL-15: a critical cytokine for the survival, proliferation and activation of both NK cells and CD8 T cells. IL-15 is most effective when trans-presented between cells while bound to the IL-15 receptor alpha (IL-15Ra), upon which it is recognized by the intermediate-affinity IL-2 receptor (IL-2Rβ/γc) and exhibits its potent effect on CD8 T and NK cells167. Taking advantage of this signaling, soluble IL-15/IL-15Ra complexes can be generated that behave as an IL-15 superagonist168. Further advancement of this tool has led to the development of N-803 (formerly known as “ALT-803”): a protein complex composed of a mutant superagonist IL-15 and a dimeric IL-15Ra sushi domain/IgG1 Fc fusion protein169. N-803 has shown great success in expanding NK cells and CD8 T cells for combatting cancer170. Interestingly, both soluble IL-

15/IL-15Ra complexes and N-803 were recently found to enhance NK cell expression of IL-10, leading to immunoregulatory effects in a mouse model of cerebral malaria110.

Of relevance to NK cell genetic or acquired defects in autoimmune disease, gene manipulation of NK cells to enhance their effects is a growing area of investigation. Expounding upon groundwork showing

40 upregulated MicroRNA-155 (miR-155) in activated human and mouse NK cells171, further investigation revealed that modifying the expression of miR-155 had profound impacts on NK cell production of

IFNγ172. In a similar study, manipulation of miR-150 altered the NK cell expression of perforin and augmented downstream cytotoxicity173. This data is representative of several studies in which targeted genetic modification of NK cells, often aiming to free them from inhibitory signals that limit their function, has therapeutic effects in inflammation and disease156.

Still other options exist to utilize NK cells therapeutically in autoimmune disease, including strategies that could circumvent inherent defects and harness the cytolytic capacity of NK cells to selectively eliminate unwanted, pathogenic cells. For example, a new technology has emerged and shown remarkable clinical success in eradicating cancer174: cytotoxic lymphocytes expressing chimeric antigen receptors (CARs). CAR specificity for tumors is classically conferred by an antibody single chain variable fragment (scFv) against a unique tumor surface antigen175. The scFv-containing recognition domain is coupled to signaling domains such as CD28 and CD3ζ that activate CAR-expressing lymphocytes and trigger an effective cytotoxic response against their tumor targets.

Though highly effective in eliminating certain neoplasms, CAR-expressing CD8 T cell therapy has significant risks that threaten its clinical utility176,177. A substantial percent of patients treated with CAR T cells have experienced toxic and even deadly side effects, including cytokine release syndrome (CRS),

GvHD, and neurotoxicity. An emerging alternative to CAR T cells is CAR expression in NK cells.

Pilot studies suggest that CAR NK cells exert potent anti-leukemic activity178, while maintaining a greatly enhanced safety profile in comparison to their CAR T cell counterparts179. Early trials using CAR NK cells have shown minimal adverse effects and almost no reported CRS or neurotoxicity180. Moreover, CAR NK

41 cells generated from cord blood, stem cells, or cell lines can be utilized therapeutically in a safe manner, thereby elevating the “off-the-shelf” potential of CAR NK cell therapies and likely reducing manufacturing costs178,181,182.

Altogether, CAR technology represents a promising utilization of NK cells to selectively target and safely eradicate pathogenic cells to improve disease. Autoimmune diseases such as SLE are also characterized by pathogenic cells (e.g. TFH cells, autoantibody-producing ASCs, etc.), and thus represent an opportunity to borrow CAR technology from the cancer field and develop promising new therapeutics for curbing autoimmunity.

Summary & overall hypotheses

In summary, available evidence supports the tenets that 1) GCs and their cellular components, including

TFH cells, greatly contribute to pathogenesis in SLE; 2) NK cells possess an inherent ability to kill autologous

TFH cells, suppress GC reactions, and reduce the generation of antibody-secreting ASCs; 3) NK cells demonstrate intrinsic or acquired deficits and defects in SLE; and 4) interventions that work to enhance

NK cell quantity and function are therapeutically valuable in several disease states.

Based upon these premises, the investigations henceforth presented in this dissertation seek to test the following hypotheses: Chapter 2 tests whether reversal of an NK cell deficit in a mouse model of SLE-like disease via in vivo NK cell expansion prevents or therapeutically treats autoimmune disease manifestations. Chapter 3 seeks to uncover whether transcriptional differences exist in SLE NK cells versus healthy controls, thus establishing potential gene targets whose manipulation could be beneficial in an autoimmune context. Finally, Chapter 4 tests whether CAR technology can be successfully utilized to

42 generate an NK cell that selectively targets and eliminates TFH cells, thus providing a possible new treatment option for autoimmune diseases such as SLE, wherein TFH cells are pathogenic.

Chapter 2. Immunomodulatory effects of IL-2Rβ/γc-mediated expansion of natural killer cells and CD8 T cells in a mouse model of

SLE-like disease

Rationale & Hypothesis

As previously discussed, therapeutics which expand the endogenous NK cell population may be beneficial in treating an autoimmune disease such as SLE, yet the effects and mechanism of such treatments require further elucidation. Herein, we test both the prophylactic and therapeutic effects of two different IL-

2Rβ/γc–targeting agents, IL-2/anti-IL-2 (S4B6) complexes and the IL-15 superagonist N-803, in an MHC class II-restricted, chronic graft-versus-host-disease (cGvHD) mouse model of SLE-like disease. In this model, lymphocytes from B6I-H2-Ab1bm12/KhEgJ (bm12) donor mice, which are genetically identical to

C57BL/6 (B6) mice other than three amino acid substitutions in the MHC class II (I-Ab)183 protein, are injected into B6 recipients, inducing the allo-activation of bm12 donor CD4 T cells by allogeneic MHC class

II molecules on host APCs. This interaction results in cGvHD with manifestations closely resembling SLE, including a critical role for type I interferon184. More specifically, host GC B cells and ASCs rapidly expand,

+ + as do the majority of engrafted bm12 donor CD4 T cells, which largely adopt a CXCR5 PD-1 TFH phenotype54. As is characteristic of human SLE, the dysregulated GC reactions in the bm12 model result in massive generation of antinuclear antibodies (ANA), including anti-dsDNA, anti-ssDNA, and anti- chromatin antibodies183.

Although several spontaneous and inducible mouse models of SLE exist (see Chapter 1), we chose to test in vivo NK cell expansion in the bm12 model for several reasons. First, the Bm12 model hinges upon TFH cells that expand and drive (autoreactive) GCs – the same immune process that NK cells disrupt in the context of vaccination and viral infection. Second, we have obtained Bm12 mice on a CD45.1 congenic background, allowing us to easily track the presence of donor TFH cells via flow cytometry and determine if expanded NK cells have a direct impact on this pathogenic population. Third, as our available methods of in vivo NK cell expansion also expand CD8 T cells, the potential confounding effects of these cells may be minimized due to the fact that disease arises from an MHC-II restricted alloreaction and appears CD8

T cell-independent185. Therefore, we operated under the assumption that any effects of IL-2 complex or

N-803 treatment would be attributable to the expansion of NK cells alone. Fourth, the Bm12 model efficiently develops autoreactive GCs in a short timeframe (e.g. two weeks), allowing us to quickly assess the impact of expanded NK cells on the pathogenic process that we are attempting to modulate. Lastly, despite its cGvHD etiology, the Bm12 model exhibits bona fide and defining SLE features (e.g. ANA, type I

IFN, deadly glomerulonephritis), and therefore represents an excellent inflammatory context in which to test whether NK cells impact disease-relevant immune processes.

We hypothesized that by expanding the endogenous NK cell pool, we may be able to amplify the inherent

GC-suppressive effects of NK cells (see Chapter 1), resulting in SLE-like disease prevention or amelioration.

Unexpectedly, both treatments were shown to exacerbate GC reactions and autoimmunity in the Bm12 mouse model, with a potential indirect pathogenic role for expanded CD8 T cells and IFNγ (see Chapter 2

Graphical Abstract, below). Our findings suggest that current methods of in vivo NK cell expansion in an

SLE-like mouse model have deleterious effects that may be due to co-expansion of indirectly disease- potentiating CD8 T cells.

Chapter 2 Graphical Abstract: In vivo expansion of NK cells and CD8 T cells in the Bm12 mouse model of SLE-like disease results in expanded GC B cells, ASCs, and increased ANA production. The expansion of CD8 T cells, specifically, and elevated levels of IFNγ are linked to the observed disease enhancement.

Results

Bm12-induced lupus-like disease is associated with phenotypic alteration of NK cells, as well as a contraction in the NK cell pool

Injection of a splenocyte graft from (CD45.1+) MHC-II-mismatched bm12 mice into (CD45.2+) host C57BL/6 recipient mice results in marked splenomegaly characterized by activation and expansion of graft PD1+

+ + + + + + + CXCR5 CD4 TFH cells, host Fas GL7 CD19 GC B cells, and host CD138 CD19 plasmablasts relative to

C57BL/6 mice receiving a control graft of (CD45.1+) BoyJ splenocytes (data not shown but mirrors findings published elsewhere54). Despite the approximate 3-fold increase in total splenocyte count that occurs during this cGvHD response, the average number of CD3- NK1.1+ NKp46+ NK cells was significantly

(p=0.0002) reduced in the spleen (Figure 1A) of bm12-engrafted mice. As early as five days after disease initiation, a significant (p=0.025) reduction in the proportion of circulating NK cells was apparent in the blood (Figure 1B). This contraction of the NK-cell compartment is consistent with that observed in other mouse models of SLE-like disease122 as well as in patients with SLE120. Furthermore, CD3- NK1.1+ NKp46+ splenic NK cells from bm12-engrafted mice exhibited significantly higher surface expression of CD27,

NKG2A, TRAIL, and CD11c at 2 weeks than mice injected with a BoyJ control graft (Figure 1C). This NK cell phenotype (CD27+, NKG2A+, TRAIL+) is consistent with a more “immature state” in mice93, while the increased expression of CD11c is noted in an atypical, proinflammatory NK cell subset observed in SLE patients and certain mouse models of SLE-like disease142,151,152.

Disease-associated attrition of NK cells is reversed with targeted cytokine treatment.

Consistent with published reports165,186, injections of IL-2/S4B6 (IL-2 complexed with S4B6 clone of an anti-

IL-2 antibody) or the IL-15 superagonist N-803 into C57BL/6 mice resulted in significant expansion of the

CD8 T cell and NK cell compartments, while CD25+ CD4+ regulatory T cell proportions remained relatively unchanged (Figure 2). Pre-treatment of C57BL/6 mice with either IL-2/S4B6 or N-803 prior to injection of

48 bm12 splenocytes resulted in maintenance of proportions of circulating NK cells on day five of disease

(Figure 1B) and significantly expanded splenic NK-cells at day 14 of disease (Figure 1A), relative to mice receiving a control graft of BoyJ splenocytes. These data suggest that the NK cell compartment contraction we observed upon disease initiation is prevented or reversed via these IL-2Rβ/γc-targeting agents.

IL-2/S4B6 or N-803 prophylactic treatment transiently exacerbates cGvHD.

Given the potent immunoregulatory potential of NK cells187 and the maintenance or marked expansion of

NK cells after IL-2/S4B6 or N-803 pre-treatment in bm12-splenocyte recipients (Figure 1), we hypothesized that the outcome of such therapy would be reduced measures of cGvHD and SLE-like disease in treated mice. In contrast to our expectations, the number of GC B cells and plasmablasts in the spleen of IL-2/S4B6 pre-treated, bm12-engrafted C57BL/6 mice two weeks after disease initiation was elevated relative to mice pre-treated with IL-2/IgG2a (non-complexed IL-2 and isotype control antibody) (Figure

3A). Total serum immunoglobulin (IgG) levels were also higher in IL-2/S4B6 pre-treated and engrafted mice. Similar elevations in GC B-cell and plasmablast counts, along with a trend towards increased sera

IgG, was observed in diseased mice pre-treated with N-803 relative to control mice treated with human

IL-15 (at a molar equivalent to N-803) (Figure 3B). These enhanced disease measures are observed concurrently with an approximate 2-fold increase in the number of splenic NK cells (Figure 1A), suggesting that an expanded pool of NK cells lacks disease-ameliorating activity at this time point in the bm12 model.

Of note, the number of GC B cells and plasmablasts in IL-2/S4B6 pre-treated mice was similar to or non- significantly trending lower than in control-treated mice at three months post-disease initiation (Figure

3C). A comparable or slightly reduced titer of antinuclear antibodies (ANA) was also observed in sera of

IL-2/S4B6 pre-treated mice relative to controls. Similarly, non-significant downward trends were observed in the number of GC B cells and ANA titers in N-803 pre-treated mice relative to control mice at the three-

49 month time point (Figure 3D). The number of plasmablasts was similar or slightly elevated at this time point in N-803 treated mice relative to controls. No clear difference in kidney glomerulus deposition of

IgG or C3 was evident between N-803 or control pre-treatment groups (Figure 3E), nor did kidney deposits differ in either group over non-engrafted, healthy mice (data not shown). Urine microalbumin was similar or non-significantly downward trending (i.e. proteinuria, Figure 3F) in N-803 pre-treated mice 3 months after disease initiation vs. control mice. Thus, exacerbated cGvHD at the two-week time point in IL-2/S4B6 or N-803-treated mice was not associated with sustained amplification of SLE-like disease. Data trends hint that the cytokine pre-treatment non-significantly reduce later manifestations of disease, but this requires further investigation. Our results suggest that if prophylactic cytokine-mediated expansion of NK cells can prevent disease, an alternative timing and/or dosage of prophylactic IL-2/S4B6 or N-803 should be considered.

Therapeutic application of IL-2/S4B6 or N-803 exacerbates SLE-like disease in mice.

To assess whether cytokine-mediated expansion of NK cells alleviates established SLE-like disease, control and bm12-recipient mice were administered therapeutic regimens of IL-2/S4B6 one month after disease initiation (once daily for three days; mirroring what was tried elsewhere166), or N-803 one week after disease initiation (once weekly for 11 weeks). Three months after bm12 engraftment, IL-2/S4B6-treated mice exhibited non-significant trends toward increased GC B-cell and plasmablast cell counts in the spleen relative to control treated mice (Figure 4A). A similar upward trend was evident in total sera IgG levels, which was associated with a significant (p=0.026), nearly 2-fold increase in ANA titers (Figure 4A). Parallel trends of enhanced GC B-cell, plasmablast cells, and sera IgG at three months were observed in mice therapeutically treated with N-803 (Figure 4B). Furthermore, bm12-engrafted mice exhibited no clearly observable difference in kidney IgG or C3 deposition between N-803-treated mice and controls (Figure

4C), nor were these deposits markedly different in either group vs. non-engrafted, healthy mice (data not

50 shown). A non-significant trend toward increased proteinuria in N-803-treated mice was been present at the three-month time point vs. control-treated mice (Figure 4D). Thus, Bm12-induced SLE-like disease is potentially intensified by therapeutic treatment with IL2Rβ-targeting cytokines like IL-2/S4B6 and N-803.

N-803-induced expansion of CD8 T cells, but not NK cells, contributes to enhanced cGvHD.

As N-803 triggers marked expansion of both CD8 T cells and NK cells (Figures 1 and 2), either cell lineage could be responsible for the enhanced disease measurements we observed (Figures 3 and 4). To evaluate the disparate contributions of each cell type, C57BL/6 mice were depleted of NK cells or CD8 T cells using anti-NK1.1 or anti-CD8α antibodies, respectively. Thereafter, depleted and non-depleted mice were treated with N-803 prior to engraftment of bm12 splenocytes. As expected, the number of splenic NK cells was elevated over baseline in non-depleted or CD8-T-cell-depleted mice following N-803-treatment, whereas NK cells were significantly reduced in the spleens of N-803-treated mice previously administered anti-NK1.1 antibody (Figure 5A). Correspondingly, the numbers of splenic CD8 T cells were similarly elevated in N-803 treated immune-replete and NK-cell depleted mice relative to mice depleted of CD8 T cells prior to treatment (Figure 5B). Importantly, the exaggerated GC B-cell responses following N-803 pre-treatment of bm12-engrafted mice (Figure 3B) were maintained in mice depleted of NK cells prior to

N-803 administration (p=0.99), but trended toward an abrogation of disease (p=0.064) in mice depleted of CD8 T cells prior to N-803 therapy (Figure 5C). Thus, CD8 T cells are likely an important contributor to enhanced cGvHD after N-803 pre-treatment in the Bm12 mouse model of SLE-like disease.

Excessive IFNγ contributes to pathologic accumulation of TFH cells and GC B cells, resulting in exacerbated

SLE-like disease in mouse models45,46. Additionally, evidence demonstrates that B cell sensing of IFNγ promotes autoimmune GC formation144. Indeed, manipulations that increase IFNγ production in the Bm12 model exacerbate disease188,189. As it is known that both IL-2/S4B6 complexes and N-803 increase the

51 secretion of IFNγ from CD8 T cells190,191, we therefore hypothesized that the CD8 T cell-dependent increase in GC B cells observed when bm12-engrafted mice are pre-treated N-803 (Figure 5C) may be due to expanded CD8 T cell production of IFNγ. We utilized an in vivo cytokine capture assay (IVCCA) to evaluate serum levels of IFNγ before and during the first two weeks of SLE-like disease in N-803 pre-treated mice that were previously depleted of either NK cells or CD8 T cells. Serum levels of IFNγ in N-803 pre-treated,

SLE-like mice were mildly heightened at days 7 and 13 over their average level prior to any treatment or manipulation. This elevation was potentially negated via depletion of CD8 T cells prior to N-803 treatment and bm12 engraftment, whereas depletion of NK cells had no observable effect on N-803 enhancement of serum IFNγ in SLE-like mice (Figure 5D). It is important to note, however, that we did not assess IFNγ sera levels in N-803-untreated, bm12-engrafted mice to directly compare the effect of this pre-treatment on IFNγ during disease. Nevertheless, our findings suggest that elevated serum IFNγ over baseline in N-

803 pre-treated SLE-like mice may be dependent upon an expanded CD8 T cell compartment, and could, at least in part, explain heightened GC responses after N-803 pre-treatment in this model.

Acknowledgements

We would like to thank J. Klarquist and E. Janssen for generating the Bm12/CD45.1 mouse line that was used for our disease model, as well as their expertise and the assistance of W. Shao with assessing SLE- like disease. We also thank M. Khodoun and the laboratory of F. Finkelman for reagents and assistance with the IVCCA performed, and H. Seelamneni for lab managerial support. Flow cytometry was accomplished with help from the Cincinnati Children’s Flow Cytometry Core. N-803 and guidance with using it were kindly provided by the team at Altor Bioscience (now affiliated with NantWorks LLC). Funding was provided by NIH grants DA038017, T32GM063483, and T32AI118697.

Figure 1: Bm12-induced lupus-like disease is associated with phenotypic alteration of NK cells, as well as a contraction in the NK cell pool that is reversed via targeted cytokine treatment. (A) Total splenic NK cells (CD3- NK1.1+ NKp46+) at 2 weeks following the injection of a control (BoyJ-derived) splenocyte graft, SLE-like disease-inducing (Bm12-derived) splenocyte graft, or Bm12 graft following pre-treatment with either IL-2/S4B6 complex or N-803. N=12-15 per group. (B) Peripheral NK cell frequency at day 5 between mice receiving a BoyJ graft, Bm12 graft, or Bm12 graft after pre-treatment with either IL-2/S4B6 complex or N-803. N=3-10 per group. (C) Host splenic NK cell expression of relevant surface markers 2 weeks following injection of either BoyJ or Bm12 graft. N=3-4 per group.

Due to unequal SDs between groups in A and B, data analyzed via Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. Data in C analyzed via multiple unpaired Student’s T tests using the Holm-Sidak method. Data are displayed as mean ± SEM.

Figure 2: IL-2/αIL-2 (S4B6) complexes and the IL-15 superagonist N-803 selectively expand peripheral CD8 T and NK cell populations. (A, B) Frequency (left) and average fold change (right) of

+ + - + + + + + CD3 CD8 T cells, CD3 NK1.1 NKp46 NK cells, and CD3 CD4 CD25 Tregs in peripheral blood of mice 2 days after treatment with either (A) IL-2/IgG2a control vs. IL-2/S4B6 complex, or (B) IL-15 control vs. N-803. N=2-3 mice per group. Frequencies each analyzed via separate unpaired Student’s

T tests and represented as mean ± SEM. Dotted lines indicate no fold-change.

Figure 3: Pretreatment with IL-2C or N-803 transiently exacerbates cGvHD. (A, B) Number of host

+ + + + + + mouse-derived (CD45.2 ) splenic Fas GL7 CD19 GC B cells and CD19 CD138 plasmablasts, as well as sera IgG levels, two weeks after disease initiation in mice pre-treated with (A) IL-2/S4B6 complex vs. IL-2/IgG2a control (N=4-8 per group), or (B) N-803 vs. IL-15 control (N=8 per group). (C,D) Number of host-derived splenic GC B cells and plasmablasts, as well as sera ANA titers three months after disease initiation in mice pre-treated with (C) IL-2/S4B6 complex vs. control (N=3-8 per group), or (D)

N-803 vs. control (N=3-4 per group). (E) Immunofluorescent staining of 2 representative kidney glomeruli (200x magnification) for IgG and C3 deposits (green) at 3 months post-disease initiation in diseased mice pre-treated with N-803 as in B & D. (F) Microalbumin detected in urine at 3 months from mice in E (N=2 per group). All bar graphs analyzed using unpaired Student’s T tests, and data shown as mean ± SEM. For sera analysis, dotted lines indicate the average analyte level in non-

+ engrafted, healthy control mice (N=2-3). Blue fluorescence in E indicates DAPI cell nuclei.

Figure 4: Therapeutic application of IL-2C or N-803 exacerbates lupus-like disease. Groups of

+ C57BL/6 mice were engrafted with CD45.1 bm12 splenocytes either (A) one month before administration of IL-2/S4B6 complex vs. IL-2/IgG2a control (daily for 3 days), or (B) one week prior to weekly administration of N-803 vs. IL-15 control. Three months after disease initiation, host-derived

+ (CD45.2 ) splenic GC B cells and plasmablasts were enumerated while total IgG and ANA titers were determined in sera. N=3-7 mice per group. (C) Immunofluorescent staining for IgG and C3 deposits

(green) in 2 representative kidney glomeruli (200x magnification) at 3 months post-disease initiation in mice treated with N-803 as in B, as well as (D) microalbumin detected in urine in these mice at 3 months (N=2-3 mice per group). All bar graphs analyzed using unpaired Student’s T tests and represented as mean ± SEM. For sera analysis, dotted lines indicate the average analyte level in non-

+ engrafted, healthy control mice (N=2-3). Blue fluorescence in C indicates DAPI cell nuclei.

Figure 5: N-803 expansion of CD8 T cells, but not NK cells, contributes to enhanced germinal center

B-cell responses in bm12 model of lupus-like disease and coincides with increased serum IFNγ. (A)

+ Host (CD45.2 ) splenic NK cell, (B) CD8 T cell, and (C) GC B cell counts two weeks following bm12 engraftment of mice pre-treated with N-803 after anti-NK1.1-mediated depletion of NK cells (“ΔNK”), anti-CD8-mediated depletion of CD8 T cells (“ΔCD8”), or IgG2a isotype control administration (n=4-

13 mice per group). (D) Serum IFNγ levels at days 7 and 13 in Bm12-engrafted mice treated as in A-C, as determined using an in vivo cytokine capture assay. Data shown as mean ± SEM. A & B analyzed using Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test due to unequal

SDs. C analyzed via ordinary one-way ANOVA using Dunnett’s test for multiple comparisons. Each day in D analyzed using a Kruskal-Wallis test for nonparametric data with Dunn’s multiple comparisons test. Dotted lines in A-C indicate the mean values in healthy, non-engrafted mice (N=2-3). Dotted line in D indicates the average serum IFNγ level in mice immediately prior to cell depletion, pre-treatment, and engraftment (N=3).

Chapter 3. Elucidating the NK cell transcriptome in SLE identifies

several therapeutic targets for disease intervention

Rationale & Hypothesis

Systemic lupus erythematosus (SLE) is a deadly and debilitating autoimmune disease with no current cure.

The widespread inflammation and profuse autoantibody production that defines SLE certainly have genetic underpinnings134. Genetic exploration of immune cells in the context of SLE has generally focused on T and B lymphocytes, while gene expression in other potentially important lymphocyte subsets, such as natural killer (NK) cells, remain underexplored22. Given that NK cells are known to have a potent immunoregulatory effect187 and are markedly dysfunctional in SLE patients121, it would be highly valuable to elucidate genetic differences in the NK cell compartment of SLE patients and thereby shed further light on pathogenic processes in this devastating disease.

Herein we sought to further elucidate transcriptomic differences between the NK cells of pediatric SLE

(pSLE) patients and healthy, age-matched controls. Using bulk RNA sequencing (RNAseq) of enriched peripheral NK cell fractions, we were able to identify a multitude of transcriptional differences in the NK cells of pSLE patients, some of which have documented ties to NK cell development and function. Using gene network analysis software (AltAnalyze), we were able to predict transcription factor (TF) changes that are relevant to both NK cell function and SLE. Altogether, this groundwork unveils potential genetic alterations of NK cells in human SLE that could represent critical therapeutic targets for disease intervention or guide the development of more effective clinical practices.

Results

Our pSLE cohort potentially exhibits fewer peripheral NK cells than healthy controls.

NK cells are reduced in number in patients with SLE, especially during disease flares120. Certain mouse models of SLE-like disease recapitulate these findings122, including our preliminary findings in the Bm12 inducible model (see Chapter 2, Figure 1).

Whole blood collected from pSLE patients (N=12) and age-matched healthy controls (N=7) was subjected to negative isolation of NK cells, achieving >90% purity as assessed by flow cytometry (data not shown).

All patients were <18 years of age, and the only exclusion criteria was the current use of corticosteroids.

Enumerated cells were divided by the volume of blood collected, revealing a non-statistically significant trend toward decreased NK cell concentrations (p=0.21) in the blood of pSLE patients compared to healthy control subjects (Figure 6). Interestingly, the only two pSLE patients with SLE Disease Activity Indices

(SLEDAI) scores above 0 at the time of blood draw exhibited the lowest NK cell concentrations among all

12 pSLE patients tested (5.56×104 and 6.82×104 per mL), and comprised two of the three lowest concentrations amongst all patients examined.

Together, our findings lends further support to published observations that peripheral NK cells are diminished in the context of SLE and negatively correlate with disease activity.

RNA sequencing of pSLE peripheral NK cells reveals several transcriptional differences critical for NK cell function.

To date, transcriptional profiling of NK cells in the context of SLE or pSLE has not been previously performed. We subjected enriched populations of peripheral NK cells from 6 pSLE patients (see details,

Table 1) and 7 healthy, age-matched controls to bulk RNA sequencing. Raw sequencing files that passed quality control testing were trimmed, aligned to the human genome, de-duplicated, and read-counted to obtain Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for comparison between groups.

We found over 2000 genes that were significantly differentially expressed between our pSLE cohort and healthy controls (data not shown). More specifically, these genes exhibited an adjusted p value < 0.05 and a log2 fold change greater than 1 or less than -1. The top 50 differentially-expressed genes (Figure 7) exhibited adjusted p-values less than 9.5×10-13 and a log2 fold change >2 or <-2. Within this set, 49 genes were upregulated and one was downregulated in pSLE NK cells vs. healthy control NK cells. Furthermore, several genes within this select group have known associations with NK cell function (see details, Table

2).

Altogether, RNAseq analysis reveals that a multitude of transcriptional differences exist in the peripheral

NK cells of pSLE patients, some of which could potentially contribute to the dysfunctional phenotype of

NK cells observed in SLE. As these NK cells were collected in patients not taking corticosteroids, we can rule out the confounding effects that steroid treatment would otherwise have had on NK cell phenotype.

Several patients were on other immune-modifying drugs at the time of specimen collection that may have affected our results (see Table 1), although the effects of these drugs on NK cell function remain ill- explored. Furthermore, our data suggests that upregulated gene expression far outweighs downregulated expression in pSLE peripheral NK cells, proposing that SLE NK cells may be more transcriptionally “active” over baseline, in general.

Gene network analysis of the pSLE NK cell transcriptome predicts the involvement of transcription factors with relevant associations to NK cells and SLE pathogenesis.

The expression of a gene does not exert its effects in a vacuum, but in a context of numerous other genes being expressed in parallel, forming a network of transcriptional interactions. Gene network analysis offers the ability to untangle these interactions and identify important genetic players (e.g. TFs) from a systematic level192. One major advantage of such analysis is to identify potential upstream genetic targets

64 that, if modified therapeutically, could result in vast transcriptional changes and achieve broader beneficial effects than manipulation of a single downstream gene alone.

In attempt to identify such targets, we used AltAnalyze193 to interpret our RNAseq results, generate gene network maps, and predict candidate TFs with known associations to genes that were transcriptional altered in pSLE NK cells. It is important to note that some of these TFs (e.g. PU.1) were not directly identified in our sequencing results, but were nonetheless predicted by AltAnalyze to be involved due to their known ability to bind promoter regions of multiple genes that were identified as altered by RNAseq.

Physiologically speaking, this could be due to one of several ways by which the activity of TFs are not always dictated by their own transcription (thus not detected via RNAseq), but can be post-translationally controlled via nuclear localization, cofactor binding, and epigenetic modification, amongst other mechanisms194. Nevertheless, network analysis of significantly upregulated genes resulted in interconnected gene clusters emanating from 10 TFs (Figure 8). Of the TFs predicted, at least three have known associations with both SLE and NK cells (see details, Table 3). Thus, our transcriptomic investigation of pSLE NK cells reveals potentially relevant TFs whose expression could be altered to achieve a broad therapeutic effect in SLE NK cells, and perhaps impact the course of disease.

Future studies will be needed to determine whether genetic manipulation of specific downstream genes

(such as those identified in Figure 7), or modification of major upstream TFs (like those predicted in Figure

8), have significant effects on human NK cell function. Furthermore, whether or not “correction” of NK cell function via this genetic manipulation has therapeutic effects in an SLE disease state will require much additional investigation. Nonetheless, the data presented herein exemplifies critical groundwork upon which a novel NK cell-centric treatment approach for SLE could be built.

Acknowledgements

We thank M. Pujato for his expertise in RNAseq analysis, as well as K. Ernst, T. Hong, and A. Ali for their assistance. We thank N. Salomonis for his expertise in using AltAnalyze, and L. Kottyan & K. Kaufman for their consultation. Specimen collection was kindly coordinated by the CCHMC Division of Rheumatology research team, with leadership from H. Brunner. RNA sequencing was performed by the University of

Cincinnati Genomics, Epigenomics and Sequencing Core (GESC) with excellent support from X. Zhang. Flow cytometry was accomplished with help from the Cincinnati Children’s Flow Cytometry Core. Funding was provided by NIH grants DA038017, T32GM063483, and T32AI118697, as well as the CCHMC Center for

Pediatric Genomics (CpG).

Figure 6: Peripheral NK cell numbers trend toward a reduction in a pediatric SLE patient cohort.

Enriched peripheral NK cells from pSLE patients (N=12, <18 years of age, not on corticosteroids) and healthy, age-matched controls (N=7) were enumerated and divided by the volume of whole blood collected. Data analyzed via unpaired Student’s T test and represented as mean ± SEM.

Table 1. Available clinical information and current medications of the 6 pediatric SLE patients from which peripheral blood NK cells were isolated and analyzed via RNAseq. Values highlighted in blue and red represent results that were below and above a standard reference range (www.mayoclinic.org), respectively. One patient (#2) was excluded due to an RNA extract that did not pass quality-control testing. For de-identification purposes, gender and age of patients were not available, although all patients were less than 18 years old. Clinical data was not collected from the 7 age/gender-matched control patients that were used, thus is not shown. WBC = white blood cell count; Neut = neutrophil count; Lymph = lymphocyte count; Mono = monocyte count; Plt = platelet count; ESR = erythrocyte sedimentation rate; SLEDAI = SLE disease activity index; MTX = methotrexate; HCQ = hydroxychloroquine; MMF = mycophenolate mofetil.

Patient WBC Neut Lymph Mono Plt ESR SLE SLEDAI ID (103/uL) (103/uL) (103/uL) (103/uL) (103/uL) (mm/hr) Medications MTX, HCQ, 1 n/a n/a n/a n/a n/a n/a n/a Belimumab

3 5.4 3.13 1.24 0.65 294 31 0 HCQ

HCQ, 4 2.4 0.82 1.32 0.24 228 21 2 Tocilizumab

5 4.2 2.56 1.22 0.29 376 43 0 HCQ, MMF

None (non- 6 6.2 n/a n/a n/a 180 87 0 adherent)

7 4.9 2.84 1.47 0.49 217 5 0 HCQ, MMF

Figure 7: RNA sequencing of pSLE peripheral NK cells reveals several transcriptional differences versus peripheral NK cells from healthy controls. Heatmap reflecting Fragments Per Kilobase of transcript per Million mapped reads (FPKM) from RNA sequencing performed on enriched healthy

(right, N=7) or pSLE (left, N=6) peripheral NK cells. Top 50 differentially-expressed genes are presented in order of lowest-to-highest adjusted p-value, ranging from 9.85×10-13 – 2.46×10-64. All genes shown have a log2 fold change >2 or <-2. Pt ID = patient identification number (refer to Table 1 for pSLE patient details). Select gene involvement in NK cell development/function is detailed in Table 2.

Table 2. Known relevance of specific genes within the top 50 most differentially-expressed genes in pSLE peripheral NK cells vs. healthy control NK cells. Genes listed in descending order of significance (adjusted p-value).

Transcriptional Gene Gene Name Change (pSLE Relevance & Citation Symbol over control) Tumor excretion of alpha-fetoprotein was shown to Alpha- AFP Upregulated directly induce an NK cell pro-inflammatory fetoprotein phenotype and reduce NK cell survival in vitro195. Platelet-derived High PDGFRA expression in human NK cell PDGFRA growth factor Upregulated lymphoma associated with increased tumor survival receptor alpha and worse patient outcomes196. Iron-saturated human transferrin at physiologically TF Transferrin Upregulated relevant levels inhibits NK cell cytotoxicity against K562 tumor cells.197 AMBP is increased in the cerebral spinal fluid of Alpha-1- patients with extranodal natural killer cell/T-cell AMBP microglobulin/ Upregulated lymphoma of nasal-type (NKTCL) following bikunin precursor chemotherapy198. Proteinase 3-stimulated endothelial cells were PRTN3 Proteinase 3 Upregulated shown to trigger NK cell degranulation in vitro199. Volunteers drinking a select mineral water, shown AQP2 Aquaporin 2 Downregulated to promote AQP2 activity in rat oocytes in vitro, were found to exhibit enhanced NK cell activity200. IL-12 triggers NK cell secretion of MPO201. MPO Myeloperoxidase Upregulated MPO reduces NK cell cytotoxicity via generation of reactive oxidative species202.

Figure 8: Gene network analysis of the pSLE NK cell transcriptome predicts the contribution of 10

TFs to observed differences in RNA expression versus control NK cells. RNA sequencing data of enriched healthy (N=7) and pSLE (N=6) peripheral NK cells was subjected to AltAnalyze analysis to generate interconnected clusters of identified upregulated genes (red dots/squares), as well as to predict the involvement of unidentified TFs (yellow squares). Three TFs (and their local gene networks) with known relevance to both NK cells and SLE are highlighted: ARNT (red), SPI1/PU.1

(blue), and STAT1 (green). Further detail on the relevance of these TFs is explained in Table 2.

Table 3. Known relevance of specific transcription factors predicted to be operating based upon gene network analysis of differentially-expressed genes in pSLE peripheral NK cells vs. healthy control NK cells.

TF Symbol TF Name/Alias Relevance & Citation  Forms a heterodimer with HIF1-alpha. A mouse strain expressing constitutively active HIF1-alpha/ARNT in Aryl hydrocarbon hematopoietic cells exhibited a hemophagocytic receptor nuclear ARNT lymphohistiocytosis-like disease that included a dysregulated translocator, and reduced NK cell compartment (including reduced HIF1-Beta production of IFNγ)203.  HIF1-alpha polymorphisms are associated with SLE204.  PU.1 is a critical regulator of NK cell differentiation and homeostasis205.  Suppressing PU.1 in human NK cells led to decreased SPI1 Spi-1 proto- cytotoxicity in vitro206. (PU.1) oncogene, PU.1  PU.1 is upregulated in PBMC and B cells of pSLE patients and directly correlates with SLEDAI score207.  Significant associations exist between SPI1 polymorphisms and SLE208.  Patients with STAT1 gain-of-function domains exhibit impaired NK cell functions, including reduced proliferation, Signal transducer decreased IFNγ production, and impaired degranulation and STAT1 and activator of cytotoxicity209. transcription 1  Activation of STAT1 is reported in both human SLE and relevant mouse models210,211.

Chapter 4. Therapeutic targeting of follicular helper T cells with

chimeric antigen receptor-expressing natural killer cells

Rationale & Hypothesis

Follicular helper T cells (TFH) are critical for vaccine and infection elicitation of long-lived humoral immunity, but exaggerated TFH responses promote autoimmunity and other pathologies. Unfortunately, no clinical interventions are currently available for selective depletion of TFH cells to alleviate these disease conditions. We engineered a novel chimeric antigen receptor (CAR) that facilitates specific targeting of cells highly expressing human programmed cell death protein 1 (PD-1), a cardinal feature of TFH cells. CAR- expressing natural killer (NK) cells robustly degranulate in response to PD-1, resulting in discriminatory elimination of human tonsil TFH cells while sparing other T and B-cell subsets. Given that CAR NK cells are emerging as a safe and effective alternative to CAR T cells in cancer immunotherapy, our results suggest a new, clinically-translatable application of CAR NK cells for selective depletion of pathogenic TFH cells in specific disease states.

We engineered and evaluated an innovative CAR NK-cell that targets PD-1-expressing cells to eliminate

TFH cells. The extracellular portion of programmed death-ligand 1 (PD-L1) was used in place of a scFv to confer selectivity of the CAR NK cells for TFH that express very high levels of surface PD-1 (see Chapter 4 graphical abstract, below). This finding establishes CAR NK cell targeting of PD-1 as a promising new approach for therapeutic culling of TFH cells that may be valuable in autoimmunity and other TFH-driven disease states.

Chapter 4 Graphical Abstract: PD-L1 CAR NK cells selectively kill PD-1-expressing TFH cells and indirectly reduce downstream B cell dynamics, including the production of antibodies. Thus, our CAR NK cells have clinical relevance in an autoimmune context during which TFH cell interaction with B cells elicits the pathogenic production of autoantibodies. TN = naïve T cells, TM = memory T cells, Treg = regulatory T cells.

Results

PD-1 is a selective marker of human TFH cells

The PDCD1 gene encoding PD-1 is expressed at low to intermediate levels on several types of leukocytes,

15 but is highly expressed by bona fide TFH cells . Flow cytometer-acquired cell-surface expression of PD-1

+ + on resting, non-inflamed human tonsil mononuclear revealed that electronically gated TFH cells (CD3 CD4

CXCR5+ ICOS+) exhibit the highest degree of PD-1 expression relative to other major tonsillar leukocyte subsets (Figure 9A). The median fluorescence intensity (MFI) of PD-1 expression on TFH cells exceeded that of regulatory T cells (Treg, CD3+ CD4+ CD25+ FoxP3+); naïve (CD45RA+ CD45ROneg) or memory (CD45RAneg

CD45RO+) T cells (CD3+, CD4+ or CD8+); immature (CD21+ CD38hi CD27neg IgM+ IgDneg), mature (CD27+ IgD+

IgM+), memory (CD21+ CD27+ CD38neg), and follicular (CD21+ CD38lo CD27neg IgD+ IgMneg) B cells (CD3neg

CD19+ CD20+); plasmablasts (CD3neg CD19+ CD27+ CD138+ CD38hi); NK (CD45+ CD3neg CD56+) or natural killer

T (NKT; CD45+ CD3+ CD56+) cells; classical (CD16neg) and non-classical (CD16+) monocytes (CD45+ CD3neg

CD19neg CD14+); and dendritic cells (CD45+ CD3neg CD19neg CD1c+ HLA-DR+) in both human tonsil (Figure

9B) and peripheral blood mononuclear cells (PBMC) (Figure 9C). PD-1 expression on tonsillar TFH cells was only surpassed by the closely-related, albeit >1,600-fold less abundant (data not shown) follicular

+ + + + + + regulatory T cell (TFR, CD3 CD4 CXCR5 ICOS CD25 Foxp3 ) population (Figure 9A, B). Thus, TFH cells demonstrated roughly 6- to 600-fold higher PD-1 expression (by MFI) than most other leukocytes.

PD-L1-based CAR generation and expression on NK cells

high Selective targeting of PD-1 TFH cells may be achieved by optimizing the affinity of a CAR to limit its activation by PD-1low cells. The anti-tumor antigen antibody-derived scFv often used in CAR design typically confer an EC50 affinity of 0.015 to 320 nM, which facilitates killing of targets exhibiting both high and low

212,213 expression levels of the target protein . In contrast, the Kd affinity of human programmed death- ligand 1 (PD-L1), also known as B7-H1 or CD274, for PD-1 is reported between 770 and 8,200 nM214,215.

Therefore, we reasoned that the lower affinity of PD-L1 for PD-1, relative to the scFv of an anti-PD-1

high low antibody, would permit more selective targeting of PD-1 TFH versus other PD-1 bystander cells.

We cloned the extracellular domain of human PD-L1 (a.a. 19-238) upstream of conventional CAR components175 (Figure 10), into a lentiviral plasmid. Empty and PD-L1-CAR-containing plasmids were packaged in vesicular stomatitis virus glycoprotein-pseudotyped viral particles and subsequently used to transduce the human NK cell-line, NK-92, which is currently being utilized for immunotherapy216. Based on fluorescent reporter expression, transduction efficiency in NK-92 cells was 6.8% and 1.3% for the empty and CAR-encoding lentiviral vectors, respectively, as measured by flow cytometry (Figure 11). After sorting for fluorescent reporter-positive cells, CAR transgene mRNA and surface PD-L1 were detected on CAR- transduced but not empty vector-transduced NK-92 (Figure 12).

PD-L1 CAR NK cells are activated by plate-bound ligands

CAR NK-92 cells in short-term culture with either plate-bound antibody specific for PD-L1 (α-PD-L1) or recombinant human PD-1-Fc fusion protein (rhPD-1-Fc) triggered degranulation, as measured by surface exposure of CD107a (Figure 13A). This response was not observed in control NK-92 cells. Neither CAR nor control NK-92 responded to IgG-Fc fusion protein or immunoglobulin isotype (negative controls), while both CAR and control NK-92 responded robustly to stimulation with (positive control) phorbol myristate acetate (PMA) and ionomycin (Figure 13A). Stimulation of CAR NK-92 over increasing concentrations of plate-bound rhPD-1-Fc (Figure 13B) revealed a response curve with an affinity of PD-L1 CAR NK-92 for rhPD-1-Fc with a calculated EC50 of 0.61 µg/mL. These experimental findings establish that our PD-L1- based CAR construct was functional and responsive to ligands that bind PD-L1.

PD-L1 CAR NK cells respond to cell-associated PD-1 via degranulation and killing

The drosophila melanogaster-derived S2 cell line lacks expression of relevant activating or inhibitory receptors for human NK cells, and therefore elicits no functional NK-cell response217. Consistent with this data, NK-92 cells did not degranulate when co-cultured with S2 cells, regardless of CAR expression (Figure

13C). In contrast, CAR NK-92 but not control NK-92 degranulated (Figure 13C) during co-culture with S2 cells engineered to express surface human PD-1 (Figure 14A, B). Moreover, CAR NK-92 cells degranulated

34% more (Figure 13D) during co-culture with sorted “PD-1Hi” S2 cells (Figure 14A) than CAR NK-92 cell co-cultured with “PD-1Lo” S2 cells. Similarly, Raji human B lymphoma cells engineered to express high levels of human PD-1 (Figure 14C, D) triggered 2.4-fold more degranulation of CAR NK-92 relative to degranulation in response to wild-type Raji cells (Figure 13E). PD-1+ Raji cells cultured in the presence of

CAR NK-92, but not those cultured with control NK-92, exhibited uptake of propidium iodide (PI) as a measure of loss of viability (Figure 13F). Furthermore, 51Cr-labeled PD-1+ Raji cells cultured with CAR NK-

92 released more radioactive chromium into culture supernatant (in an effector cell concentration- dependent manner) than PD-1+ Raji cultured with control NK-92 (Figure 13G). Thus, CAR NK-92 recognize and respond to cell-surface PD-1 expressed on otherwise NK-cell refractory target cells.

PD-L1 CAR NK cells selectively kill TFH cells and suppress TFH-induced B-cell responses.

To test the function of PD-L1 CAR NK-92 cells in response to bona fide human TFH, bulk CD4 T cells

(including TFH, TFR, and Treg) were magnetically-purified from non-inflamed human tonsil. Co-culture of tonsil CD4 T cells with CAR-expressing NK-92 for 4 hours resulted in greater than a 7-fold reduction in recoverable TFH cells relative to cultures of CD4 T cells alone (Figure 15A). This was not observed when

CD4 T cells were co-cultured with control NK-92 (Figure 15A). Correspondingly, remaining TFH cells after culture with CAR NK-92 exhibited 9-fold more PI uptake than those cultured with control NK-92 or without added NK cells (Figure 15B), as well as enhanced Zombie viability dye staining (Figure 16A). Non-TFH CD4 memory T cells also exhibited increased loss of viability when cultured with CAR NK-92, though overall PI

79 uptake in this population was miniscule relative to that of TFH cells (Figure 16B). Naïve CD4 T cells (Figure

15B) and Treg cells (Figure 15C) demonstrated no such loss of viability over background after co-culture with either type of NK-92.

To determine capacity of CAR NK cells to target TFH in a more complex cellular milieu reminiscent of

neg lymphoid follicles, we generated co-cultures of human tonsillar lymphocytes enriched for TFH cells (CD19

CD3+ CD4+ CXCR5+) and memory B cells (CD3neg CD19+ CD27+) added in a 1:2 ratio, respectively.

Staphylococcal enterotoxin B (SEB), which crosslinks the T-cell receptor with MHCII218, was added to these co-cultures to trigger mutual cell proliferation. At an effector (NK-92) to target (TFH) ratio (E:T) of 5:1, CAR

NK-92 demonstrated 12-fold more degranulation than control NK-92 (Figure 17A). Calcein-AM labeled TFH and B cells co-cultured with CAR-expressing but not control NK-92 released calcein into culture supernatant in an effector cell concentration-dependent manner (Figure 17B). PI staining of target lymphocytes revealed greater loss of viability in TFH cells cultured with CAR NK-92 cells compared to control NK-92 (Figure 17C). B cells, conversely, exhibited no increased PI uptake following addition of either CAR-expressing or control NK-92 (Figure 17C). In a separate assay, we cultured enriched tonsillar

CD4 T cells in the presence of control or CAR NK-92 for 4 hours (followed by subsequent magnetic depletion of CD56+ NK cells), then co-cultured 3×10^4 residual CD4 T cells with autologous, CellTrace

Violet (CTV)-labeled memory B cells (in a 1:2 ratio) in the presence of SEB. We observed less proliferation of memory B cells (Figure 17D), decreased prevalence of plasmablasts (Figure 17E), and less supernatant

IgG (Figure 17F) in wells containing CD4 T cells pre-cultured with CAR NK-92. Together, these results demonstrate that PD-L1 CAR-expressing NK-92 promote death of TFH cells while sparing other lymphocyte populations. Consequently, this killing suppresses TFH cell-mediated effects on memory B cells, including the production of immunoglobulin.

Acknowledgements

We would like to acknowledge: D. Krishnamurthy, D. Ohayon, and M. Khodoun for technical assistance;

H. Seelamneni for lab managerial support; E. Fjellman and C. Forney for helping to obtain and process tissues; D. Miller and A. Sauder for cloning support; L. Ding for statistical expertise; and E. Long, S. Borrow,

D. Peppa, and I. Pedroza-Pacheco for expert advice. Funding was provided by the Dr. Ralph and Marian

Falk Medical Research Trust Catalyst Awards Program and NIH grants DA038017, T32GM063483,

T32AI118697, and AR073228. Support was also provided by Cincinnati Children’s Viral Vector Core, Flow

Cytometry Core, and Functional Genomics Core (NIH grants AR070549 & S10OD023410), as well as the

Cincinnati Children’s Research Foundation.

Figure 9: PD-1 is a selective marker of human TFH cells. (A) Representative PD-1 expression levels determined by flow cytometry on electronically gated subsets of leukocytes in non-inflamed human tonsil. (B) Median PD-1 expression (median fluorescence intensity) across major leukocytes subsets identified in two independent non-inflamed human tonsils. (C) Median PD-1 expression across major leukocytes subsets identified in three healthy human PBMC.

Figure 10: PD-L1 CAR design. (A) Coding sequence for CAR construct, color-coded by domain. (B)

Graphical depiction of CAR protein expressed on cell surface (dashed line), with specific domains.

Figure 11: Generation of CAR NK-92 via lentiviral transduction. Percent transduction efficiency of

NK-92 with empty lentiviral vector (top-left) and CAR-containing lentiviral vector (bottom-left) as measured by reporter fluorescence. Images of the corresponding sorted fluorescent NK-92 (right, top and bottom) following ~1 week of culture (growing in characteristic clumps).

Figure 12: CAR-transduced NK-92 express CAR-specific mRNA and surface PD-L1. (A) Graphical depiction of RNA alignment of CAR-specific qPCR primer/probe sets (top) and the corresponding fold CAR mRNA expression (bottom) in control vs. CAR NK-92. mRNA expression normalized to

GAPDH. (B) PD-L1 expression of empty-vector-transduced vs. CAR-lentivirus-transduced NK-92, including PD-L1 fluorescence-minus-one (FMO) control.

Figure 13: PD-L1 CAR NK cell responses to plate- and cell-associated PD-1. (A) Degranulation (surface exposure of CD107a) of control (left) or CAR (right) NK-92 following a 4-hour incubation in the presence of either anti-PD-L1 IgG vs. goat IgG control (20 µg/mL), or rhPD-1-Fc vs. IgG-Fc control (10µg/mL). (B)

Degranulation (as in A) in response to increasing concentrations of rhPD-1-Fc or IgG1-Fc, or (C) in the presence of either control or PD-1+ drosophila S2 cells (or PMA/Ionomycin) at an effector:target ratio of

1:5 for four hours. (D) Fold degranulation (over control NK-92 with control S2) of CAR NK-92 in the presence of either control, PD-1Lo, or PD-1Hi drosophila S2 cells at a 1:5 E:T for four hours (n=2-3). Dotted line represents average degranulation of control NK-92 cultured with control S2 cells. Data were analyzed via 1-way ANOVA with multiple comparisons. (E) CAR NK-92 degranulation in the presence of control or

PD-1+ Raji cells for four hours at a 1:5 E:T (n=4). Data analyzed using Student’s t-test. (F) Control or PD-1+

Raji uptake of propidium iodide following 4-hour co-culture with either control or CAR NK-92 at a 20:1 E:T

(n=2-3). Data analyzed using 1-way ANOVA. (G) Percent lysis of chromium-51(51Cr)-labeled PD-1+ Raji cells after incubation with control or CAR NK-92 for four hours over an increasing E:T ratio. Percent lysis calculated as [(sample release – spontaneous release)/(maximum release – spontaneous release)] for 4 biological replicates. Error bars (B and G) denote standard deviation. Representative data from one of two

(B,D-G) or three (A,C) experimental replicates is shown.

Figure 14: Generation of target cell lines expressing human PD-1. (A) Contour plots showing PD-1 expression in pAc5/V5-His-PD1-transfected S2 cells, and electronic gates indicating sorting parameters for PD-1+ (left) or PD-1Hi and PD-1Lo (right) cell populations. (B) Resulting PD-1 expression on sorted populations in A, after 1 week of culture vs. empty-vector-transfected control S2 cells. (C)

Contour plots showing percent PiggyBac transposition efficiency of Raji cells with PB-513 empty- vector (left) and PD-1-containing PB-513 vector (right) as measured by reporter fluorescence. (D)

Resulting PD-1 expression in the fluorescently-sorted Raji cells in C after 1 week of culture.

Figure 15: PD-L1 CAR NK cells selectively kill TFH cells. (A) Representative contour plots of enriched tonsillar CD4 T cells, with circle denoting electronic gating of TFH cells (CXCR5+ PD-1+) following a 4- hour incubation either alone (left, n=1) or in the presence of control NK-92 (middle, n=5) or CAR NK-

92 (right, n=5). Percentages indicate frequency of all CD4 T cells ±SEM. (B) Enriched tonsillar CD4

Naïve T, non-TFH memory T, or TFH cell uptake of PI following 4-hour incubation with either control or

CAR NK-92 (n=8). Dotted lines represent PI uptake in the absence of NK-92. Data analyzed using

Student’s t-test with Welch’s correction. (C) CD4+ Foxp3+ CD25+ CD127lo Treg uptake of Zombie viability dye following 4-hour incubation of enriched tonsillar CD4 T cells without NK-92 or with either control or CAR NK-92 (n=4-8). Dotted line indicates the average Zombie dye uptake of TFH cells incubated with CAR NK-92 (n=8) in the same experiment. Data analyzed using 1-way ANOVA. A and B represent one of two experiments, whereas C includes pooled data from two patient tonsils.

+ + + Figure 16: PD-L1 CAR NK cells kill both tonsil and peripheral TFH cells. (A) CD4 CXCR5 ICOS TFH cell uptake of Zombie viability dye following 4-hour incubation of enriched tonsillar CD4 T cells with either

control or CAR NK-92 (n=4). Dotted line indicates the average Zombie dye uptake of TFH cells alone

+ (n=2). Data analyzed using unpaired Student’s T test. (B) PI uptake in sorted, SEB-stimulated CD4

+ CXCR5 peripheral blood T cells co-cultured with either control or CAR NK-92 at a 5:1 E:T ratio for 4 hours, or a 10:1 ratio for 20 hours (n=2-4). 20-hour co-culture analyzed via unpaired Student’s T test.

4-hour co-culture not analyzed due to low N. Results are representative of two independent experiments.

Figure 17: PD-L1 CAR NK cells kill TFH cells in T:B co-cultures and suppress TFH-dependent B-cell responses. (A) Control or CAR NK-92 degranulation following 4-hour co-culture with SEB-stimulated

+ tonsillar TFH and CD27 B cells at an NK:T:B cell ratio of 5:1:2 (n=5). Representative one of three experiments is shown. Data analyzed via Student’s t-test. (B) Percent lysis of calcein-AM (CAM)-

+ labeled, SEB-stimulated tonsillar TFH and CD27 B cells after incubation with control or CAR NK-92 for four hours over an increasing E:T ratio, as measured by calcein fluorescence in supernatant. Shaded area is ≤ percent background lysis of T and B cells alone. Error bars denote standard deviation. Percent lysis calculated as [(sample fluorescence – minimum fluorescence)/(maximum fluorescence –

+ minimum fluorescence)] for 3 biological replicates. (C) TFH and CD27 B cell PI uptake following 4-hour incubation with control or CAR NK-92 (n=4). Uptake in each cell population alone represented by dotted line. Data analyzed via Student’s t-test. (D) Frequency of proliferating (CTV-) CD27+ B cells (n=3-

8) and (E) prevalence of CD19+ CD27+ CD38+ plasmablasts (n=3-8) after 7 days of SEB-stimulated co- culture with tonsillar CD4 T cells that were pre-cultured either without (“No NK-92”) or with control or CAR NK-92. (F) Total supernatant IgG at day 7 in the co-culture assay used in D and E (n=3-8).

Dotted lines in D-F represent the average value of wells containing CD4 T cells not pre-cultured with

NK-92 and not stimulated with SEB (n=2). D-F were analyzed via 1-way ANOVA with multiple comparisons (comparing each group to “No NK-92” group”). One of two similar experiments is shown.

Chapter 5. Summary, discussion, & future directions

Overall summary of findings

This dissertation represents an effort to cultivate and test novel NK cell-based therapeutic options for the devastating autoimmune disease SLE. The work herein took a three-pronged approach to developing such therapies: expanding endogenous NK cells to potentially achieve an immunosuppressive outcome

(Chapter 2), interrogating the SLE NK cell transcriptional profile to identify functional defects that might be correctible and beneficial for disease (Chapter 3), and engineering an NK cell that could selectively eliminate pathogenic cells underlying SLE (Chapter 4).

Though some of our endeavors were more successful than others, much has been learned in this undertaking. To briefly summarize, both therapeutic and prophylactic administration of cytokine-based therapeutics to expand endogenous NK cells in SLE-like mice (at least transiently) enhanced disease measures. Although this result was unexpected, further analysis revealed that the concomitant expansion of CD8 T cells and increased serum IFNγ levels may be at least partially culpable for disease exacerbation, while expansion of NK cells alone had negligible effects. Transcriptional characterization of NK cells in pSLE revealed a multitude of RNA expression changes versus healthy controls, some of which have known relevance to NK cell function. Based upon our transcriptional findings, gene pathway analysis predicted changes in other, broader-acting genes (e.g. PU.1) with documented associations to both NK cells and SLE, thereby identifying potential genetic targets whose modification in NK cells could have a therapeutic benefit. Finally, engineering NK cells to express a novel CAR construct resulted in successful, targeted elimination of human TFH cells in vitro, leading to indirect suppression of downstream ASC generation and antibody production. Our CAR NK cell represents a potent new therapeutic opportunity to be further advanced and heavily considered in autoimmune diseases such as SLE.

Discussion of results and outstanding questions

Is endogenous NK cell expansion a worthwhile therapeutic avenue for treating SLE?

Mirroring (and adding to) data reported both in human SLE and relevant mouse models120,122,129,131 (see

Chapter 1), we observe that the Bm12 inducible model of SLE-like disease also exhibits a contraction of the NK cell pool shortly after disease induction (Figure 1A, B). Though the cause of this contraction remains unclear and should be further explored, it may be related to elevated levels of type I IFN associated with the Bm12 model54, as type I IFN effects are directly linked to a reduced NK cell compartment125.

Nevertheless, it is evident that treatment of SLE-like mice with IL-2Rβ/γc-targeted cytokines (either IL-

2/anti-IL-2 (S4B6) complexes or the IL-15 superagonist N-803) counteracts NK cell contraction. Both treatments result in similar or above-average levels of NK cells (as compared to healthy controls) despite ongoing disease (Fig. 1A, B).

Upon examination of NK cells in the Bm12 model, we observed elevated surface expression of CD27,

TRAIL, and NKG2A as compared to BoyJ-engrafted control mice (Fig. 1C). The expression of these markers on mouse NK cells is associated with a more immature phenotype93, and may mirror impaired differentiation and the abundance of immature NK cells that is seen in SLE patients and other relevant mouse models121,131. As the Bm12 model exhibits a type I IFN signature54, and IFNβ is known to expand

“immature” CD56bright NK cells in MS patients101, it is possible that type I IFN drives a more immature NK cell state in this model. We also observed increased CD11c on the NK cells of mice receiving a bm12 graft

(Fig. 1C). This marker is part of a phenotypic profile that identifies a proinflammatory NK cell subset associated with human SLE and specific mouse models142,151,152. Although more characterization should be done to confirm whether our observed NK cells represent this subset, it is possible that the change in

CD11c reflects a shift toward a more proinflammatory and disease-aggravating NK cell phenotype in the

Bm12 model. An elevated type I IFN signature in our model may play a role, as IFNα is shown to induce

CD11c+ NK cells in vitro142. Our depletion experiments, however, suggest that NK cells are not actively contributing to disease in bm12-engrafted mice (Fig. 5C, D), and are therefore not likely proinflammatory.

Future experiments should seek to test whether the NK cells in bm12 SLE-induced mice are actually less functional, thus mirroring the NK cells of SLE patients. For example, NK cells could be magnetically isolated from bm12-engrafted mice after disease initiation and tested ex vivo in standard killing assays (e.g. chromium release assays), or in activation assays to examine their production of IFNγ or TNFα (as compared to NK cells from BoyJ-engrafted control mice). Even more relevant, to test their killing efficacy against autologous and activated TFH cells (and potential downstream regulatory effects on the GC), NK cells isolated from bm12-engrafted mice could be added to SEB-stimulated (mouse-derived) T:B co- cultures in assays similar to those used in Chapter 4. Reduced or absent ability of bm12-engrafted mouse

NK cells to kill activated TFH and indirectly suppress downstream B cell dynamics in this co-culture system may suggest that the bm12-induced SLE-like disease process invokes reduced regulatory function in host

NK cells, thus contributing to disease pathogenesis and possibly causing the altered phenotype we observed in Fig. 5C.

Studies have shown that SLE patients have a slightly elevated overall risk of malignancy, including a three- fold higher risk of hematologic cancers such as leukemia and lymphoma219. Although several factors likely contribute to the association between SLE and malignancy (e.g. genetics, chronic inflammation, and immunosuppressive drug treatments), the relevance and potential consequences of a concurrent NK cell deficit in an SLE patient with cancer remains unexplored. If future studies reveal that an SLE-related NK cell defect underlies the observed increased cancer risk, our findings suggest that the immunotherapeutic agents tested herein may successfully counter an NK cell insufficiency in SLE patients and potentially offer a valuable cancer treatment option (albeit with risks and limitations, as discussed below).

SLE often manifests as debilitating flares of disease activity – episodes which are known to coincide with rampant GC reactions49. Thus, the rapid GC kinetics and expansion of autoantibody-producing ASCs that define the Bm12 SLE mouse model provide an opportunity to test whether treatment prior to disease induction prevents or suppresses an inflammatory context closely resembling an SLE flare. We prophylactically treated B6 mice with either IL-2/anti-IL-2 (S4B6) complexes or N-803 before injecting a

Bm12 splenocyte graft in the hope that an expanded pool of host NK cells might quell the ensuing GC reactions. Contrary to our hypothesis, pre-treated mice exhibit enhanced GC activity at two weeks than control-treated groups, as indicated by increased GC B cells, plasmablasts and total serum IgG (Fig. 3A, B).

At three months, however, prophylactic treatment has no lasting effects on GCs or other disease measures, including serum ANA (Fig. 3C-F). Together, these data suggest that prophylactic treatment of an SLE patient with treatments such as IL-2/anti-IL-2 (S4B6) complexes or N-803 may in fact worsen GC reactions during a flare, though this effect appears to dissipate without resulting in subsequent autoimmune consequences (presumably due to treatment effects that wane by 3 months).

Our findings are in concurrence with those published in a study by Heiler et. al. wherein the prophylactic effect of IL-2/anti-IL-2 (S4B6) complexes in a similar cGvHD mouse model of SLE-like disease was found to exacerbate disease166. They theorize that IL-2/anti-IL-2 (S4B6) pre-treatment caused an off-target expansion of graft CD4 TFH cells (which is unexpected and never fully explained) that subsequently enhanced host GC reactions. However, the Bm12 model used in our investigation is defined by graft TFH cells intensifying host GCs, yet we observe no significant difference in graft TFH expansion between both cytokine pre-treatment groups and their respective controls (data not shown). Nonetheless, Heiler and colleagues observed the opposite effect when their SLE-like mice were treated with IL-2/anti-IL-2 (S4B6) complexes after disease was initiated166. Thus, we sought to determine whether this therapeutic effect

97 would take place in our model as well, going so far as to specifically match our treatment schema (see methods) to theirs to allow for better comparison.

Unlike Heiler et. al., we observe that treatment of mice with IL-2/anti-IL-2 (S4B6) complexes or N-803 after initiating SLE-like disease exacerbates GC reactions and increases measures of autoimmunity (Fig. 4).

Although we again anticipated a suppressive effect of expanded NK cells on GC reactions, we find that GC

B cell and plasmablast populations, in addition to total IgG production and proteinuria all exhibit a (non- significant) trend toward increase by three months post-disease. Presumably, these immunologic changes may explain an observed significant increase in serum ANA following treatment with either cytokine complex versus control-treated groups. We observed no difference in IgG or C3 kidney deposits between groups, but this may be reflective of disease not being severe enough by 3 months. Regardless, our findings suggest that (as was found using a prophylactic approach) therapeutic treatment of an SLE patient with IL-2/anti-IL-2 (S4B6) complexes or N-803 could worsen GC reactions and aggravate their autoimmune disease.

Our findings are contrary to Heiler and colleagues potentially due to a critical difference between the disease models that were used. Whereas their DBA/2 into B6 × DBA/2 F1 model manifests from MHC class I and II mismatch166, our Bm12 model is MHC class II-restricted. Although Heiler et al surmise that the disease amelioration they observe after therapeutic treatment with IL-2/anti-IL-2 (S4B6) complexes could be due to expanded CD8 T cell graft allo-elimination of MHC class I-mismatched host B cells166, the same alloreaction should not apply to the class II-restricted Bm12 model185. Further manipulation of our investigation, however, revealed a potential and more indirect role for expanded CD8 T cells in SLE-like disease exacerbation.

To further elucidate whether expanded NK cells or CD8 T cells underlie the enhanced GC kinetics observed in our pre-treatment approach (Figure 3), we performed a series of experiments in which either NK cells or CD8 T cells were antibody-depleted prior to prophylactic treatment with N-803 and before SLE-like disease induction. A pattern emerged in which the depletion of CD8 T cells, but not NK cells, reduces GC

B cells to a level closer to non-diseased mice (Fig. 5C). This data suggests that expanded CD8 T cells may be necessary for N-803-mediated GC enhancement in SLE-like mice, while N-803-expanded NK cells exhibit little (if any) appreciable effect on disease. Of relevance however, in a similar experiment that did not incorporate in vivo NK cell expansion, we depleted NK cells prior to bm12 graft injection and observed increased GC B cells at two weeks that approached statistical significance (Figure 18, below). This finding leaves open the possibility that endogenous NK cells may still exert a suppressive effect on GCs in an autoimmune context, even if cytokine-mediated expansion of NK cells lacks an observable therapeutic benefit. This could be due to IL-2Rβ/γc-mediated effects on specific NK cell phenotype and function, which is reported elsewhere220, but was not specifically examined in our experiments. However, more investigation is needed in the Bm12 mouse model to confirm the disease consequences of each expanded cell population independently of one another (further discussed below).

45 IFNγ excess causes pathologic accrual of TFH cells and GCs . IFNγ acting specifically on B cells promotes autoimmune GC formation144. Moreover, both IL-2/anti-IL-2 (S4B6) complexes and N-803 increase the secretion of IFNγ from CD8 T cells190,191. We therefore tested the hypothesis that N-803-mediated enhancement of GC reactions in bm12-injected SLE-like mice may be due to expanded CD8 T cell production of IFNγ by measuring serum IFNγ during the first two weeks of disease in N-803 pre-treated mice that were depleted of either NK cells or CD8 T cells. Our (non-statistically significant) observation that IFNγ may be elevated over baseline in N-803 pre-treated SLE-like mice at 1 and 2 weeks post-disease initiation, except when CD8 T cell expansion is neutralized (Fig. 5D), posits that elevated serum IFNγ in

99 this disease context is dependent upon the CD8 T cell pool and independent of the NK cell compartment.

Given the known potential of IFNγ to exacerbate bm12-induced SLE-like disease188,189, this data suggests that expanded CD8 T cells producing excess IFNγ may be responsible for the exacerbated autoimmunity we observed following N-803 pre-treatment, potentially via direct aggravation of the autoreactive GCs that underlie disease in the Bm12 model. Of relevance, increased cell secretion of IFNγ is pathogenic in human SLE as well24.

Collectively, our data suggests that treatment of SLE with current immunotherapeutics that selectively expand endogenous CD8 T and NK cells should be approached with caution and may not achieve the therapeutic benefits of similar agents (i.e. daclizumab) that are observed in autoimmune diseases like MS.

In fact, our results imply that treatment of SLE patients with compounds such as IL-2/anti-IL-2 (S4B6) complexes or N-803 may actually worsen disease symptoms, likely as a result of expanded CD8 T cells and increased production of IFNγ. Furthermore, as the bm12 mouse model of SLE-like disease also models cGvHD183, the therapeutics we tested should also be considered carefully before using in patients with cGvHD.

Returning briefly to the discussion of co-occurring SLE and malignancy, our results suggest that using current interventions like IL-2/anti-IL-2 (S4B6) complexes or N-803 to battle cancer in SLE patients may be complicated by a potential trade-off of their effects. We demonstrate that these immunotherapeutics can indeed reverse an NK cell deficit and expand CD8 T cells in SLE-like mice (Fig. 1), thus achieving the necessary effect for combatting cancer. However, the worsening of SLE-like disease in the bm12 model

(Figs. 3 & 4) suggests that treating an SLE patient’s cancer with such therapies may only be valuable if the benefit against malignancy outweighs the potential risk of exacerbated autoimmune symptoms. Future experiments could test this by observing the effects of IL-2Rβ/γc-mediated NK cell/CD8 T cell expansion in

100 mice induced to SLE-like disease (via bm12-engraftment or pristane) and given a tumor xenograft. If in vivo expansion of NK cells and CD8 T cells helps to eradicate the tumor but worsens autoimmunity, this hypothesis would be supported. Nevertheless, our results suggest that treating an SLE patient’s malignancy with immunotherapeutics that expand NK cells and CD8 T cells should be approached with caution.

It is important to note that aspects of our experimental design could limit the interpretation of some of our findings. For example, we used antibodies against CD8α and NK1.1 to deplete CD8 T cells and NK cells, respectively (Fig. 5). As CD8α is also expressed on a specific subsets of DCs, we likely removed this leukocyte population as well in these experiments. However, because mouse splenic CD8α+ DCs are best known for cross-presenting exogenous antigens to CD8 T cells via MHC Class-I221, their elimination should have little if any impact in the MHC-II restricted bm12 model. Indeed, an unchanged graft TFH cell population following anti-CD8α depletion (data not shown) provides further evidence that MHC-mediated activation of graft CD4 T cells by host DCs was unaltered by any depletion of the CD8α+ DC subset.

Nonetheless, DCs are known to secrete a multitude of cytokines and inflammatory mediators221, thus it remains possible that unanticipated depletion of CD8α+ DCs impacted our results shown in Figure 5.

Similarly, NK1.1 is expressed not only on mouse NK cells, but also on most ILC subsets222 that are likely depleted by anti-NK1.1 antibody as well. As the exact role of ILCs in mouse SLE models is still poorly understood, the off-target effect of this depleting antibody is difficult to speculate at this time. In addition, it is important to mention that, because whole Bm12 splenocytes were injected to induce SLE-like disease, we observed expansion of both graft and host CD8 T cell populations upon IL-2/anti-IL-2 (S4B6) complexes and N-803 treatment. Thus, potential additive effects of an expanded and activated exogenous (albeit

MHC class-I-matched) CD8 T cell population should be kept in mind when interpreting our findings, especially when considering that only an endogenous CD8 T cell population exists in human SLE. To

101 address some of these concerns, future experiments could activate and expand B6 CD8 T cells ex vivo using IL-2 and/or IL-15, then adoptively transfer them into the bm12-engrafted B6 hosts. If disease is worsened, this would further implicate activated CD8 T cells in disease aggravation. Moreover, if CD8 T cells are isolated and transferred from an IFNγ-deficient mouse strain on a B6 background (e.g. B6.129S7-

Ifngtm1Ts/J mice), an absence of disease aggravation would further connect IFNγ production to the findings we observed.

Because we could not fully conclude whether or not an expanded NK cell population (by itself) provided a therapeutic benefit in SLE-like disease, in combination with other preliminary data in the same mouse model suggesting that NK cells may play a protective role against autoimmune-relevant GC reactions

(Figure 18), efforts should be considered to develop and test drugs (or drug combinations) that specifically expand endogenous NK cells while leaving CD8 T cells unaffected. Such a treatment approach has the potential to benefit patients with autoimmune diseases such as SLE, or perhaps have previously unforeseen advantages as an antiviral or anti-cancer therapeutic (especially in patients with both SLE and cancer). How such a treatment could be achieved is discussed more later (see “Future Directions”). Yet another option exists (and should not be dismissed) to expand an SLE patient’s NK cells ex vivo223, then infuse them back into the patient in hopes of achieving a GC-suppressive effect. This approach would bypass the detrimental co-expansion of CD8 T cells, but would likely be more time-consuming, costly, and risky than administering a drug that specifically expands NK cells in vivo. Additionally, testing this approach in a mouse model has its own limitations, as we personally know from several attempts (data not shown) that mouse NK cells are much less amenable to ex vivo expansion and are quite difficult to adoptively transfer.

102

Figure 18: Depletion of NK cells prior to induction of SLE-like disease may increase GC B cells and

plasmablast populations. Host splenic GC B cells and plasmablasts following bm12 engraftment in

mice pre-treated with either the NK cell-depleting antibody αNK1.1 or an IgG2a isotype control (N=

4-10 per group). Data analyzed using unpaired Student’s T test and shown as mean ± SEM.

What does transcriptomic analysis reveal about NK cells in pSLE, and can this information be utilized for therapeutic benefit?

Through RNA sequencing, we uncovered a multitude of transcriptional changes in pSLE NK cells versus those of healthy controls, the top 50 most-significant of which are shown in Figure 7. Fittingly, some of the top 50 differentially-expressed genes have documented roles in NK cells function (Table 2). For example, alpha-fetoprotein (AFP) expression was the most upregulated genes in pSLE NK cells over controls. As alpha-fetoprotein is known to both induce a pro-inflammatory phenotype in and reduce the survival of human NK cells (when secreted by tumor cells) in vitro195, it can be postulated that increased intrinsic production of alpha-fetoprotein by NK cells could have similar detrimental effects, perhaps by acting in an autocrine manner. We also observed upregulated transferrin (TF) in pSLE NK cells. As this

103 protein is known to inhibit NK cell cytotoxicity via interruption of target cell synapse formation and through other, less clear effects on NK cells themselves197, a similar scenario could be hypothesized in which NK cells producing excess transferrin may be negatively regulating their own functionality.

Upregulated proteinase 3 (PRTN3) expression was observed in pSLE NK cells, and the enzyme encoded by this gene is known to stimulate endothelial cells to subsequently trigger NK cell degranulation in vitro199.

If circulating SLE NK cells are producing and secreting proteinase 3, one could imagine a scenario in which the exposure of nearby endothelial cells (i.e. lining the blood vessels) could become stimulated and, in turn, cause abnormal degranulation of NK cells. This could potentially lead to a hyperactivated and/or exhausted NK cell phenotype. In fact, endothelial cell activation is highly implicated in SLE224, and NK cells play a role in endothelial inflammation in the kidneys of SLE-prone mice149. The potential role of PRTN3 in this process, however, remains unexplored.

Both AMBP and PDGFRA were upregulated in pSLE NK cells and have known ties to NK cell lymphoma196,198. As in SLE NK cells, some cancer-derived human NK cell lines exhibit maturation and cytolytic defects225. Thus the change in AMBP and PDGFRA expression that we observed may reflect NK cell dysfunction in pSLE and could potentially be used as disease biomarkers.

AQP2, the gene encoding aquaporin 2, was the most significantly downregulated gene found in our pSLE

RNAseq analysis. Researchers testing for therapeutic benefits among mineral waters found upregulated

AQP2 in rat oocytes when exposed to one specific mineral water in vitro. Oddly enough, when human volunteers drank this same mineral water exclusively, their NK cells were found to exhibit enhanced cytolytic activity200, although no mechanism for this finding was explored. As aquaporins are known to facilitate transmembrane diffusion of hydrogen peroxide226, and NK cell cytotoxicity and cytokine

104 secretion are modified following the uptake of hydrogen peroxide227,228, it stands to reason that downregulated AQP2 activity in pSLE NK cells may contribute to a dysfunctional phenotype.

Sharing similarities to this finding, we also observed upregulated expression of MPO, the gene encoding the enzyme myeloperoxidase, in pSLE NK cells over control cells. Myeloperoxidase is known for its ability to break down hydrogen peroxide into harmful reactive oxidative species (ROS), and is strongly implicated in autoimmune pathogenesis229. The SLE-associated cytokine IL-12230 is known to enhance NK cell secretion of MPO201, which could cause direct negative effects in an autoimmune state. Furthermore, given that MPO inhibits NK cell cytotoxicity via ROS production202, overexpression of MPO in SLE NK cells could elicit self-suppression of their own ability to kill pathogenic cells.

Although genetic modification of any of the above-mentioned genes in NK cells could potentially enhance their function, more power may lie in manipulation of more governing, “master” regulators of gene expression such as TFs. By applying gene network analysis (AltAnalyze) to our RNAseq data, we identified several TFs that were predicted to be differentially expressed in pSLE NK cells versus controls (Figure 8).

Interestingly, three of those TFs have documented roles in both NK cell function and SLE (Table 3). More specifically, ARNT encodes a protein that forms a heterodimer with HIF1-α to exert its effects. HIF1-α polymorphisms are associated with SLE204. Overexpression of this ARNT/HIF1-α dimer in mice leads to a hemophagocytic lymphohistiocytosis-like disease, including an NK cell compartment that is diminished and less cytotoxic203. Thus, the predicted increase in ARNT signaling we observed may help to explain NK cell reduction and impaired killing in SLE.

In a similar fashion, STAT1 is overexpressed in human SLE211 and exhibits relevance in SLE-like mouse models as well210. STAT1 gain-of-function mutations in humans results in compromised NK cell function,

105 including reduced proliferation, decreased IFNγ production, and impaired degranulation and cytotoxicity209. On the other hand, the STAT1 increase we observed in NK cells may be a result of known elevated levels of type I IFN in pSLE34 and subsequent downstream STAT1-mediated signaling through

IFNAR231. Prior studies suggest that an elevated type I IFN signature is almost certainly present in our pSLE cohort232. However, expression differences in genes encoding IFNα or IFNβ were not directly detected, possibly due to the relatively stable disease status of our cohort (see SLEDAI scores, Table 1). If this is the case, then elevated STAT1 signaling could be inducing a pro-inflammatory, antiviral-like state in pSLE NK cells that aggravates disease. Thus, therapies that modify STAT1 expression, which are already being explored in the cancer field233, may impact the NK cell compartment in SLE and elicit a therapeutic effect.

PU.1 (a.k.a. SPI1) was another upregulated TF predicted by our gene network analysis that has several ties to NK cells and SLE. As discussed in Chapter 1, PU.1 is known to regulate NK cell differentiation and homeostasis205. The transition of NK cell precursors to immature NK cells requires PU.1, as suppression of this TF resulted in more NK cells arrested at this stage95. As our analysis predicted increased PU.1 activity in pSLE NK cells, it begs the question as to how PU.1 overexpression beyond the immature stage of NK cell maturation may effect phenotype and function. Suppression of PU.1 was found to reduce NK cell cytotoxicity in vitro206, although this was performed on NK cells isolated from patients with chronic hepatitis C infection and may not be applicable to overactive PU.1 in SLE. One potential effect of prolonged

PU.1 excess could be a surplus of immature NK cells, or an immature phenotype that lingers beyond their exodus from the bone marrow. Both of these findings were observed in human SLE and relevant mouse models, including our own findings in the Bm12 model (see Fig. 1C). Indeed, PU.1 is upregulated in lymphocytes of pSLE patients and directly correlates with their SLEDAI scores207, while polymorphisms in

PU.1 are significantly associated with SLE208. What’s more, preliminary evidence from our colleagues

(laboratory of M. Weirauch), which was obtained using SLE chromatin immunoprecipitation sequencing

106

(ChIP-Seq) datasets and the cutting-edge computational method known as Regulatory Element Locus

Intersection (RELI), suggests that PU.1 binds to SLE risk loci in human NK cells more so than any of the other 220 cell types interrogated (data not shown). Altogether, this data insinuates a strong link between

SLE, NK cells, and PU.1, suggesting a potential PU.1-dependent regulatory mechanism spanning multiple genetic loci and involving several downstream genes that may additively or synergistically promote SLE.

Thus, targeted manipulation of PU.1 in NK cells (similar to what was accomplished elsewhere using

RNAi206) could have profoundly beneficial effects upon disease in SLE patients. Other future endeavors that explore the role and therapeutic potential of PU.1 in NK cells and SLE are further discussed below

(see “Future Directions”).

It is important to note that several limitations exist in our transcriptomic interrogation of pSLE NK cells that should be mentioned. For example, although our pSLE cohort excluded patients currently on corticosteroids, other disease-modifying drugs were being taken by patients (e.g. hydroxychloroquine, tocilizumab, mycophenolate mofetil, methotrexate, and belimumab) that could have imparted unknown confounding effects on NK cell transcriptional profiles (see Table 1). As previously mentioned, the enrichment method we used to isolate NK cells from whole blood achieved >90% purity by flow cytometry

(data not shown), which implies that non-NK cells were almost certainly a contaminating factor in our RNA sequencing and could have resulted in some misleading gene hits. In addition, it is vital to stress that we transcriptionally assessed the NK cells of patients with pediatric, and not adult SLE. This has the potential advantage of better assessing underlying genetic anomalies in SLE NK cells at what could represent an earlier stage of disease development, in hopes of developing therapeutics that may halt disease progression at an early age. However, as SLE is frequently diagnosed in adulthood19, aspects of our findings may not be as applicable to an adult SLE population. Moreover, reports suggest that pSLE has a more prevalent type I IFN signature than SLE in adult cohorts232, which may be reflected in the predicted

107 upregulation of the STAT1 gene network in our results (Fig. 8). Type I IFN signaling is known to activate NK cells, which may have contributed to the bias toward gene upregulation that was evident in our transcriptomic analysis (Fig. 7). Lastly, we acknowledge that our sample size (6 pSLE patients vs. 7 healthy controls) is relatively small and should be increased to better power our study and strengthen our findings.

Altogether, the relevance of top differentially-expressed genes and predicted altered TFs in pSLE NK cells portrays this cellular population as rather proinflammatory and hyperactive (i.e. elevated AFP, PRTN3,

STAT1, ARNT), with potential developmental abnormalities (i.e. increased PU.1) and decreased cytotoxic ability (i.e. upregulated transferrin, MPO, STAT1) – all findings that mirror the SLE NK cell phenotype that is reported in a significant body of literature121,131,140. Contrary to reports of immunoregulatory NK cells in autoimmune contexts101,155,234, we did not observe major differences in the expression of previously identified genes (e.g. adenosine, CD38, TRAIL, IL-10, etc.) that may be reflective of immunoregulatory dysfunction in pSLE NK cells. To better explore this, future experiments could isolate NK cells from a pSLE cohort and interrogate their immune-regulatory function (compared to NK cells from healthy controls) using the SEB-stimulated T:B co-culture system (as used in Chapter 4), thus demonstrating whether inherent defects alter their ability to kill autologous lymphocytes and limit downstream antibody production.

Nevertheless, the RNA sequencing and transcriptional analysis of enriched pSLE NK cells presented herein is, to our knowledge, the first of its kind in this disease context. Because of known NK cell defects in SLE

(discussed in detail in Chapter 1), the gene expression differences we uncovered may further elucidate underlying causes of this dysfunction. As NK cells exhibit immunoregulatory behavior, modification of the genetic pathways we identified could correct a previously-unknown pathway of NK cell regulatory function in SLE with potential therapeutic benefit. Alternatively, as NK cells in SLE exhibit a

108 proinflammatory phenotype, the transcriptional changes we found could further explain this cellular behavior and provide new targets for disease intervention.

Is TFH targeting using CAR NK cells a reasonable therapy for autoimmune diseases such as SLE?

We designed a PD-L1-based CAR that permits NK cells to selectively and efficiently target TFH cells based upon their markedly elevated expression of PD-1 relative to other leukocytes in human blood or tonsil.

Expression of this CAR construct on human NK-92 cells conferred a capacity for cytolytic degranulation in response to PD-1 presented on the surface of a tissue-culture plates, insect cells, Raji tumor cells, or bona fide human TFH. CAR-expressing NK cells selectively eliminated TFH but not B cells or naïve T cells during short-term, in vitro co-cultures. Importantly, TFH culling by CAR NK cells led to a reduction in memory B cell proliferation, differentiation, and immunoglobulin secretion in vitro. These results form the foundation of translational development of PD-L1-based methodologies for clinical elimination of TFH cells in autoimmune diseases such as SLE, and potentially other pathologies as well.

A limitation in developing TFH-targeting strategies is the broad expression of many TFH-associated markers on other leukocytes. In the context of PD-1, we observe low but not negligible expression of this receptor on some non-TFH cells, including subpopulations of NKT, CD8 and CD4 T cells. Given that these non-TFH lineages are variably implicated in aggravation or dampening of diseases such as SLE22,235,236, the desirability of off-target depletion of any of these cells via PD-L1 CAR-expressing cells needs further evaluation in a disease-specific context. Peripheral blood TFH cells exhibited the highest PD-1 expression of peripheral cells surveyed, though roughly six-fold less than that of their tonsillar counterparts, a finding that parallels observations made elsewhere237. We observed that our CAR NK cells preferentially kill stimulated peripheral TFH-like cells, albeit less efficiently than tonsillar TFH cells (Fig. 16B). Given that

52 circulating TFH cells are associated with SLE and behave like GC TFH cells , our proposed CAR NK cell

109 therapy may be constrained by the reduced ability to target these peripheral cells. Nonetheless, PD-L1

CAR NK cells demonstrated more robust degranulation against PD-1high cell types (e.g. “PD-1Hi” S2 cells and human tonsillar TFH cells), and exhibited a measurable cytolytic preference for TFH cells relative to other T cell subsets. Thus, our results demonstrate that the PD-L1 CAR confers a substantial degree of specificity for target cells based on PD-1 expression levels.

Of note, PD-1 expression in TFH cells was exceeded only by TFR cells, a lymphocyte population known to

238 curtail humoral immune responses arising from GC reactions . Thus, elimination of TFR cells is a likely off- target effect of our proposed therapy that requires further exploration. However, given the relatively low abundance of tonsillar TFR cells compared to TFH cells observed (more than 1,600-fold less; data not shown), we predict that any ill-effects of TFR elimination would be largely offset by the benefits of pathogenic TFH cell removal. This idea is supported by assays in which we enrich for CD4 TFH cells without excluding TFR, yet still see reduced memory B cells responses when these CD4 T cells are pre-cultured with

CAR NK cells. Thus, the effects of targeted TFH killing appears to outweigh the concomitant death of TFR

239 that is likely occurring. In addition, as TFR differentiate from Treg cells and we show that this population is unaltered by our CAR NK cells, the TFR pool would replenish faster following CAR NK cell treatment than would be expected if they developed from TFH precursors. Moreover, the suppressive effects of TFR cells

240 on B-cell responses are mediated via durable epigenetic changes that persist in absence of TFR , further limiting negative consequences of potential off-target depletion of these cells.

Another important consideration for selective PD-L1 CAR targeting of TFH is the potential for altered expression of PD-1 on various leukocytes in the context of inflammation and disease. PD-1 expression is elevated (over baseline) on highly-activated T and B lymphocytes241, particularly in the context of chronic inflammation or infection. These cell subsets have putative pathogenic roles in autoimmunity 242 and may

110 represent advantageous “off-target” cell populations to eradicate via CAR NK cell therapy. Changes in PD-

1 expression by TFH and other immune cell subsets must be subsequently evaluated in disease-specific contexts. Such analyses would increase confidence in the capacity of PD-L1 CAR-based strategies to selectively eliminate TFH or a combination of TFH and other pathogenic cell types in a particular human disease state.

The choice of PD-L1 as a targeting molecule may result in additional off-target effects due to low affinity interactions between PD-L1 and CD80/B7-1215. We observed that, when compared to PD-1, much higher concentrations of CD80 are necessary to trigger degranulation of PD-L1 CAR-expressing NK cells (data not shown). Moreover, CD80 expression levels on select subpopulations of leukocytes, including B cells and other antigen-presenting cells243, are relatively low. Although unlikely to occur, off-target PD-L1 CAR NK cell killing of CD80-expressing cells in vivo could potentially help to lessen disease by reducing CD80- mediated co-stimulation of immune responses. In fact, targeting of CD80 ameliorates lupus-like disease

244 in mice . Nevertheless, TFH are also characterized by elevated surface expression of other markers, such as ICOS15, which could be similarly targeted in next generation CAR constructs. Alternatively, PD-1 is also recognized by PD-L2, a ligand with higher affinity for PD-1 but no reported off-target binding to CD80215.

Numerous other enhancements could be adopted from the rapidly evolving CAR field to increase the safety and functional capacity of our TFH-targeted CAR NK cells. For example, the “second generation” CAR used in our study contains signaling domains of CD28 and CD3ζ that are optimized for CAR T cell function.

Recent studies reveal that inclusion of “third generation” components to specifically enhance persistence

(e.g. IL-15) or co-stimulation (e.g. NKG2D) of NK cells elevates CAR efficacy178,179. Though the potential toxicity of selectively eliminating TFH remains unexplored, the preservation of naïve and memory CD4 T cells as well as B cells and other types of immune cells suggests that the state of immunodeficiency

111 induced by the PD-L1 CAR NK cells will be far less severe than other immunotherapeutic strategies applied to autoimmune disease (i.e. rituximab). Moreover, the fact that PD-L1 CAR NK spare the Treg compartment should prevent major immune-pathologic reactions with this therapy.

In addition, CAR-mediated elimination of TFH cells likely will require efficient localization of CAR-expressing

NK or T cells in the follicular regions of secondary, and even tertiary lymphoid structures (TLS). Of note,

245 246,247 TFH cells are found in the TLS of patients with SLE and other autoimmune diseases , where they exhibit high expression of PD-1 and may contribute to pathogenesis. Engineered expression of CXCR5 on

PD-L1 CAR NK cells is likely to improve trafficking to sites of TFH residence throughout the body. This would mirror observed migration and CXCR5 expression patterns of CXCR5+ T or NK cells naturally present248,249 or artificially engineered250 during chronic virus infection, and may provoke beneficial consequences for patients with TFH-driven diseases.

CAR NK cells in general have an impressive safety profile despite their robust ability to eradicate malignant lymphocyte populations. Nevertheless, efforts are being made to introduce “suicide switches” into CAR

NK cells178 – a feature that may be critical to the safety of our PD-L1 CAR NK cell for safe clinical use. We were able to detect populations of PD-L1 CAR NK cells in the small vasculature of the lung, liver, and kidneys following injection into humanized mice in pilot studies (data not shown). Given that these locations are often the site of antibody complex deposition in SLE, it follows that CAR NK cell treatment may increase inflammation at these sites due to CD16 engagement of antibody Fc. This would be less concerning when using CAR-expressing NK-92 that are known to not express CD16251, but of more concern if using primary CAR NK cells. Therefore, implementing a “suicide switch” to turn off PD-L1 CAR NK cell activity, engineering them to lack CD16 expression, or enhancing their ability to home away from these

112 sites and toward lymphoid tissues may all be paramount to their safe clinical utilization. The inherent short-lived nature of CAR NK cells, however, may actually be an advantage in this context.

If it is determined that PD-L1 CAR NK cells are safe in SLE, then engineering them to persist for extended periods (e.g. via autocrine production of IL-15178) might be advantageous and require fewer cell infusions.

This approach could be particularly useful if CAR elimination of TFH cells is short-lived due to rapid reconstitution of the TFH cell pool from CD4 T cell precursors. This is of real concern in SLE, as upregulated

58 OX40L-mediated differentiation of CD4 T cells into TFH cells is implicated in disease pathogenesis . As an alternative approach to prevent rapid TFH cell reconstitution in SLE patients, PD-L1 CAR NK cells could be used in combination with OX40L blockade, which is currently being utilized successfully in transplant models to prevent rejection252.

A caveat to long-lived PD-L1 CAR NK cells in an SLE patient, however, is the potential for immune- suppressive effects, especially on GC reactions that are not aberrant and critical for protective humoral responses. As discussed in Chapter 1, SLE patients are often immunocompromised at baseline for various reasons, adding to the potential risks of sustained PD-L1 CAR treatment. Thus, the possibility exists that

PD-L1 CAR NK cells may need to be used sparingly and particularly during SLE flares, when it is known that new pools of autoreactive ASCs are generated through GC reactions49. In addition, the treatment of SLE patients with our CAR NK cells to suppress autoantibody production might need to be accompanied by infusions of protective antibodies via intravenous immunoglobulin – a treatment already known to have benefits in SLE253. Planned testing of our CAR NK cells in humanized mouse models of SLE (see “Future

Directions”) may help to identify CAR-mediated immunosuppressive effects, especially if these mice are challenged with an infection in parallel.

113

As discussed in Chapter 1, it is known that SLE autoantibodies can arise from processes outside the GC. It stands to reason that our PD-L1 CAR NK cell therapy, which is highly selective at targeting TFH cells, would therefore not prevent or quell such extrafollicular autoantibody production. More specifically, long-lived

B-1 cells bypass GC reactions, differentiate into ASCs, and generate weakly autoreactive antibodies that are of low affinity and infrequently class-switched6. However, the role of B-1 cells in SLE pathogenesis is appears limited at best, as one study revealed that they are neither necessary nor sufficient in specific

SLE-prone mice9. In addition, given that antibodies from B-1 cells aid in the clearance of autoantigens and apoptotic debris6, a lack of impact on this B cell population by PD-L1 CAR NK cells may actually be beneficial.

MZ B cells contribute to the autoantibody pool in SLE-like mice8, but what is known about this B cell subset in humans remains limited24. MZ B cells mainly generate short-lived ASCs that produce non-class-switched

IgM, and recent studies actually suggest a protective role for this B cell subset (as well as B-1 cells) in autoimmune vasculitis254. Thus, the likely persistence of MZ B cells following PD-L1 CAR treatment may be beneficial or result in relatively minor consequences.

Other extrafollicular B cells that bypass GCs, receiving help from MZ DCs and non-TFH CD4 T cells along the way, tend to differentiate into relatively short-lived ASCs that secrete low affinity (albeit class-switched) antibodies8. The role of such B cells may be significant in SLE7,49, and our CAR NK cell’s inability to affect them could reduce its efficacy. Thus, PD-L1 CAR therapy may be best suited toward patient populations whose disease is highly characterized by autoantibodies that originate from GC reactions (i.e. high-affinity, class-switched, etc.). Patients with LN may represent one such population, as anti-dsDNA antibodies in LN display high antigen affinity and are IgG-dominant24,26. Furthermore, treating some CAR-refractory SLE

114 patients with B-cell modifying agents such as rituximab or belimumab, in parallel with PD-L1 CAR therapy, may be necessary to reduce the impact of extrafollicular-derived autoantibodies.

The role of ASC populations such as plasma cells is substantial in SLE, as they are the cellular source of autoantibodies8. While our PD-L1 CAR NK cells should limit the GC-derived addition of new ASCs to the autoreactive pool, they are unlikely to have any effect on long-lived, autoantibody-producing ASCs residing in the bone marrow. The downside of overlooking plasma cells in SLE was potentially demonstrated by the failings of rituximab, and a similar fate could befall PD-L1 CAR therapy as well (in certain patient populations). In such refractory patients, treatment with PD-L1 CAR NK cells could require parallel treatment with a plasma cell-targeting therapeutic such as bortezomib. Furthermore, several plasma cell-specific CAR therapies are being tested and developed for use in multiple myeloma255, and could prove effective when given in tandem with PD-L1 CAR NK cells.

Elucidating the potential effects of persisting extrafollicular B cell subtypes and long-lived bone marrow- residing plasma cells following PD-L1 CAR treatment, in addition to combination therapies that may help to quell them, may be most easily achieved in a mouse model of SLE-like disease that incorporates these humoral immune features. NZB/W SLE-prone mice exhibit the components of autoreactive GCs (i.e.

41 involvement of TFH cells, generation of long-lived autoantibody-producing ASCs) , yet also harbor a known role for autoreactive B-1 cells9. Although it would likely require engineering mouse NK (or T) cells to express a murine version of our PD-L1 CAR, the NZB/W mouse represents an excellent model to test whether PD-L1 CAR therapy reduces autoantibodies via elimination of TFH cells, while simultaneously assessing the impact of any autoreactive ASCs and extrafollicular B cells that may persist. The relative contribution of GC-derived autoantibodies vs. those that were extrafollicular-derived could be measured by comparing autoantibodies that are IgG vs. IgM, specific vs. polyreactive, and high-affinity vs. low-

115 affinity. Furthermore, combination therapy approaches could be tested in CAR-treated NZB/W mice through parallel treatment with anti-CD20 (i.e. rituximab), anti-BAFF (i.e. belimumab), and/or proteasome inhibitors (i.e. bortezomib). This model is known for LN-like manifestations, thus relevant disease outcomes such as proteinuria, kidney pathology, and death could be measured and compared. Although generating and testing a murine PD-L1 CAR may seem like a step away from the clinic, we feel that it opens up vast opportunities for testing the efficacy of our PD-L1 CAR in vivo, and should be pursued.

256 As previously mentioned, angioimmunoblastic T-cell lymphoma (AITL) cells exhibit a TFH cell phenotype and thus represent a potential cancer target for our PD-L1 CAR NK cells. Using this treatment for AITL would likely have its caveats, however, due to the fact that our CAR may exhibit (yet-to-be-determined) immunosuppressive effects through its PD-L1 extracellular domain, which could dampen the ability of the endogenous immune system to control the lymphoma’s growth and spread. Furthermore, concomitant use of anti-PD-1 checkpoint inhibitors in AITL may render our PD-L1 CAR NK cell useless via target domain blockade. Future experiments could test this effect by infusing PD-L1 CAR NK cells into an established mouse model of AITL257 and gauging their effect on lymphoma clearance, endogenous immune function, and potential interference by checkpoint inhibitors.

The use of the extracellular domain of PD-L1 in the targeting region of our CAR to selectively eliminate

high PD-1 TFH represents a novel repurposing of CAR technology for use in autoimmunity and other human diseases. Importantly, the initial success of our strategy illuminates a new avenue for the development of therapeutics for targeting TFH or other pathogenic leukocytes that lack specific target molecules for antibody-mediated targeting. PD-L1-based targeting of TFH could be incorporated into CAR T cell, bi- specific killer engager (BiKE), or other immunotherapeutic modalities178. Though the effects herein are achieved via a human NK-cell line, we envision a therapeutic approach that employs (autologous) primary

116 or induced pluripotent stem cell-derived NK cells engineered to express our PD-L1 CAR via mRNA transfection, transposon technology, or viral delivery. Such innovative strategies represent a readily amenable platform for the treatment of deadly and intractable diseases like SLE.

Of further relevance to PD-L1, NK cells, and autoimmune disease, IL-18-treatment of mouse NK cells naturally enhances their expression of PD-L1 in vitro. When transferred into mouse models of autoimmune diabetes, IL-18-stimulated NK cells exhibited a therapeutic suppression of pathogenic CD8 T cells that occurred in a PD-1/PD-L1-dependent manner258. In a separate line of investigation (with funding support from the Lupus Foundation of America), we attempted a similar treatment approach in the bm12 inducible model of SLE-like disease. The rationale, design, and results of this endeavor is summarized in the attached Appendix, below.

Future directions

Revisiting in vivo NK cell expansion in SLE-like mice

As previously mentioned, our results point to concomitant expansion of CD8 T cells and elevated serum

IFNγ in aggravated SLE-like disease upon IL-2/anti-IL-2 (S4B6) or N-803 treatment, whereas expanded NK cells exhibit little observable effect. Nonetheless, it still may be worthwhile to explore a treatment option that either expands endogenous NK cells while neutralizing the negative effects of expanded CD8 T cells

(i.e. excess IFNγ production), or an approach that bypasses CD8 T cell expansion altogether. The former route could be achieved by co-administering IL-2/anti-IL-2 (S4B6) or N-803 with an IFNγ-blocking agent (a drug that is already being explored therapeutically in SLE259), although such blockade would be broadly- acting and its effects may be difficult to interpret. The latter route could be accomplished by creating an

IL-15 superagonist (similar to N-803) that also contains a single-chain antibody region that is specific for an NK cell receptor such as NKp46. This would be similar to diphtheria-toxin based antibody-linked

117 immunotoxins that were successfully used to deplete specific cellular subsets in vivo260. One can envision such a compound directing strong IL-15-mediated effects specifically to cells that highly express NKp46

(i.e. NK cells), while potentially having negligible effects on NKp46-lacking CD8 T cells. A potential disadvantage of this approach could be the consequences of engaging a receptor like NKp46 (known for its various roles in NK cell activation261) in an SLE-like context, such as eliciting a pro-inflammatory NK cell phenotype. Further exploration of either of these treatment strategies in the Bm12 SLE-like mouse model

(or perhaps a spontaneous model that also exhibits elevated GC reactions53) is something that we may pursue.

It is certainly possible that pathogenic TFH cells in the bm12 model are inherently protected from autologous NK cell killing, which occurs via type I IFN-sensing by T cells262,263. Moreover, unpublished work by our colleagues (J. Klarquist, C. Hennies, M. Lehn, & E. Janssen) demonstrates IFNα-sensing does indeed protect CD4 T cells from NK cell killing in the bm12 mouse model of SLE-like disease. Therefore, expanding

NK cells (even if achieved specifically) may represent a futile endeavor in this model, unless type I IFN signaling is blocked in parallel with NK cell expansion. Future experiments could test this treatment strategy by expanding NK cells in bm12-engrafted mice and co-treating them with an anti-IFNAR blocking antibody that is similar to Anifrolumab, which is currently in early clinical trials for SLE39. If anti-IFNAR treatment plus in vivo NK cell expansion reduces TFH cells and ameliorates SLE-like disease more than either treatment alone, this finding would support a combination therapy approach in SLE of blocking

IFNAR to expose TFH cells to killing by an expanded pool of NK cells.

Attempting to “correct” NK cell function as a therapy for SLE

As previously mentioned, we have sufficient evidence to hypothesize that modifying expression of the transcription factor PU.1 in NK cells may have a profound effect on their function, especially in a disease

118 context such as SLE. Continuing this line of investigation, we have designed and received multiple lentiviral vectors encoding short-hairpin RNAs (shRNAs) that specifically target and theoretically suppress expression of the human PU.1 gene. Future experiments will entail transducing the human NK cell line

KHYG1 (previously confirmed to express PU.1 by Western blot; data not shown) with these shRNA- encoding lentiviruses and subsequently testing their function in vitro. Functional assays could include degranulation, proliferation, cytotoxicity against tumor cells, cytokine production, and “regulatory” function, which could be measured in SEB-stimulated T:B co-cultures (as utilized in Fig. 17). Given available published data205,206, we expect to observe several changes in NK cells that exhibit PU.1 suppression, including reduced activation and altered cytotoxic and regulatory function. Such a finding would further insinuate PU.1 as a source of NK cell dysfunction in SLE and elevate its priority as a gene to target for therapeutic benefit, possibly via a lentiviral or RNAi treatments that modulate PU.1 expression.

Other future plans for examining the role of PU.1 in NK cells and SLE includes the generation of an Ncr1-

Cre+ PU.1FL/FL (Ncr1ΔPU.1) conditional knockout mouse line and subsequent evaluation of induced SLE-like disease. This mouse line will harbor a PU.1 deletion in cells expressing NCR1 (NKp46), the vast majority of which are NK cells. As PU.1 expression in NK cells is known to precede expression of NKp46 during development93, we do not expect our mutant mice to exhibit a disruption in NK cell differentiation, but rather demonstrate an inability to express PU.1 following maturation, which will be confirmed via NK cell phenotyping. SLE-like disease induction by pristane injection87 into Ncr1ΔPU.1 mice would elucidate the in vivo effect of PU.1 deletion in NK cells in an autoimmune context. Specifically, pristane-injected Ncr1ΔPU.1

+ FL/FL mice (compared to pristane-injected Ncr1-Cre and PU.1 control strains) will be monitored over the course of several weeks for serum concentrations of ANA and type I IFNs, as well as proteinuria. NK cell phenotype will be assessed in diseased Ncr1ΔPU.1 mice and control strains to identify changes that may reflect what was observed in the NK cells of our pSLE cohort (e.g. markers of maturity and activation,

119 differential expression of genes related to function, etc.). In addition, later disease manifestations will be compared (glomerulonephritis, lymphoid tissue frequency of germinal center components and ASCs, etc.) between Ncr1ΔPU.1 mice and control strains. Because of PU.1 overexpression and the associated transcriptional phenotype that we observed in pSLE NK cells (see Figures 7 & 8), we predict that PU.1 deletion in the NK cells of SLE-like mice will result in a cellular phenotype that is potentially more mature

(as assessed by CD27/CD11b staining) and less pro-inflammatory (as assessed by activation markers and cytokine production) than NK cells in diseased control strains. Furthermore, we expect that Ncr1ΔPU.1 mice will exhibit reduced measures of SLE-like disease due to an NK cell phenotype that is less inflammatory and aberrant.

Driving the PD-L1 CAR NK cell closer to the clinic

Our novel PD-L1 CAR NK cell exhibits encouraging effectiveness in vitro, but several limitations exist that hamper its relevance and clinical application. First of all, we demonstrated effective TFH cell elimination through the use of a CAR-expressing NK-92 cell line, representing critical proof-of-concept groundwork.

Though use of this (irradiated) cell line in clinical trials for cancer has shown surprising safety180, it is understandable that the rheumatology clinic may be less inclined to try such a risky approach to combat

SLE. Thus it will be critical to demonstrate that our CAR construct is effective when expressed on primary, autologous NK cells. Better yet, generating CAR NK cells from an SLE patient’s own iPSC would be an innovative and more cost-effective way to establish an “infinite” pool of effector cells180,182.

Preliminary efforts using IL-21-expressing feeder cell-expanded NK cells from a patient’s blood were successful in generating PD-L1 CAR-expressing primary NK cells (Figure 19A, below). When these CAR NK cells were co-cultured with tonsillar CD4 T cells from the same patient (i.e. autologous), we observed a pattern of selective TFH killing by the CAR NK cells (Figure 19B). This pilot data needs to be further repeated

120 but represents encouraging results that our PD-L1 CAR construct is nevertheless effective in primary NK cells against autologous TFH cells.

In addition, the usefulness of generating infinite PD-L1 CAR NK cells from an SLE patient’s iPSC is nullified if functional NK cells cannot be made due to a disease-related genetic defect. Using published protocols264,265, however, we were able to successfully differentiate a pSLE patient’s iPSC into phenotypically normal NK cells (Figure 20A). What’s more, these NK cells exhibited typical killing of tumor cells when tested in a radioactive chromium-release assay (Figure 20B). Efforts are currently underway to engineer these iPSC to express our unique CAR, then differentiate them into TFH-culling NK cells.

The fact that a beneficial effect of our PD-L1 CAR NK cells has only been demonstrated in vitro also represents a large barrier to its progress toward clinical utilization. Seeking to validate its therapeutic potential in vivo, we tested our CAR-expressing NK-92 cells in a humanized mouse model of SLE-like disease88, albeit in a slightly different immunocompromised mouse strain (NSGS mice266). We found that humanized NSGS mice treated with pristane exhibited engrafted human T cells that were highly activated and expressed PD-1 at high levels (Figure 21A). We were able to successfully transfer irradiated PD-L1 CAR

NK-92 into SLE-like humanized mice and find them in tissues such as the liver and spleen one day later

(Figure 21B). In addition, humanized SLE-like mice treated with a regimen of PD-L1 CAR NK-92 cells exhibited a trend toward decreased human CD4 T cells, but no observable change in their human B cell pool (which does not highly express PD-1). As engrafted PD-1+ CD4 T cells in this model lack expression of

CXCR5 (data not shown), they cannot be considered bona fide TFH cells. Nonetheless, these results are encouraging and suggest that PD-L1 CAR NK cells may selectively reduce PD-1+ cell populations in vivo, bringing this technology one step closer to clinical use for SLE.

121

In future experiments, we plan to treat pristane-injected humanized NSGS mice with (irradiated) PD-L1

CAR NK cells (vs. non-CAR control NK cells) over the course of several weeks while monitoring their serum levels of ANA, type I IFNs, and other proinflammatory cytokines known to be elevated in this model (e.g.

IFNγ, IL-6, etc.). Later disease measures will be compared, including glomerulonephritis and tissue frequencies of human memory B cells, ASCs, and memory T cells (including changes in PD-1-expressing lymphocytes). If PD-L1 CAR-treated mice exhibit decreases in PD-1+ T cell populations, memory B cells,

ASCs, ANA, pro-inflammatory cytokines, and/or kidney disease, this would provide strong in vivo evidence that PD-L1 CAR therapy may be beneficial in human SLE as well. Fluorescent microscopy can be used to determine whether CAR NK cells co-localize (and potentially form synapses) with PD-1+ T cells in lymphoid tissues, thus strengthening the hypothesis that specific CAR targeting of this cell population underlies subsequent disease amelioration.

122

Figure 19: PD-L1 CAR-expressing primary human NK cells selectively kill autologous TFH cells in vitro.

(A) PD-L1 expression of empty-vector-transduced vs. CAR-lentivirus-transduced primary NK cells. (B)

Enriched tonsillar CD4 Naïve T, non-TFH memory T, or TFH cell uptake of PI following 4-hour incubation with either control or CAR autologous primary NK cells obtained from same patient (n=2-3). Dotted lines represent PI uptake in the absence of NK cells. Data analyzed using unpaired Student’s t-test and represented as mean ± SEM.

123

Figure 20: Functional NK cells can be differentiated from SLE patient-derived iPSC. (A) Day 12 of SLE patient-derived iPSC differentiation showing the appearance of CD34+ CD45+ hematopoietic stem cells, and day 54 demonstrating the presence of CD45+ CD56+ NK cells. (B) SLE iPSC-derived NK cells exhibit increasing cytotoxicity toward chromium-labeled K562 target cells in an effector:target ratio- dependent manner.

124

Figure 21: CAR NK-92 potentially reduce PD-1-expressing human CD4 T cells in a humanized mouse model of SLE-like disease. (A) Human CD4 T cells (CD19-CD3+) engrafted in the spleens of NSGS mice

(n=3) 16wks after pristane injection exhibit high expression of PD-1 (B) Irradiated CD56+ ZsGreen+

CAR NK-92 cells (boxed) are detected in the spleen and liver of humanized NSGS mice 24h after injection of 107 CAR cells. (C) Potentially reduced frequency of PD-1high human CD4 T cells, but not B cells, following three injections of 5x106 CAR NK-92 into pristane-treated, humanized NSGS mice

(n=2). Data represented as mean + SEM.

125

Putting it all together: the therapeutic potential of NK cells in autoimmune disease

Altogether, the findings presented in this dissertation mostly portray NK cells as a promising new treatment opportunity for autoimmune diseases such as SLE. Although currently available tools to increase NK cell (and CD8 T cell) numbers in vivo had a detrimental effect in an SLE-like mouse model, there is still hope that a modified approach that more specifically expands NK cells (or enhances their effects) may have a suppressive effect on the immune processes underlying SLE. Nevertheless, our data reveals that the immunotherapeutic agents we tested should be used with caution in SLE patients who may be considering them for treatment of malignancy. Our transcriptomic interrogation of pSLE NK cells identified many genes and predicted several TFs that are differentially-expressed compared to healthy control NK cells. One TF in particular, PU.1, was highlighted by our analysis and has deep ties to NK cell function and SLE, thereby representing a genetic flaw that may (at least partially) explain the various NK cell defects often observed in SLE. Manipulating the expression of PU.1 (or other genes that we identified) could re-establish critical NK cell regulatory functions and lead to the development of new treatment approaches for SLE. Finally, our attempt to harness CAR NK cell technology as a way to treat the autoimmune underpinnings of diseases like SLE was perhaps the greatest success of our efforts to utilize NK cells therapeutically. Bypassing any defects or deficits that characterize the NK cell pool in SLE, we engineered an NK cell that specifically targets TFH cells – key players in the aberrant GC reactions that are strongly associated with the pathogenesis of SLE. Our PD-L1 CAR NK cell specifically eliminates TFH cells while leaving bystander cells intact, and have an indirect effect on the downstream generation of

ASCs and antibody production – a critical feature for the treatment of SLE. Our efforts have generated a potentially safe and powerful new therapeutic option for thousands of patients who suffer the consequences of SLE, and could prove effective in other autoimmune disease contexts as well.

Altogether, the NK cell represents a wealth of therapeutic opportunity that should not be overlooked when considering new treatment approaches for autoimmunity.

126

Chapter 6. Materials & Methods

127

Cell culture

The NK-92 (ATCC CRL-2407™) and Raji (ATCC CCL-86™) cell lines were obtained from ATCC and cultured according to their standard protocols using the appropriate media as described on the ATCC website. S2 cells were obtained from ThermoFisher (R69007) and cultured according to their standard protocol217 in

Schneider’s Drosophila Media (ThermoFisher) containing 10% heat-inactivated fetal bovine serum (HI-

FBS, Gibco). IL-21-expressing K562 used for ex vivo expansion of human NK cells were a gift from the laboratory of K. Rezvani, and were cultured in cRPMI (see below). When necessary for selection, blasticidin or puromycin were added at a concentration of 25µg/mL and 2µg/mL, respectively.

All cultures involving primary human lymphocytes were performed in RPMI-1640 (HyClone SH30255) supplemented with 10% HI-FBS, 100U/mL penicillin, 100µg/mL streptomycin, 1mM sodium pyruvate,

10mM HEPES buffer, 1X MEM non-essential amino acids, and 0.1mM 2-beta-mercaptoehtanol

(henceforth referred to as “cRPMI”).

Microscopy

Fluorescent cell images were taken using a Nikon Eclipse Ti microscope, equipped with Zyla sCMOS camera (Andor) and 488nm & 395nm filter cubes. Obtained images were processed using NIS-Elements

Imaging software (Nikon).

Cell enumeration

Human/mouse primary cells and all cell lines were enumerated with a hemocytometer using either Trypan

Blue exclusion or 3% acetic acid with methylene blue (StemCell Technologies).

Study participants & acquisition of human tissue samples

128

For tonsillar tissue (see Chapter 4), male and female children 9 years old or younger requiring tonsillectomy were recruited to a prospective study at a tertiary academic care center through the Division of Pediatric Otolaryngology-Head and Neck Surgery at the Cincinnati Children’s Hospital Medical Center

(CCHMC). Criteria for enrollment in the study included a history of sleep-disordered breathing or recurrent or chronic tonsillitis requiring removal of the tonsillar tissue. Consent was obtained from parents in the perioperative suite on the day of the procedure. Children were excluded from the study if the tonsillar tissue was acutely infected or if anatomic abnormalities were present requiring a more detailed pathologic evaluation. Institutional review board (IRB) approval at CCHMC was obtained prior to initiation of this project. After recruitment, patients underwent tonsillectomy as part of the standard of practice. Tonsillar tissue not used for research was sent to pathology for gross evaluation as part of the routine clinical care.

Pediatric SLE patient peripheral NK cells (see Chapter 3) were isolated from 8-10mL of whole blood drawn from male and female children 18 years old or younger through the Division of Pediatric Rheumatology at

CCHMC. Criteria for enrollment in the study included a diagnosis of systemic lupus erythematosus. Healthy control blood samples were obtained from male & female children 18 years old or younger that were personally known to the pSLE patients (so-called “best-friend” controls) and who were also recruited by

CCHMC Rheumatology. Consent was obtained from parents on the day of blood draw. Children were excluded from the study if they were currently taking corticosteroids. Two pSLE patients has SLEDAI scores of 2 at the time of blood draw, while the rest has scores of 0. Of the pSLE blood samples used for RNAseq, all were currently taking hydroxychloroquine, more than half were taking mycophenolate mofetil, one was on methotrexate, and one was using leflunomide. IRB approval at CCHMC was obtained prior to initiation of this project.

129

PBMC used in Chapter 4 were obtained by thorough PBS-washing of deidentified leukocyte reduction filters obtained from the University of Cincinnati Hoxworth Blood Center with IRB approval at CCHMC.

Processing of tonsillar tissue

After removal, approximately ½ of each of the bilateral tonsils were transected and placed in RPMI-1640 media with 10% human AB serum. Samples were labeled with a deidentified barcode and transferred to the research team for further processing. Tonsil tissue was processed via mincing with scissors followed by transfer of up to 4g of tissue to a gentleMACS C tube (Miltenyi Biotec) containing 8mL of phosphate- buffered saline (PBS) with 0.5mg/mL collagenase D and 3000U/mL DNaseI, then dissociated on a

GentleMACS Octo Dissociator (Miltenyi Biotec) using “program C”. Homogenates were incubated in a 37°C water bath for 15 minutes, then dissociated again using “program C” and transferred through a 100um cell strainer into cRPMI. This cell suspension was then layered over Ficoll-Paque PLUS (GE Healthcare) and subjected to density-gradient centrifugation to isolate tonsillar mononuclear cells, as described below.

Isolation of human PBMC

Whole blood (diluted 1:1 in PBS) or PBS-washed reduction-filter cells were carefully layered over a 50% volume of Ficoll-Paque PLUS, then centrifuged at 1000xG for 20 minutes at RT with the centrifuge brake turned off. The leukocyte monolayer at the interphase was carefully collected, PBS-washed, & enumerated before further use. When isolating NK cells from whole blood (as in Chapter 3), the

RosetteSep NK cell isolation kit (StemCell Technologies) was used according to manufacturer’s instructions.

Cell cryopreservation & thawing

130

All PBMC and tonsil mononuclear cells were cryopreserved in FBS containing 10% dimethyl sulfoxide and frozen @ -1°C/min using CoolCell freezing containers to -80°C, then stored long-term in vapor phase of liquid nitrogen. These cryopreserved cells were rapidly thawed in a 37°C water bath and transferred into warm cRPMI (and washed twice) before use.

Flow cytometry & cell sorting

Fluorescently-conjugated antibodies used were obtained from either Biolegend, Ebioscience

(ThermoFisher), Invitrogen, or BD Biosciences and used at manufacturer’s recommended concentrations.

For surface staining, cells were resuspended at a concentration of 1-2×106/mL in 50-100µL of cold Hank’s

Buffered Salt Solution (HBSS) containing 5% HI-FBS, 100U/mL penicillin, and 100µg/mL streptomycin,

(henceforth referred to as “FACS buffer”). Fluorescently-conjugated antibodies were each added at the manufacturer’s recommended concentration. Cells were incubated at 4°C for 20 minutes, then washed twice with FACS buffer, and either analyzed fresh (same-day), fixed using 100µL BD Cytofix for 20 minutes at 4C, or stained for intracellular markers. Intracellular staining was performed by fixation/permeabilization of surface-stained cells in 100µL Cytofix/Cytoperm (BD Biosciences) for 20 minutes at 4⁰C, followed by staining in 100µL 1x Perm/Wash buffer (BD Biosciences) containing fluorescently-conjugated antibodies, each at the manufacturer’s recommended concentration, for 20 minutes at 4⁰C. All fixed cells were washed twice with FACS buffer to remove fixative, and kept at 4⁰C in

FACS buffer until analysis (1-3 days later).

Acquisition of stained cells was performed using a Fortessa or LSRII cytometer (BD Biosciences) with

FACSDiva software (BD Biosciences). Flow cytometric cell sorting was performed using an SH800S cell sorter (Sony) with the accompanying Sony acquisition software.

131

Design & generation of PD-L1 CAR vector & PD-1-containing vectors

In general, plasmid amplifications were performed by transformation and expansion of the competent E. coli strains DH5α or XL10-Gold (for pUC57 plasmids), and Stbl3 or StellarComp (for pLVX-IRES-ZsGreen1,

PiggyBac, and pAc/V5-His plasmids), grown in LB broth/agar plates containing the appropriate selective antibiotic (50µg/mL ampicillin or 100µg/mL kanamycin). Digestions were performed according to the relevant New England BioLabs online NEBcloner protocol. Digestion products were resolved using 1% agarose gel electrophoresis. DNA-containing gel fragments were purified using the GeneJET Gel Extraction kit (ThermoFisher). Ligations were performed for two hours at room temperature using T4 DNA ligase

(ThermoFisher). Final plasmids were purified using QIAprep Mini, Midi, or Maxi kits (Qiagen) and stored at -20°C. Plasmid insertions were verified via Sanger sequencing using custom primers (IDT Technologies or Invitrogen). Sequence files were aligned using Snapgene and/or Benchling.

The 2nd generation CAR (PDL1-CD28-CD3ζ) was designed by splicing the PD-L1 signal and extracellular domains (a.a. 1-238, NP_054862) to typical 2nd generation CAR domains 175,267,268, including the leader and hinge regions of CD8α, CD28 transmembrane & intracellular domains, and CD3ζ intracellular domain

(Supp. Fig. 1). The CAR sequence was synthesized by Genewiz into a pUC57 vector. The CAR construct was excised from pUC57 and ligated into the multiple cloning site (MCS) of the lentiviral vector pLVX-IRES-

ZsGreen1 (Clontech). Clones were screened for the correct restriction digestion pattern and sequence- verified prior to being amplified and purified.

The full length, human PD-1 protein sequence was obtained from UniProt (a.a. 1-288). An open reading frame (ORF) for this amino acid sequence was optimized for a Drosophila expression system using the

“codon optimization tool” (IDT Technologies), then this DNA sequence was synthesized by Genewiz into

132 a Puc57 plasmid. The PD-1 ORF was excised from pUC57 and ligated into the MCS of a pAc/V5-His A vector

(Invitrogen), then verified via sequencing, amplified, and purified (referred to as “pAc/V5-His-PD1”).

The human PD-1 ORF was excised from the aforementioned pUC57 plasmid using and ligated into the

MCS of a PiggyBac Transposon, Cloning and Expression Vector (System Biosciences, PB513B-1), then amplified, purified, and verified via sequencing (referred to as “PB513-PD1”).

Lentiviral generation and transduction of NK cells

After sequence verification the CAR-containing pLVX-IRES-ZsGreen plasmid was given to the Cincinnati

Children’s Viral Vector Core for lentivirus production. Briefly, lentivirus was packaged by transfection of

293-T cells with a 3rd generation packaging system: pCDNA3.g/p.4xCTE plasmid (GagPol, 8µg/plate), pRSV rev plasmid (Rev, 6.5µg/plate), vector plasmid (8µg/plate), and m75-VSVG plasmid (VSV-G envelope,

2µg/plate). Viral supernatant from four 10-cm plates was collected 24-48 hours post-transfection, purified via sucrose-gradient, and titer analysis was performed. Viral supernatant was concentrated to 350 µL and stored at -80°C in 25 µL aliquots. Titer was determined by transfection of control cell line and flow cytometry analysis.

NK-92 were transduced in 48 or 96-well flat bottom plates, previously coated overnight with human fibronectin at 20 µg/mL. Cells were transduced with either CAR-containing pLVX-IRES-ZsGreen lentiviral vector or an empty-vector control lentivirus at a multiplicity of infection (MOI) of 5 in 8 µg/mL protamine sulfate for 4-6 hours at 37°C. After 48 hours, cells were sorted based on fluorescent reporter expression.

Primary NK cells were transduced as above but without the use of fibronectin, at an MOI of 20, and in the presence of 6 µg/mL DEAE dextran instead of protamine sulfate. After 24 hours, NK cells were expanded using irradiated IL-21-expressing K562 feeder cells in a manner fully described elsewhere223. After a week

133 of expansion, cells were sorted based on fluorescent reporter expression, then subjected to another week of feeder cell-expansion prior to evaluation by flow cytometry and subsequent use.

Generation of PD-1-expressing target cell lines

S2 cells were chemically transfected (CaCl2) using the DES Blasticidin Support Kit (Invitrogen) protocol.

Briefly, the pAc/V5-His-PD1 plasmid (or empty-vector control plasmid) was introduced at a ratio of ≥19:1 to the pCoBlast plasmid vector (Invitrogen). Cells were cultured for a week in the presence of blasticidin to select for pCoBlast-expressing clones. Blasticidin-resistant cells transfected with pAc/V5-His-PD1 were stained for PD-1 expression and sorted for PD-1+ cells (see Supp. Fig. 4A). Following >2 weeks of culture, a portion of PD-1+ S2 cells were stained for PD-1 expression and further sorted into “PD-1Hi” and “PD-1Lo” populations (see Supp. Fig. 4A). All sorted S2 cells were cultured for >2 weeks prior to co-culture with NK-

92 to minimize any effects of residual αPD-1 antibody still bound to S2 cell surfaces.

Raji cells were co-transfected via electroporation with either the empty PiggyBac plasmid (PB513) or

PB513-PD1 in addition to the Super PiggyBac transposase plasmid (System Biosciences, PB210PA-1) at a

1:2.5 ratio using the Neon Transfection System (Invitrogen, MPK5000). Electroporation parameters and cell concentration were obtained from the Neon Transfection System standard protocol for Raji cells

(using 100µL tips). Transfected Raji cells were cultured in 6-well, low adherent plates prior to fluorescent sorting and subsequent puromycin selection.

qPCR

NK-92 total RNA was extracted per RNeasy Mini Kit manufacturing protocol (Qiagen). Reverse transcription was performed using iScript Reverse Transcriptase and iScript Reaction mix per iScript cDNA

Synthesis Kit manufacturing protocol (Bio-Rad). Real-Time PCR reactions were carried out using a

134

PrimeTime Gene Expression Master Mix and custom PrimeTime Std qPCR assay primer/probe sets (IDT

Technologies), as well as a TaqMan Gene Expression Assay (ThermoFisher). An ABI 7500 Real-Time PCR

Thermal Cycler (ThermoFisher) was used under the PCR polymerase activation and amplification conditions of 95°C for 3 minutes and 40 cycles (95°C for 15s and 60°C for 1 minute).

Plate-bound activation assay rhPD-1-Fc, anti-PD-L1, IgG1-Fc, and Goat IgG (R&D systems) were solubilized in PBS and added at varying concentrations in 100µL/well to 96-well MaxiSorp plates (ThermoFisher) before overnight incubation at

4⁰C. Plates were washed with PBS before counted NK-92 cells were added at a concentration of 4×104 cells/mL in 200µL per well and incubated for 4 hours at 37⁰C. Degranulation was measured via addition of 2µg/mL of anti-CD107a PE-Cy7 (Biolegend) for the duration of the assay. PMA and ionomycin were added at 1µg/mL each for the duration of the assay.

NK-92: target cell co-cultures

For NK-92:S2 co-culture assays, NK-92 and S2 cells were combined at a ratio of 1:5 (2×104 and 1×105, respectively) in 200µL of S2 media (without blasticidin) in 96-well round-bottom plates, then incubated at room temperature for 4 hours prior to analysis.

For NK-92:Raji co-culture assays, NK-92 and Raji cells were combined at either a ratio of 1:5 (2×105 and

1×105, respectively) to assess degranulation, or a ratio range of 0.0625-20:1 (with targets seeded at 1×104) to assess killing. Assays were carried out in in 200µL of Raji media (without puromycin) in 96-well round- bottom plates, then incubated at 37⁰C for 4 hours prior to analysis.

135

For NK-92:CD4 co-culture assays, human tonsil cells were subjected to CD4 T cell negative magnetic isolation (StemCell Technologies), achieving 88% purity, on average (data not shown), then counted. This isolated CD4 fraction was counted and cultured in 96 well round-bottom plates at 30,000 cells/well in

100µL of cRPMI. 1.5×105 control or CAR NK-92 were added in 100ml cRPMI to each well for a 5:1 ratio of

NK:T cells, whereas control wells received 100µL of cRPMI alone. Cells were then incubated for 4 hours at

37⁰C prior to collection and analysis.

For NK-92:TFH:B cell co-culture assays in Figure 17 A-C, human tonsil cells were subjected to either CD4 T cell or B cell negative magnetic isolations (StemCell Technologies), achieving >80% purity (data not shown). The isolated CD4 T cell fraction was stained with fluorescently-conjugated antibodies to identify

- + + + and sort for live (Zombie ) CD3 CD4 CXCR5 TFH cells. Similarly, isolated and stained B cells were sorted

- - + + to obtain live (Zombie ) CD3 C19 CD27 memory B cells. Sorted TFH and B cells were cultured separately overnight (at 37⁰C) in 96 well round-bottom plates at 3×104 cells/well and 6×104 cells/well, respectively, in 100µL of cRPMI containing 1µg/mL SEB (EMD Millipore). The next day, each well of TFH cells was combined with a single well of memory B cells (achieving a 1:2 ratio of T:B cells) and further cultured. At days 3-4 post initial cell culture, 1.5×105 control or CAR NK-92 were added in 50ml cRPMI to each well to achieve a 5:1 ratio of NK:TFH cells, whereas control wells received 50µL of cRPMI alone. Co-cultures were then incubated for 4 hours at 37⁰C prior to collection and analysis.

For NK-92:TFH:B cell co-culture assays used in Figure 17 D-F, experiments were carried out as above with the following changes: Purified tonsillar CD4 T cells were not further sorted, but were co-cultured without or with NK-92 at a 1:5 T:NK cell ratio for 4 hours at 37°C. Co-cultured cells were then subjected to NK cell depletion using anti-CD56 microbeads and MS magnetic columns (Miltenyi Biotec) according to the manufacturer’s protocol. A portion of each collected fraction was analyzed by flow cytometry to verify

136 the depletion of NK-92 cells. Collected CD4 T cells were re-enumerated and co-cultured at a 1:2 T:B ratio with autologous sorted tonsillar memory B cells in the presence of SEB (as detailed above), and supernatants were collected at days 5 and 7 to test for the secretion of IgG.

NK cell activation & cytotoxicity assays

For calcein AM release assays, sorted TFH and CD27+ B cell co-cultures (at a 1:2 T:B ratio; previously stimulated with SEB for 4 days as described above) were labeled with 1:300 dilution (volume:volume) of

1mg/mL Calcein AM solution according a published protocol 223. Labeled cells were transferred to 96 well round-bottom plates at 1×104 cells/well in 100µL cRPMI. Control or CAR NK-92 were added in 100ml cRPMI to wells at varying E:T ratios, whereas control wells received 100µL of cRPMI alone. These co-cultures were carried out for 4 hours at 37⁰C prior to collection and analysis.

51 For chromium release assays, target cells were labeled with 50 µCi of Na2 CrO4 and added to a 96 well plate at 104 target cells per well. NK cells were added at different effector to target ratios and incubated at 37˚C for 4 hours. Spontaneous (no stimulus for cell death) and Maximum Release (lysis of target cells with triton X-100 detergent) controls were used to calculate the relative percent specific lysis of target cells cultured with NK cells. (Percent Specific Lysis = [(Experimental Release – Spontaneous

Release)/(Maximum Release – Spontaneous Release)] x100.

Degranulation was assessed by addition of 2µg/mL of anti-CD107a PE-Cy7 (Biolegend) for the duration of the assay. When used, PMA and ionomycin were added at 1µg/mL each for the duration of the assay.

Propidium iodide (Biolegend) was added at a 1:10 ratio (volume: volume) with stained, unfixed cells approximately 10-15 minutes prior to cytometric acquisition. Reconstituted Zombie UV (Biolegend) was added at a 1:400 ratio in antibody-containing FACS buffer mix for flow cytometry.

137

Bm12 cGvHD model of inducible SLE-like disease

Model was carried out in similar fashion to what is described elsewhere in more detail54. Spleens were harvested from B6(C)-H2-Ab1bm12/KhEgJ (bm12) on a BoyJ (CD45.1+) background donor mice and passed through 70um nylon strainers to obtain single-cell suspensions. Following enumeration using 3% acetic acid with methylene blue (i.e. no red blood cell lysis performed), splenocytes were resuspended in PBS and 4-6×107 cells were injected either intraperitoneally or intravenously (via retro-orbital injection under isoflurane anesthesia) into each recipient C57BL/6 (B6) mouse. Donor and recipient mice were age and gender-matched. When necessary, whole blood was obtained via laceration of the tail vein and collected into either EDTA or SST Microtainer tubes (BD Biosciences). Prior to staining for flow cytometry, whole blood from EDTA tubes was subjected to red blood cells lysis via ACK Lysing Buffer (Thermo Fisher).

Following euthanasia, mouse spleens were weighed and processed into single-cell suspensions (as above), splenic red blood cells were lysed (as above), and remaining splenocytes were enumerated prior to staining for flow cytometry. Whole blood was collected through the inferior vena cava and transferred to

SST Microtainer tubes for serum isolation.

Targeted cytokine treatment

Per mouse, 1ug of recombinant murine IL-2 (PeproTech) was combined with 10ug anti-mouse IL-2 (clone

S4B6; BioXCell) in 200uL Hank’s Balanced Salt Solution (HBSS), then incubated overnight at 4°C while mixing (as described elsewhere158. For each control mouse, 1ug of recombinant murine IL-2 was incubated overnight with 10ug rat IgG2a (BioXCell) in 200uL HBSS. As a pre-disease treatment, each mixture was intraperitoneally injected every other day for a total of 3 injections, after which a Bm12 splenocyte graft was injected the following day (see above). As a post-disease treatment, injections were administered

138 once daily for three days beginning one month after graft injection (mirroring a treatment schema elsewhere described166).

N-803 was provided via collaboration with Altor Bioscience, and dosing was recommended by their team of experts. Per mouse, 200ug/kg of N-803 in PBS was injected subcutaneously, while control mice received a molar equivalent of recombinant human IL-15 (Peprotech) in PBS. As a pre-disease treatment, two injections were administered, once every other day, finishing one day prior to Bm12 graft injection. As a post-disease treatment, injections were administered once weekly over the course of 3 months, beginning

1 week after Bm12 graft injection (for a total of 11 treatments).

Administration of cell-depleting antibodies

For NK cell, CD8 T cell, or mock depletion, mice were intraperitoneally injected once with either 25ug

InVivoMAb anti-mouse NK1.1 (BioXCell, clone PK136), 100ug InVivoMAb anti-mouse CD8α (BioXCell, clone

YTS169.4), or 25ug InVivoMAb mouse IgG2a isotype control (BioXCell, clone BE0085) in HBSS, respectively.

Antibodies were administered one day prior to the first treatment with either IL-2/αIL-2 complexes, N-

803, or their respective controls.

ELISA assays

Mouse serum or plasma was assessed via the IgG (Total) Mouse ELISA Kit (Thermo Fisher), according to the manufacturer’s directions, using 96-well Nunc Maxisorp plates (Thermo Fisher) with samples diluted

1:20,000 – 1:40,000 and plated in duplicate. Serum or plasma was assessed for ANA using the Mouse ANA

Total Ig ELISA Kit (Alpha Diagnostic International), according to the manufacturer’s directions, with samples diluted 1:50 and plated in duplicate. Mouse urine was collected via restraining animals over plastic wrap, then frozen at -80C until tested. Urine samples were diluted 1:20 and tested in duplicate

139 using the Mouse Microalbumin ELISA kit (Fisher Scientific) according to manufacturer’s instructions. All plates were read using a GlowMax Discover Microplate Reader (Promega) on the absorbance setting at

450nm. See below for details of IFNγ IVCCA ELISA.

In vitro cytokine capture assay (IVCCA)

To capture serum IFNγ in vivo, mice were intravenously injected with 0.4mg of biotinylated anti-mouse

IFNγ (a gift from the laboratory of Fred Finkelman). 24 hours later, mice were bled via tail vein into SST tubes for subsequent serum isolation. For assessment of in vivo-captured serum IFNγ, Nunc Maxisorp plates were coated overnight at 4°C with 0.5ug per well of anti-mouse IFNγ antibody clone AN18 (also a gift from the Finkelman lab). The following day, plates were washed 4 times using TBS + 0.05% Tween 20 and serum samples were diluted 1:2-1:-24 in TBS + 0.05% Tween 20 + 10% SuperBlock (ThermoFisher) and aliquoted at 30uL per well in duplicate, then incubated for two hours at RT. To generate a standard curve, dilutions of anti-IFNγ biotin bound to known concentrations of mouse IFNγ were also plated in duplicate.

Plates were washed 4 times, and 30uL of streptavidin-HRP (ThermoFisher; diluted 1:20,000) was added to each well and incubated at RT for 20 minutes. After washing again 4 times, 150uL of SuperSignal ELISA

Femto substrate (ThermoFisher) was added to each well and incubated at RT for 10 minutes, then immediately read using a GlowMax Discover Microplate Reader (Promega) on the luminescence setting.

Kidney fixation & sectioning

Mouse kidneys were manually decapsulated and fixed in 4% formalin for 24 hours, then dehydrated in

30% sucrose for up to 3 days. Kidneys were embedded in OCT compound and quickly frozen in a dry ice/ethanol slurry. 8um sections were cut using a cryostat and placed on microscope slides before being fixed with acetone and stained with immunofluorescent antibodies according to manufacturer’s

140 instructions. Finally, slides were cured and protected using Prolong Gold (ThermoFisher) and imaged via fluorescent microscopy.

RNA isolation & sequencing

RNA was extracted from RosetteSep-purified peripheral NK cells using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions and quantified using a NanoDrop. Frozen extracts were then taken to the UC Genomics, Epigenomics and Sequencing Core, where QC was peformed via Agilent

Bioanalyzer and RNA further quantified using Qubit assay. Samples that passed QC were then subjected to Illumina next generation (bulk) RNA sequencing.

Analysis of RNAseq data

All transcriptional analysis was performed within Google Chrome Secure Shell Application using the

CCHMC Biomedical Informatics Computational cluster. Fastq files were subjected to QC analysis using the

“fastqc” module, then trimmed using “trimgalore” and “cutadapt”. Sequences were then aligned to the human genome (hg19) using the “hisat2” module. Sequences were sorted using “samtools” and duplicate sequences were removed using “Picard”. Aligned reads were then counted using “FeatureCounts” and

FPKMs calculated via R software using the “DESeq2” and “calculateFPKMs” modules. Finally, a heatmap was generated with FPKM files via the software found here: https://tfwebdev.research.cchmc.org/shiny/PlotsREasy/.

AltAnalyze was downloaded (http://www.altanalyze.org/) and raw sequencing (fastq) files were inputted as suggested by the accompanying user’s guide.

Evaluating CAR NK-92 function in humanized mice

141

Eight-week-old male and female human IL-3/SCF/GM-CSF transgenic mice on an immune-deficient

NOD/LtSz-SCID IL-2RG−/− (NSGS mice) background, bred in-house, were conditioned via i.p. injections of

30 mg/kg busulfan (Sigma Aldrich) and kept on doxycycline chow throughout experiments. Twenty-four hours post-conditioning, mice were humanized via i.v. injection of 10-15 million density-gradient purified

(RBC-depleted), T cell-depleted (magnetic column; Miltenyi Biotec) cord blood leukocytes. After 4-5 weeks of reconstitution, mice were given an intraperitoneal injection of 0.5 mL pristane (Sigma Aldrich). A bolus of 107 irradiated (10 Gy,γ-ray) CAR-expressing NK-92 cells was introduced into these mice by intravenous injection. Alternatively, we intravenously injected either 5x106 irradiated control or CAR NK-

92 three times over 10 days.

iPSC differentiation into NK cells iPSC were generated from SLE patient peripheral blood by the CCHMC Pluripotent Stem Cell Core. These iPSC were cultured in mTeSR (StemCell Technologies) with media refreshment and removal of differentiating colonies performed every weekday. Differentiation of iPSC into NK cells was performed via an embryoid body-based method as described in detail elsewhere264,269, using cytokines and growth factors purchased from StemCell Technologies.

Statistical Analysis

All flow cytometric analysis was performed using FlowJo v.10 software. Compensation was performed using single-color-stained cells and/or beads (ThermoFisher), while Zombie dye single-color controls were generated using cells pre-heated on a heat block at 70°C for 10 minutes. All electronic gating was performed downstream of a FSC-H x FSC-A “singlet” gate.

142

All statistical analysis was performed using GraphPad Prism 8. Data was analyzed using one of the following tests (specifically noted in figure legends). An unpaired, two-tailed, parametric Student’s t test with a 95% confidence interval was used in cases of normal data distribution. Welch’s correction was used if heteroscedasticity was observed. If comparisons were made across more than two conditions, data was analyzed using ordinary one-way ANOVA. Accompanying ANOVA, Tukey’s multiple comparison test with a single pooled variance was used to compare each column to every other column. Dunnett’s multiple comparison test with a single pooled variance was used to compare each column to a control column.

Multiple Student’s T test using the Holm-Sidak method was performed to compare multiple columns individually. Data points were only excluded from figures due to identification as an outlier using ROUT method (Q=1%).

EC50 of rhPD-1-Fc for PD-L1 CAR NK cells was determined using the GraphPad Prism analysis “Nonlinear

Regression (curve fit): [Agonist] vs. response - Variable slope (four parameters)” as described here: https://www.graphpad.com/guides/prism/7/curve-fitting/index.htm?REG_DR_stim_variable_2.htm.

Nucleic acid oligomers used

Sequencing primers to verify PD-L1 CAR insertion into pLVX-IRES-ZsGreen plasmid

• 5’-GCACACCGGCCTTATTCCAA-3’ (Rev 1)

• 5’-CATTCAACAGACCTTGCATTCC-3’ (Rev 2)

• 5’-CTACTAGAGGATCTATTTCCGG-3’ (Fwd)

Sequencing primers to verify PD-1 insertion into pAc/V5-His plasmid

• 5’-TAGAAGGCACAGTCGAGG-3’ (Fwd)

• 5’-ACACAAAGCCGCTCCATCAG-3’ (Rev)

143

Sequencing primers to verify PD-1 insertion into PB513 plasmid

• 5’-AGAGCTCGTTTAGTGAACCGTC-3’ (Fwd)

• 5’-AACTCCTCGGGGACTGTG-3’ (Rev)

Primer/probe sets to verify CAR expression via qPCR

 PD-L1 CAR Set A

o Probe: 5’-/56-FAM/ATCGCTCCA/ZEN/GAGTGAAGTTCAGCA/3IABkFQ/-3’

o Primer 1: 5’-GCAAGCATTACCAGCCCTAT-3’

o Primer 2: 5’-TTCTGGCCCTGCTGGTA-3’

 PD-L1 CAR Set B

o Probe: 5’-/56-FAM/CCAGGCCGA/ZEN/TGAGGATATTTGCTGT/3IABkFQ/-3’

o Primer 1: 5’-CTTACCAGTGACCGCCTTG-3’

o Primer 2: 5’-CTTGGGAACCGTGACAGTAAA-3’

144

References

1. Charles A Janeway, Jr, Paul Travers, Mark Walport, and M. J. S. Immunobiology: The Immune

System in Health and Disease. (Garland Science, 2001).

2. LeBien, T. W. & Tedder, T. F. B lymphocytes: how they develop and function. Blood 112, 1570–80

(2008).

3. Nemazee, D. Mechanisms of central tolerance for B cells. Nat. Rev. Immunol. 17, 281–294 (2017).

4. Eibel, H., Kraus, H., Sic, H., Kienzler, A. K. & Rizzi, M. B cell biology: An overview. Curr. Allergy

Asthma Rep. 14, (2014).

5. Victora, G. D. & Nussenzweig, M. C. Germinal Centers. Annu. Rev. Immunol. 30, 429–457 (2012).

6. Hardy, R. R. & Hayakawa, K. B cell development pathways. Annu. Rev. Immunol. 19, 595–621

(2001).

7. Jenks, S. A., Cashman, K. S., Woodruff, M. C., Lee, F. E. H. & Sanz, I. Extrafollicular responses in

humans and SLE. Immunol. Rev. 288, 136–148 (2019).

8. Malkiel, S., Barlev, A. N., Atisha-Fregoso, Y., Suurmond, J. & Diamond, B. Plasma cell

differentiation pathways in systemic lupus erythematosus. Front. Immunol. 9, (2018).

9. Duan, B. & Morel, L. Role of B-1a cells in autoimmunity. Autoimmun. Rev. 5, 403–408 (2006).

10. Harris, D. P. et al. Reciprocal regulation of polarized cytokine production by effector B and T cells.

Nat. Immunol. 1, 475–482 (2000).

11. Mauri, C. & Ehrenstein, M. R. The ‘short’ history of regulatory B cells. Trends Immunol. 29, 34–40

(2008).

12. Mesin, L., Ersching, J. & Victora, G. D. Germinal Center B Cell Dynamics. Immunity 45, 471–482

(2016).

13. Dogan, I. et al. Multiple layers of B cell memory with different effector functions. Nat. Immunol.

10, 1292 (2009).

145

14. Cerutti, A., Puga, I. & Cols, M. New helping friends for B cells. Eur. J. Immunol. 42, 1956–1968

(2012).

15. Crotty, S. Follicular Helper CD4 T Cells (TFH). Annu. Rev. Immunol. 29, 621–663 (2011).

16. Vinuesa, C. G., Linterman, M. A., Yu, D. & MacLennan, I. C. M. Follicular Helper T Cells. Annu. Rev.

Immunol. 34, 335–368 (2016).

17. Lupus. Centers for Disease Control and Prevention Available at:

https://www.cdc.gov/lupus/index.htm. (Accessed: 9th May 2019)

18. Lupus Research Alliance. (2019). Available at: https://www.lupusresearch.org/. (Accessed: 9th

May 2019)

19. Systemic Lupus Erythematosus. US National Library of Medicine (2019). Available at:

http://www.nlm.nih.gov/medlineplus/ency/article/000435.htm. (Accessed: 9th May 2019)

20. National Institute of Diabetes and Digestive and Kidney Diseases. Glomerular Diseases. (2014).

Available at: http://www.niddk.nih.gov/health-information/health-topics/kidney-

disease/glomerular-diseases/Pages/facts.aspx. (Accessed: 9th May 2019)

21. Carter, E. E., Barr, S. G. & Clarke, A. E. The global burden of SLE: Prevalence, health disparities and

socioeconomic impact. Nat. Rev. Rheumatol. 12, 605–620 (2016).

22. Tsokos, G. C., Lo, M. S., Reis, P. C. & Sullivan, K. E. New insights into the immunopathogenesis of

systemic lupus erythematosus. Nat. Rev. Rheumatol. 12, 716–730 (2016).

23. Oku, K. & Atsumi, T. Systemic lupus erythematosus: nothing stale her infinite variety. Mod.

Rheumatol. 28, 758–765 (2018).

24. Nashi, E., Wang, Y. H. & Diamond, B. The role of B cells in lupus pathogenesis. Int. J. Biochem. Cell

Biol. 42, 543–550 (2010).

25. Fei, Y. et al. Death causes and pathogens analysis of systemic lupus erythematosus during the

past 26 years. Clin. Rheumatol. 33, 57–63 (2014).

146

26. Cameron, J. S. Lupus Nephritis. J. Am. Soc. Nephrol. 10, 413 LP-424 (1999).

27. Wen, J., Stock, A. D., Chalmers, S. A. & Putterman, C. The role of B cells and autoantibodies in

neuropsychiatric lupus. Autoimmun. Rev. 15, 890–895 (2016).

28. Tedeschi, S. K. et al. Developing and Refining New Candidate Criteria for Systemic Lupus

Erythematosus Classification: An International Collaboration. Arthritis Care Res. (Hoboken). 70,

571–581 (2018).

29. Morawski, P. A. & Bolland, S. Expanding the B Cell-Centric View of Systemic Lupus

Erythematosus. Trends Immunol. xx, 1–10 (2017).

30. Lee, K. H. et al. Neutrophil extracellular traps (NETs) in autoimmune diseases: A comprehensive

review. Autoimmun. Rev. 16, 1160–1173 (2017).

31. Mistry, P. & Kaplan, M. J. Cell death in the pathogenesis of systemic lupus erythematosus and

lupus nephritis. Clin. Immunol. 185, 59–73 (2017).

32. Hasham, M. G. et al. Systemic autoimmunity induced by the TLR7/8 agonist Resiquimod causes

myocarditis and dilated cardiomyopathy in a new mouse model of autoimmune heart disease.

Dis. Model. Mech. 10, 259–270 (2017).

33. Yokogawa, M. et al. Epicutaneous application of toll-like receptor 7 agonists leads to systemic

autoimmunity in wild-type mice: A new model of systemic lupus erythematosus. Arthritis

Rheumatol. 66, 694–706 (2014).

34. Rose, T. & Dörner, T. Drivers of the immunopathogenesis in systemic lupus erythematosus. Best

Pract. Res. Clin. Rheumatol. 31, 321–333 (2017).

35. Liu, Z. & Davidson, A. IFNalpha Inducible Models of Murine SLE. Front. Immunol. 4, 306 (2013).

36. Han, L. et al. Epstein-Barr virus infection and type I interferon signature in patients with systemic

lupus erythematosus. Lupus 961203317753069 (2018). doi:10.1177/0961203317753069

37. Ascherio, A. & Munger, K. L. EBV and Autoimmunity. Curr. Top. Microbiol. Immunol. 390, 241–65

147

(2015).

38. McClain, M. T. et al. Early events in lupus humoral autoimmunity suggest initiation through

molecular mimicry. Nat. Med. 11, 85–89 (2005).

39. Murphy, G. & Isenberg, D. A. New therapies for systemic lupus erythematosus — past imperfect,

future tense. Nat. Rev. Rheumatol. (2019). doi:10.1038/s41584-019-0235-5

40. Mizui, M. et al. IL-2 protects lupus-prone mice from multiple end-organ damage by limiting CD4-

CD8- IL-17-producing T cells. J. Immunol. 193, 2168–77 (2014).

41. Li, W., Titov, A. A. & Morel, L. An update on lupus animal models. Curr. Opin. Rheumatol. 29,

434–441 (2017).

42. Lee, Y. H. & Song, G. G. Associations between the FAS -670 A/G, -1377 G/A, and FASL -844 T/C

polymorphisms and susceptibility to systemic lupus erythematosus: a meta-analysis. Clin. Exp.

Rheumatol. 34, 634–640 (2016).

43. Liu, Y. et al. Lupus susceptibility genes may breach tolerance to DNA by impairing receptor

editing of nuclear antigen-reactive B cells. J. Immunol. 179, 1340–1352 (2007).

44. Gao, N. et al. Impaired suppressive capacity of activation-induced regulatory B cells in systemic

lupus erythematosus. Arthritis Rheumatol. (Hoboken, N.J.) 66, 2849–2861 (2014).

45. Lee, S. K. et al. Interferon-γ Excess Leads to Pathogenic Accumulation of Follicular Helper T Cells

and Germinal Centers. Immunity 37, 880–892 (2012).

46. Haas, C., Ryffel, B. & Le Hir, M. IFN-gamma is essential for the development of autoimmune

glomerulonephritis in MRL/Ipr mice. J. Immunol. 158, 5484–5491 (1997).

47. Hutloff, A. T Follicular Helper-Like Cells in Inflamed Non-Lymphoid Tissues. Front. Immunol. 9,

(2018).

48. William, J., Euler, C., Christensen, S. & Shlomchik, M. J. Evolution of autoantibody responses via

somatic hypermutation outside of germinal centers. Science 297, 2066–2070 (2002).

148

49. Tipton, C. M. et al. Diversity, cellular origin and autoreactivity of antibody-secreting cell

population expansions in acute systemic lupus erythematosus. Nat. Immunol. 16, 755–765

(2015).

50. Vinuesa, C. G., Sanz, I. & Cook, M. C. Dysregulation of germinal centres in autoimmune disease.

Nat. Rev. Immunol. 9, 845–857 (2009).

51. Linterman, M. a et al. Follicular helper T cells are required for systemic autoimmunity. J. Exp.

Med. 206, 561–576 (2009).

52. Zhang, X. et al. Circulating CXCR5+ CD4+ helper T cells in systemic lupus erythematosus patients

share phenotypic properties with germinal center follicular helper T cells and promote antibody

production. Lupus 909–917 (2015). doi:10.1177/0961203314567750

53. Kim, Y. U., Lim, H., Jung, H. E., Wetsel, R. a. & Chung, Y. Regulation of Autoimmune Germinal

Center Reactions in Lupus-Prone BXD2 Mice by Follicular Helper T Cells. PLoS One 10, e0120294

(2015).

54. Klarquist, J. & Janssen, E. M. The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in

C57BL/6 Mice. J. Vis. Exp. 1–11 (2015). doi:10.3791/53319

55. Gensous, N., Schmitt, N., Richez, C., Ueno, H. & Blanco, P. T follicular helper cells, interleukin-21

and systemic lupus erythematosus. Rheumatology (Oxford). (2016).

doi:10.1093/rheumatology/kew297

56. De Franco, A. L. Germinal centers and autoimmune disease in humans and mice. Immunol. Cell

Biol. 94, 918–924 (2016).

57. Witte, T. IgM antibodies against dsDNA in SLE. Clin. Rev. Allergy Immunol. 34, 345–347 (2008).

58. Blanco, P., Ueno, H. & Schmitt, N. T follicular helper (Tfh) cells in lupus: Activation and

involvement in SLE pathogenesis. Eur. J. Immunol. 46, 281–290 (2016).

59. Choi, J.-Y. et al. Circulating Follicular Helper-Like T Cells in Systemic Lupus Erythematosus:

149

Association With Disease Activity. Arthritis Rheumatol. 67, 988–999 (2015).

60. Vinuesa, C. G. et al. A RING-type ubiquitin ligase family member required to repress follicular

helper T cells and autoimmunity. Nature 435, 452–458 (2005).

61. Craft, J. E. Follicular helper T cells in immunity and systemic autoimmunity. Nat. Rev. Rheumatol.

8, 337–347 (2012).

62. Crotty, S. T Follicular Helper Cell Biology: A Decade of Discovery and Diseases. Immunity 50,

1132–1148 (2019).

63. Kobayashi, T., Iijima, K., Dent, A. L. & Kita, H. Follicular helper T cells mediate IgE antibody

response to airborne allergens. J. Allergy Clin. Immunol. 139, 300–313 (2017).

64. Ahearne, M. J., Allchin, R. L., Fox, C. P. & Wagner, S. D. Follicular helper T-cells: Expanding roles in

T-cell lymphoma and targets for treatment. Br. J. Haematol. 166, 326–335 (2014).

65. Murphy, G., Lisnevskaia, L. & Isenberg, D. Systemic lupus erythematosus and other autoimmune

rheumatic diseases: Challenges to treatment. Lancet 382, 809–818 (2013).

66. Sawada, T., Fujimori, D. & Yamamoto, Y. Systemic lupus erythematosus and immunodeficiency.

Immunol. Med. 1–9 (2019). doi:10.1080/25785826.2019.1628466

67. Ponticelli, C. & Moroni, G. Hydroxychloroquine in systemic lupus erythematosus (SLE). Expert

Opin. Drug Saf. 16, 411–419 (2017).

68. Lee, Y. H., Woo, J. H., Choi, S. J., Ji, J. D. & Song, G. G. Induction and maintenance therapy for

lupus nephritis: a systematic review and meta-analysis. Lupus 19, 703–710 (2010).

69. MedlinePlus. Cyclophosphamide: MedlinePlus Drug Information. U.S. National Library of

Medicine (2011). Available at:

https://www.nlm.nih.gov/medlineplus/druginfo/meds/a682080.html. (Accessed: 20th May 2019)

70. Mok, C. C. Current role of rituximab in systemic lupus erythematosus. Int. J. Rheum. Dis. 18, 154–

163 (2015).

150

71. Dörner, T. & Lipsky, P. E. Beyond pan-B-cell-directed therapy — new avenues and insights into

the pathogenesis of SLE. Nat. Rev. Rheumatol. (2016). doi:10.1038/nrrheum.2016.158

72. Ehrenstein, M. R. & Wing, C. The BAFFling effects of rituximab in lupus: danger ahead? Nat. Rev.

Rheumatol. 12, 367–372 (2016).

73. Matsuki-Muramoto, Y., Nozawa, K., Uomori, K., Sekigawa, I. & Takasaki, Y. Bortezomib treatment

prevents glomerulosclerosis associated with lupus nephritis in a murine model through

suppressive effects on the immune and renin-angiotensin systems. Mod. Rheumatol. 27, 77–86

(2017).

74. Alexander, T. et al. The proteasome inhibitior bortezomib depletes plasma cells and ameliorates

clinical manifestations of refractory systemic lupus erythematosus. Ann. Rheum. Dis. 74, 1474–

1478 (2015).

75. Alexander, T. et al. Proteasome inhibition with bortezomib induces a therapeutically relevant

depletion of plasma cells in SLE but does not target their precursors. Eur. J. Immunol. 48, 1573–

1579 (2018).

76. Khodadadi, L. et al. Bortezomib Plus Continuous B Cell Depletion Results in Sustained Plasma Cell

Depletion and Amelioration of Lupus Nephritis in NZB/W F1 Mice. PLoS One 10, e0135081 (2015).

77. Ishii, T. et al. Multicenter double-blind randomized controlled trial to evaluate the effectiveness

and safety of bortezomib as a treatment for refractory systemic lupus erythematosus. Mod.

Rheumatol. 28, 986–992 (2018).

78. Sawaf, M., Dumortier, H. & Monneaux, F. Follicular Helper T Cells in Systemic Lupus

Erythematosus: Why Should They Be Considered as Interesting Therapeutic Targets? J. Immunol.

Res. 2016, (2016).

79. Faliti, C. E. et al. P2X7 receptor restrains pathogenic Tfh cell generation in systemic lupus

erythematosus. J. Exp. Med. jem.20171976 (2019). doi:10.1084/jem.20171976

151

80. Choi, S.-C. et al. Inhibition of glucose metabolism selectively targets autoreactive follicular helper

T cells. Nat. Commun. 9, 4369 (2018).

81. Glatigny, S. et al. Abatacept Targets T Follicular Helper and Regulatory T Cells, Disrupting

Molecular Pathways That Regulate Their Proliferation and Maintenance. J. Immunol. ji1801425

(2019). doi:10.4049/jimmunol.1801425

82. Pimentel-Quiroz, V. R., Ugarte-Gil, M. F. & Alarcon, G. S. Abatacept for the treatment of systemic

lupus erythematosus. Expert Opin. Investig. Drugs 25, 493–499 (2016).

83. Coquery, C. M. et al. BAFF regulates follicular helper t cells and affects their accumulation and

interferon-gamma production in autoimmunity. Arthritis Rheumatol. (Hoboken, N.J.) 67, 773–784

(2015).

84. Perry, D., Sang, A., Yin, Y., Zheng, Y.-Y. & Morel, L. Murine models of systemic lupus

erythematosus. J. Biomed. Biotechnol. 2011, 271694 (2011).

85. Morel, L. et al. Genetic reconstitution of systemic lupus erythematosus immunopathology with

polycongenic murine strains. Proc Natl Acad Sci U S A 97, 6670–6675 (2000).

86. Hsu, H. C. et al. Production of a novel class of polyreactive pathogenic autoantibodies in BXD2

mice causes glomerulonephritis and arthritis. Arthritis Rheum. 54, 343–355 (2006).

87. Reeves, W. H., Lee, P. Y., Weinstein, J. S., Satoh, M. & Lu, L. Induction of autoimmunity by

pristane and other naturally occurring hydrocarbons. Trends Immunol. 30, 455–464 (2009).

88. Gunawan, M. et al. A Novel Human Systemic Lupus Erythematosus Model in Humanised Mice.

Sci. Rep. 7, 16642 (2017).

89. Eisenberg, R. a. & Via, C. S. T cells, murine chronic graft-versus-host disease and autoimmunity. J.

Autoimmun. 39, 240–247 (2012).

90. Waggoner, S. N. et al. Roles of natural killer cells in antiviral immunity. Curr. Opin. Virol. 16, 15–

23 (2016).

152

91. Kannan, G. S., Aquino-Lopez, A. & Lee, D. A. Natural Killer Cells in Malignant Hematology: A

Primer for the Non-Immunologist. Blood Rev. (2016). doi:10.1016/j.blre.2016.08.007

92. Vivier, E., Tomasello, E., Baratin, M., Walzer, T. & Ugolini, S. Functions of natural killer cells. Nat.

Immunol. 9, 503–510 (2008).

93. Geiger, T. L. & Sun, J. C. Development and maturation of natural killer cells. Curr. Opin. Immunol.

39, 82–89 (2016).

94. Montaldo, E., Vacca, P., Moretta, L. & Mingari, M. C. Development of human natural killer cells

and other innate lymphoid cells. Semin. Immunol. 26, 107–113 (2014).

95. Hesslein, D. G. T. & Lanier, L. L. Transcriptional Control of Natural Killer Cell Development and

Function. Advances in Immunology 109, (Elsevier inc., 2011).

96. Robinette, M. L. et al. Transcriptional programs define molecular characteristics of innate

lymphoid cell classes and subsets. Nat. Immunol. 16, (2015).

97. Yu, J., Freud, A. G. & Caligiuri, M. A. Location and cellular stages of natural killer cell

development. Trends Immunol. 34, 573–582 (2013).

98. Orr, M. T. & Lanier, L. L. Natural Killer Cell Education and Tolerance. Cell 142, 847–856 (2010).

99. Orr, M. T. & Lanier, L. L. Inhibitory Ly49 receptors on mouse natural killer cells. Curr. Top.

Microbiol. Immunol. 350, 67–87 (2011).

100. Cichocki, F., Sitnicka, E. & Bryceson, Y. T. NK cell development and function - Plasticity and

redundancy unleashed. Semin. Immunol. 26, 114–126 (2014).

101. Poli, A. et al. CD56bright natural killer (NK) cells: An important NK cell subset. Immunology 126,

458–465 (2009).

102. Ferlazzo, G. et al. The Abundant NK Cells in Human Secondary Lymphoid Tissues Require

Activation to Express Killer Cell Ig-Like Receptors and Become Cytolytic. J. Immunol. 172, 1455 LP-

1462 (2004).

153

103. Hayakawa, Y. & Smyth, M. J. CD27 dissects mature NK cells into two subsets with distinct

responsiveness and migratory capacity. J. Immunol. 176, 1517–1524 (2006).

104. Marquardt, N., Wilk, E., Pokoyski, C., Schmidt, R. E. & Jacobs, R. Murine CXCR3+CD27bright NK

cells resemble the human CD56bright NK-cell population. Eur. J. Immunol. 40, 1428–1439 (2010).

105. Cooper, M. A. et al. Human natural killer cells: A unique innate immunoregulatory role for the

CD56bright subset. Blood 97, 3146–3151 (2001).

106. Li, Z., Lim, W. K., Mahesh, S. P., Liu, B. & Nussenblatt, R. B. Cutting edge: in vivo blockade of

human IL-2 receptor induces expansion of CD56(bright) regulatory NK cells in patients with active

uveitis. J. Immunol. 174, 5187–91 (2005).

107. Hayakawa, Y., Huntington, N. D., Nutt, S. L. & Smyth, M. J. Functional subsets of mouse natural

killer cells. Immunol. Rev. 214, 47–55 (2006).

108. Müller, J. R., Waldmann, T. A. & Dubois, S. Loss of cytotoxicity and gain of cytokine production in

murine tumor-activated NK cells. PLoS One 9, (2014).

109. El-Zayadi, A. A. et al. Interleukin-22 drives the proliferation, migration and osteogenic

differentiation of mesenchymal stem cells: a novel cytokine that could contribute to new bone

formation in spondyloarthropathies. Rheumatology kew384 (2016).

doi:10.1093/rheumatology/kew384

110. Burrack, K. S. et al. Interleukin-15 Complex Treatment Protects Mice from Cerebral Malaria by

Inducing Interleukin-10-Producing Natural Killer Cells. Immunity 48, 760–772.e4 (2018).

111. Zhang, C., Zhang, J. & Tian, Z. The regulatory effect of natural killer cells: do ‘NK-reg cells’ exist?

Cell. Mol. Immunol. 3, 241–254 (2006).

112. Waggoner, S. N., Cornberg, M., Selin, L. K. & Welsh, R. M. Natural killer cells act as rheostats

modulating antiviral T cells. Nature 481, 394–398 (2011).

113. Crouse, J., Xu, H. C., Lang, P. a. & Oxenius, A. NK cells regulating T cell responses: mechanisms

154

and outcome. Trends Immunol. 36, 49–58 (2015).

114. Rydyznski, C. et al. Generation of cellular immune memory and B-cell immunity is impaired by

natural killer cells. Nat. Commun. 6, 6375 (2015).

115. Rydyznski, C. E. et al. Affinity Maturation Is Impaired by Natural Killer Cell Suppression of

Germinal Centers. Cell Rep. 24, 3367–3373.e4 (2018).

116. Brown, M. G. & Erickson, L. D. Editorial: NK cell reaping of Tfh cells: reckless slaughter or sensible

pruning? J. Leukoc. Biol. 98, 139–142 (2015).

117. Perricone, R., Perricone, C., De Carolis, C. & Shoenfeld, Y. NK cells in autoimmunity: A two-edg’d

weapon of the immune system. Autoimmun. Rev. 7, 384–390 (2008).

118. Zitti, B. & Bryceson, Y. T. Natural killer cells in inflammation and autoimmunity. Cytokine Growth

Factor Rev. 42, 37–46 (2018).

119. Bentham, J. et al. Genetic association analyses implicate aberrant regulation of innate and

adaptive immunity genes in the pathogenesis of systemic lupus erythematosus. Nat. Genet.

(2015). doi:10.1038/ng.3434

120. Hervier, B. et al. Phenotype and function of natural killer cells in systemic lupus erythematosus:

Excess interferon-γ production in patients with active disease. Arthritis Rheum. 63, 1698–1706

(2011).

121. Park, Y.-W. et al. Impaired differentiation and cytotoxicity of natural killer cells in systemic lupus

erythematosus. Arthritis Rheum. 60, 1753–1763 (2009).

122. Takeda, K. & Dennert, G. The development of autoimmunity in C57BL/6 lpr mice correlates with

the disappearance of natural killer type 1-positive cells: evidence for their suppressive action on

bone marrow stem cell proliferation, B cell immunoglobulin secretion, and autoimmune sy. J.

Exp. Med. 177, 155–64 (1993).

123. Kim, J. R., Mathew, S. O., Patel, R. K., Pertusi, R. M. & Mathew, P. A. Altered expression of

155

signalling lymphocyte activation molecule (SLAM) family receptors CS1 (CD319) and 2B4 (CD244)

in patients with systemic lupus erythematosus. Clin. Exp. Immunol. 160, 348–358 (2010).

124. Hagberg, N. et al. Systemic lupus erythematosus immune complexes increase the expression of

SLAM family members CD319 (CRACC) and CD229 (LY-9) on plasmacytoid dendritic cells and

CD319 on CD56(dim) NK cells. J. Immunol. 191, 2989–98 (2013).

125. Schepis, D. et al. Increased proportion of CD56bright natural killer cells in active and inactive

systemic lupus erythematosus. Immunology 126, 140–146 (2009).

126. Shi, F.-D. & Van Kaer, L. Reciprocal regulation between natural killer cells and autoreactive T cells.

Nat. Rev. Immunol. 6, 751–60 (2006).

127. Liu, R. et al. Autoreactive T Cells Mediate NK Cell Degeneration in Autoimmune Disease. J.

Immunol. 176, 5247–5254 (2006).

128. Hagberg, N. et al. Functional Anti-CD94/NKG2A and Anti-CD94/NKG2C Autoantibodies in Patients

With Systemic Lupus Erythematosus. Arthritis Rheumatol. 67, 1000–1011 (2015).

129. Fogel, L. A., Yokoyama, W. M. & French, A. R. Natural killer cells in human autoimmune disorders.

Arthritis Res. Ther. 15, 216 (2013).

130. Baxter, A. G. & Smyth, M. J. The role of NK cells in autoimmune disease. Autoimmunity 35, 1–14

(2002).

131. Zahran, A. M. et al. Natural Killer and Natural Killer T Cells in Juvenile Systemic Lupus

Erythematosus: Relation to Disease Activity and Progression. Arch. Immunol. Ther. Exp. (Warsz).

(2019). doi:10.1007/s00005-019-00537-6

132. Löfgren, S. E. et al. A 3′-untranslated region variant is associated with impaired expression of

CD226 in T and natural killer T cells and is associated with susceptibility to systemic lupus

erythematosus. Arthritis Rheum. 62, 3404–3414 (2010).

133. Wandstrat, A. E. et al. Association of Extensive Polymorphisms in the SLAM/CD2 Gene Cluster

156

with Murine Lupus. Immunity 21, 769–780 (2004).

134. Graham, R. R., Hom, G., Ortmann, W. & Behrens, T. W. Review of recent genome-wide

association scans in lupus. J. Intern. Med. 265, 680–688 (2009).

135. Sibbit, W. L., Mathews, P. M. & Bankhurst, D. Natural Killer Cell in Systemic Lupus Erythematosus.

J. Clin. Invest. 71, 1230–1239 (1983).

136. Lin, S.-J. et al. Activating and inhibitory receptors on natural killer cells in patients with systemic

lupus erythematosis-regulation with interleukin-15. PLoS One 12, e0186223 (2017).

137. Lin, Y.-L. & Lin, S.-C. Analysis of the CD161-expressing cell quantities and CD161 expression levels

in peripheral blood natural killer and T cells of systemic lupus erythematosus patients. Clin. Exp.

Med. 17, 101–109 (2017).

138. Sourour, S. K. et al. Unraveling the expression of microRNA-27a* & NKG2D in peripheral blood

mononuclear cells and natural killer cells of pediatric systemic lupus erythematosus patients. Int.

J. Rheum. Dis. 20, 1237–1246 (2017).

139. Suarez-Fueyo, A. et al. Downregulation of CD3zeta in NK Cells from Systemic Lupus

Erythematosus Patients Confers a Proinflammatory Phenotype. J. Immunol. 200, 3077–3086

(2018).

140. Henriques, A. et al. NK cells dysfunction in systemic lupus erythematosus: Relation to disease

activity. Clin. Rheumatol. 32, 805–813 (2013).

141. Lin, S.-J. et al. Cytotoxic Function and Cytokine Production of Natural Killer Cells and Natural Killer

T-Like Cells in Systemic Lupus Erythematosis Regulation with Interleukin-15. Mediators Inflamm.

2019, 4236562 (2019).

142. Voynova, E. N., Skinner, J. & Bolland, S. Expansion of an Atypical NK Cell Subset in Mouse Models

of Systemic Lupus Erythematosus. J. Immunol. 194, 1503–1513 (2015).

143. Hudspeth, K. et al. Natural Killer cell expression of Ki67 is associated with elevated serum IL-15,

157

disease activity and nephritis in Systemic Lupus Erythematosus. Clin. Exp. Immunol. 0–2 (2019).

doi:10.1111/cei.13263

144. Jackson, S. W. et al. B cell IFN-γ receptor signaling promotes autoimmune germinal centers via

cell-intrinsic induction of BCL-6. J. Exp. Med. 213, 733–750 (2016).

145. Lee, S. W. et al. iNKT Cells Suppress Pathogenic NK1.1+CD8+ T Cells in DSS-Induced Colitis. Front.

Immunol. 9, 1–14 (2018).

146. Wild, J. et al. Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores

sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis. Leukemia

29, 1676–1683 (2015).

147. Yuan, D., Thet, S., Zhou, X. J., Wakeland, E. K. & Dang, T. The role of NK cells in the development

of autoantibodies. Autoimmunity 44, 641–651 (2011).

148. Spada, R. et al. NKG2D ligand overexpression in lupus nephritis correlates with increased NK cell

activity and differentiation in kidneys but not in the periphery. J. Leukoc. Biol. 97, 583–598

(2015).

149. Uchida, T. et al. Roles of Natural Killer T Cells and Natural Killer Cells in Kidney Injury. Int. J. Mol.

Sci. 20, 2487 (2019).

150. Huang, Z. et al. Involvement of CD226+ NK cells in immunopathogenesis of systemic lupus

erythematosus. J. Immunol. 186, 3421–3431 (2011).

151. Cruz-González, D. de J. et al. Analysis of the regulatory function of natural killer cells from

patients with systemic lupus erythematosus. Clin. Exp. Immunol. 191, 288–300 (2018).

152. Voynova, E., Qi, C.-F., Scott, B. & Bolland, S. Cutting Edge: Induction of Inflammatory Disease by

Adoptive Transfer of an Atypical NK Cell Subset. J. Immunol. 195, 806–809 (2015).

153. Bielekova, B. et al. Regulatory CD56(bright) natural killer cells mediate immunomodulatory

effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis. Proc. Natl. Acad. Sci. U.

158

S. A. 103, 5941–5946 (2006).

154. Search of_ daclizumab - List Results - ClinicalTrials.

155. Morandi, F. et al. CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated

Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation. J. Immunol.

195, 965–972 (2015).

156. Guillerey, C., Huntington, N. D. & Smyth, M. J. Targeting natural killer cells in cancer

immunotherapy. Nat. Immunol. 17, 1025 (2016).

157. Boyman, O. & Sprent, J. The role of interleukin-2 during homeostasis and activation of the

immune system. Nat. Rev. Immunol. 12, 180 (2012).

158. Tomala, J., Chmelova, H., Mrkvan, T., Rihova, B. & Kovar, M. In vivo expansion of activated naive

CD8+ T cells and NK cells driven by complexes of IL-2 and anti-IL-2 monoclonal antibody as novel

approach of cancer immunotherapy. J. Immunol. 183, 4904–4912 (2009).

159. Tomala, J. & Kovar, M. IL-2/anti-IL-2 mAb immunocomplexes: A renascence of IL-2 in cancer

immunotherapy? Oncoimmunology 5, e1102829–e1102829 (2015).

160. Gold, R. et al. Daclizumab high-yield process in relapsing-remitting multiple sclerosis (SELECT): a

randomised, double-blind, placebo-controlled trial. Lancet 381, 2167–2175 (2013).

161. Martin, J. F., Perry, J. S. A., Jakhete, N. R., Wang, X. & Bielekova, B. An IL-2 paradox: blocking

CD25 on T cells induces IL-2-driven activation of CD56(bright) NK cells. J. Immunol. 185, 1311–

1320 (2011).

162. Kappos, L. et al. Daclizumab HYP versus Interferon Beta-1a in Relapsing Multiple Sclerosis. N.

Engl. J. Med. 373, 1418–1428 (2015).

163. End of the road for daclizumab in multiple sclerosis. Lancet 391, 1000 (2018).

164. Boyman, O., Kovar, M., Rubinstein, M., Surh, C. D. & Sprent, J. Selective Stimulation of T Cell

Subsets with Antibody-Cytokine Immune Complexes. Science 1924, 1–5 (2006).

159

165. Hao, J. et al. IL-2/IL-2 Ab Therapy Induces Target Organ NK Cells that Inhibit CNS Inflammation.

Ann. Neurol. 29, 997–1003 (2012).

166. Heiler, S. Prophylactic and Therapeutic effects complexes in systemic lupus erythematosus-like

chronic graft-Versus-host Disease. Front. Immunol. 9, 1–15 (2018).

167. Budagian, V., Bulanova, E., Paus, R. & Bulfone-Paus, S. IL-15/IL-15 receptor biology: A guided tour

through an expanding universe. Cytokine Growth Factor Rev. 17, 259–280 (2006).

168. Rubinstein, M. P. et al. Converting IL-15 to a superagonist by binding to soluble IL-15Ra. Proc.

Natl. Acad. Sci. U. S. A. 103, 9166–71 (2006).

169. Xu, W. et al. Efficacy and mechanism-of-action of a novel superagonist interleukin-15:

interleukin-15 receptor αSu/Fc fusion complex in syngeneic murine models of multiple myeloma.

Cancer Res. 73, 3075–3086 (2013).

170. Rhode, P. R. et al. Comparison of the Superagonist Complex, ALT-803, to IL15 as Cancer

Immunotherapeutics in Animal Models. Cancer Immunol. Res. 4, 49–60 (2016).

171. Sullivan, R. P., Leong, J. W. & Fehniger, T. a. MicroRNA regulation of natural killer cells. Front.

Immunol. 4, 1–12 (2013).

172. Sullivan, R. P. et al. MicroRNA-155 tunes both the threshold and extent of NK cell activation via

targeting of multiple signaling pathways. J. Immunol. 191, 5904–13 (2013).

173. Kim, N. et al. MicroRNA-150 regulates the cytotoxicity of natural killers by targeting perforin-1. J.

Allergy Clin. Immunol. 134, 195–203.e4 (2014).

174. Barrett, D. M. et al. Chimeric Antigen Receptor Therapy for Cancer. Anu. Rev Med 368, 333–347

(2014).

175. Guedan, S., Calderon, H., Posey, A. D. & Maus, M. V. Engineering and design of chimeric antigen

receptors. Mol. Ther. Methods Clin. Dev. 12, 145–146 (2018).

176. Anwer, F. et al. Donor origin CAR T cells: Graft versus malignancy effect without GVHD, a

160

systematic review. Immunotherapy 9, 123–130 (2017).

177. Neelapu, S. S. et al. Chimeric antigen receptor T-cell therapy-assessment and management of

toxicities. Nat. Rev. Clin. Oncol. 15, 47–62 (2018).

178. Daher, M. & Rezvani, K. Next generation natural killer cells for cancer immunotherapy: the

promise of genetic engineering. Curr. Opin. Immunol. 51, 146–153 (2018).

179. Li, Y., Hermanson, D. L., Moriarity, B. S. & Kaufman, D. S. Human iPSC-Derived Natural Killer Cells

Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity. Cell Stem Cell 23, 181–

192 (2018).

180. Tang, X. et al. First-in-man clinical trial of CAR NK-92 cells: safety test of CD33-CAR NK-92 cells in

patients with relapsed and refractory acute myeloid leukemia. Am. J. Cancer Res. 8, 1083–1089

(2018).

181. Zhang, L. et al. Chimeric antigen receptor (CAR) T cells targeting a pathogenic MHC class

II:peptide complex modulate the progression of autoimmune diabetes. J. Autoimmun. 1–9

(2018). doi:10.1016/j.jaut.2018.08.004

182. Siegler, E. L., Zhu, Y., Wang, P. & Yang, L. Off-the-Shelf CAR-NK Cells for Cancer Immunotherapy.

Cell Stem Cell 23, 160–161 (2018).

183. Morris, S., Cohen, P. L. & Eisenberg, R. Experimental induction of systemic lupus erythematosus

by recognition of foreign Ia. Clin. Immunol. Immunopathol. 57, 263–73 (1990).

184. Klarquist, J. et al. STING-mediated DNA sensing promotes antitumor and autoimmune responses

to dying cells. J. Immunol. 193, 6124–6134 (2014).

185. Chen, F., Maldonado, M. a, Madaio, M. & Eisenberg, R. a. The role of host (endogenous) T cells in

chronic graft-versus-host autoimmune disease. J. Immunol. 161, 5880–5885 (1998).

186. Gaither, K. A. et al. The immunomodulatory, antitumor and antimetastatic responses of

melanoma-bearing normal and alcoholic mice to sunitinib and ALT-803: a combinatorial

161

treatment approach. Cancer Immunol. Immunother. 65, 1123–1134 (2016).

187. Tian, Z., Gershwin, M. E. & Zhang, C. Regulatory NK cells in autoimmune disease. J. Autoimmun.

39, 206–15 (2012).

188. Wang, N. et al. Slamf6 negatively regulates autoimmunity. Clin. Immunol. 173, 19–26 (2016).

189. Ling, G. S. et al. C1q restrains autoimmunity and viral infection by regulating CD8+ T cell

metabolism. Science (80-. ). 360, 558–563 (2018).

190. Mostböck, S. et al. IL-2/Anti-IL-2 Antibody Complex Enhances Vaccine-Mediated Antigen-Specific

CD8<sup>+</sup> T Cell Responses and Increases the Ratio of Effector/Memory

CD8<sup>+</sup> T Cells to Regulatory T Cells. J. Immunol. 180, 5118 LP-5129 (2008).

191. Wong, H. C., Jeng, E. K. & Rhode, P. R. The IL-15-based superagonist ALT-803 promotes the

antigen-independent conversion of memory CD8+ T cells into innate-like effector cells with

antitumor activity. Oncoimmunology 2, e26442 (2013).

192. Liu, B.-H. Differential Coexpression Network Analysis for Gene Expression Data BT -

Computational Systems Biology: Methods and Protocols. in (ed. Huang, T.) 155–165 (Springer

New York, 2018). doi:10.1007/978-1-4939-7717-8_9

193. Emig, D. et al. AltAnalyze and DomainGraph: analyzing and visualizing exon expression data.

Nucleic Acids Res. 38, W755–W762 (2010).

194. Filtz, T. M., Vogel, W. K. & Leid, M. Regulation of transcription factor activity by interconnected

post-translational modifications. Trends Pharmacol. Sci. 35, 76–85 (2014).

195. Vujanovic, L. et al. Tumor-Derived a -Fetoprotein Directly Drives Human Natural Killer – Cell

Activation and Subsequent Cell Death. Cancer Immunol. Res. 493–503 (2017). doi:10.1158/2326-

6066.CIR-16-0216

196. Lu, L. et al. Platelet-derived growth factor receptor alpha ( PDGFR a ) is overexpressed in NK / T-

cell lymphoma and mediates cell survival. Biochem. Biophys. Res. Commun. 504, 525–531 (2018).

162

197. Baines, M. G., Lafleur, F. L. & Holbein, B. E. Involvement of transferrin and transferrin receptors in

human natural killer effector:Target interaction. Immunol. Lett. 7, 51–55 (1983).

198. Gong, Y. et al. Quantitative proteomics of CSF reveals potential predicted biomarkers for

extranodal NK- / T-cell lymphoma of nasal-type with ethmoidal sinus metastasis. Life Sci. 198, 94–

98 (2018).

199. Tognarelli, S. et al. Tissue-Specific Microvascular Endothelial Cells Show Distinct Capacity To

Activate NK Cells: Implications for the Pathophysiology of Granulomatosis with Polyangiitis. J.

Immunol. 192, 3399–3408 (2014).

200. Kitagawa, Y., Liu, C. & Ding, X. The influence of natural mineral water on aquaporin water

permeability and human natural killer cell activity. Biochem. Biophys. Res. Commun. 409, 40–45

(2011).

201. R. Fuerstenberg, S. Anderson, K. Brumbaugh, J. S. Multiplex Analysis of Secreted Biomarkers from

Stimulated Natural Killer Cells. R&D Systems Available at:

https://www.rndsystems.com/resources/posters/multiplex-analysis-secreted-biomarkers-

stimulated-natural-killer-cells. (Accessed: 7th January 2019)

202. Betten, A., Dahlgren, C., Hermodsson, S. & Hellstrand, K. Serotonin protects NK cells against

oxidatively induced functional inhibition and apoptosis. J. Leukoc. Biol. 70, 65–72 (2001).

203. Huang, R. et al. HIF1A is a critical downstream mediator for hemophagocytic lymphohistiocytosis.

Haematologica 102, 1956–1968 (2017).

204. Gladek, I., Ferdin, J., Horvat, S., Calin, G. A. & Kunej, T. HIF1A gene polymorphisms and human

diseases: Graphical review of 97 association studies. Genes. Chromosomes Cancer 56, 439–452

(2017).

205. Colucci, F. et al. Differential requirement for the transcription factor PU.1 in the generation of

natural killer cells versus B and T cells. Blood 97, 2625 LP-2632 (2001).

163

206. Elemam, N. M. et al. Repressing PU.1 by miR-29a∗ in NK cells of HCV patients, diminishes its

cytolytic effect on HCV infected cell models. Hum. Immunol. 76, 687–694 (2015).

207. Aboelenein, H. R. et al. Reduction of CD19 autoimmunity marker on B cells of paediatric SLE

patients through repressing PU.1/TNF-α/BAFF axis pathway by miR-155. Growth Factors 35, 49–

60 (2017).

208. Hikami, K. et al. Association of a functional polymorphism in the 3′-untranslated region of SPI1

with systemic lupus erythematosus. Arthritis Rheum. 63, 755–763 (2011).

209. Tabellini, G. et al. Impaired natural killer cell functions in patients with signal transducer and

activator of transcription 1 (STAT1) gain-of-function mutations. J. Allergy Clin.

Immunol. 140, 553–564.e4 (2017).

210. Shao, W.-H. et al. Stat1 Regulates Lupus-like Chronic Graft-versus-Host Disease Severity via

Interactions with Stat3. J. Immunol. 195, 4136 LP-4143 (2015).

211. Patel, Z. H. et al. A plausibly causal functional lupus-associated risk variant in the STAT1–STAT4

locus. Hum. Mol. Genet. 27, 2392–2404 (2018).

212. Chmielewski, M., Hombach, A., Heuser, C., Adams, G. P. & Abken, H. T Cell Activation by

Antibody-Like Immunoreceptors: Increase in Affinity of the Single-Chain Fragment Domain above

Threshold Does Not Increase T Cell Activation against Antigen-Positive Target Cells but Decreases

Selectivity. J. Immunol. 173, 7647–7653 (2004).

213. Caruso, H. G. et al. Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue

while maintaining potent antitumor activity. Cancer Res. 75, 3505–3518 (2015).

214. Cheng, X. et al. Structure and interactions of the human programmed cell death 1 receptor. J.

Biol. Chem. 288, 11771–11785 (2013).

215. Butte, M. J., Pena-cruz, V., Kim, M., Freeman, G. J. & Sharpe, A. H. Interaction of Human PD-L1

and B7-1. Mol Immunol 45, 3567–3572 (2013).

164

216. Klingemann, H., Boissel, L. & Toneguzzo, F. Natural Killer Cells for immunotherapy – Advantages

of the NK-92 Cell Line over Blood NK Cells. Front. Immunol. 7, 1–7 (2016).

217. March, M. E., Gross, C. C. & Long, E. O. Use of transfected Drosophila S2 cells to study NK cell

activation. Methods Mol. Biol. 612, 67–88 (2010).

218. Herman, A., Kappler, J. W., Marrack, P. C. & Pullen, A. M. Superantigens: mechanism of T-cell

stimulation and role in immune responses. Annu. Rev. Immunol. 9, 745–72 (1991).

219. Choi, M. Y., Flood, K., Bernatsky, S., Ramsey-Goldman, R. & Clarke, A. E. A review on SLE and

malignancy. Best Pract. Res. Clin. Rheumatol. 31, 373–396 (2017).

220. Lin, S.-J. et al. Effect of Interleukin-15 on CD11b, CD54, and CD62L Expression on Natural Killer

Cell and Natural Killer T-Like Cells in Systemic Lupus Erythematosus. Mediators Inflamm. 2016, 1–

8 (2016).

221. Mildner, A. & Jung, S. Development and Function of Dendritic Cell Subsets. Immunity 40, 642–656

(2014).

222. Halim, T. Y. F. & Takei, F. Isolation and characterization of mouse innate lymphoid cells. Curr.

Protoc. Immunol. 106, 3.25.1-3.25.13 (2014).

223. Somanchi, S. S., Senyukov, V. V, Denman, C. J. & Lee, D. a. Expansion, purification, and functional

assessment of human peripheral blood NK cells. J. Vis. Exp. 48, 2540 (2011).

224. Tanasescu, C. et al. Endothelial dysfunction in inflammatory rheumatic diseases. Rom. J. Intern.

Med. 47, 103–108 (2009).

225. Gunesch, J. T. et al. Genome-wide analyses and functional profiling of human NK cell lines. Mol.

Immunol. (2018). doi:10.1016/j.molimm.2018.07.015

226. Bienert, G. P. & Chaumont, F. Aquaporin-facilitated transmembrane diffusion of hydrogen

peroxide. Biochim. Biophys. Acta 1840, 1596–1604 (2014).

227. Munakata, T. et al. Induction of interferon-gamma production by human natural killer cells

165

stimulated by hydrogen peroxide. J. Immunol. 134, 2449–2455 (1985).

228. El-Hag, A. & Clark, R. A. Down-regulation of human natural killer activity against tumors by the

neutrophil myeloperoxidase system and hydrogen peroxide. J. Immunol. 133, 3291–3297 (1984).

229. Strzepa, A., Pritchard, K. A. & Dittel, B. N. Myeloperoxidase: A new player in autoimmunity. Cell.

Immunol. 317, 1–8 (2017).

230. Larosa, M. et al. IL-12 and IL-23/Th17 axis in systemic lupus erythematosus. Exp. Biol. Med.

(Maywood). 244, 42–51 (2019).

231. Hervas-Stubbs, S. et al. Direct Effects of Type I Interferons on Cells of the Immune System. Clin.

Cancer Res. 17, 2619 LP-2627 (2011).

232. Luo, S., Wang, Y., Zhao, M. & Lu, Q. The important roles of type I interferon and interferon-

inducible genes in systemic lupus erythematosus. Int. Immunopharmacol. 40, 542–549 (2016).

233. Meng, C., Guo, L.-B., Liu, X., Chang, Y.-H. & Lin, Y. Targeting STAT1 in Both Cancer and Insulin

Resistance Diseases. Curr. Protein Pept. Sci. 18, 181–188 (2017).

234. Nielsen, N., Ødum, N., Ursø, B., Lanier, L. L. & Spee, P. Cytotoxicity of CD56 bright NK cells

towards autologous activated CD4 + T cells is mediated through NKG2D, LFA-1 and TRAIL and

dampened via CD94/NKG2A. PLoS One 7, 1–10 (2012).

235. Blanco, P. et al. Increase in activated CD8+ T lymphocytes expressing perforin and granzyme B

correlates with disease activity in patients with systemic lupus erythematosus. Arthritis Rheum.

52, 201–211 (2005).

236. Wu, L. & Van Kaer, L. Natural killer T cells in health and disease. Front. Biosci. (Schol. Ed). 3, 236–

251 (2011).

237. Locci, M. et al. Human Circulating PD-1+CXCR3−CXCR5+ Memory Tfh Cells Are Highly Functional

and Correlate with Broadly Neutralizing HIV Antibody Responses. Immunity 39, 758–769 (2014).

238. Linterman, M. A. et al. Foxp3+follicular regulatory T cells control the germinal center response.

166

Nat. Med. 17, 975–982 (2011).

239. Essig, K. et al. Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell

Differentiation and Conversion of Treg to Tfr Cells. Immunity 47, 1067–1082.e12 (2017).

240. Sage, P. et al. Suppression by Tfr cells leads to durable and selective inhibition of B cell effector

function. Nat. Immunol. 17, 1436–1446 (2016).

241. Yamazaki, T. et al. Expression of Programmed Death 1 Ligands by Murine T Cells and APC. J.

Immunol. 169, 5538–5545 (2002).

242. Curran, C. S., Gupta, S., Sanz, I. & Sharon, E. PD-1 immunobiology in systemic lupus

erythematosus. J. Autoimmun. 97, 1–9 (2019).

243. Liu, S. M. et al. Immune cell transcriptome datasets reveal novel leukocyte subset-specific genes

and genes associated with allergic processes. J. Allergy Clin. Immunol. 118, 496–503 (2006).

244. Shi, Q. et al. A novel monoclonal antibody against human CD80 and its immune protection in a

mouse lupus-like disease. Int. J. Immunopathol. Pharmacol. 24, 583–593 (2011).

245. Liarski, V. M. et al. Cell Distance Mapping Identifies Functional T Follicular Helper Cells in

Inflamed Human Renal Tissue. Sci. Transl. Med. 6, 230–246 (2014).

246. Rao, D. A. et al. Pathologically expanded peripheral T helper cell subset drives B cells in

rheumatoid arthritis. Nature 542, 110–114 (2017).

247. Taylor, D. K. et al. T follicular helper–like cells contribute to skin fibrosis. Sci. Transl. Med. 10,

eaaf5307 (2018).

248. He, R. et al. Follicular CXCR5-expressing CD8+ T cells curtail chronic viral infection. Nature 537,

412–416 (2016).

249. Huot, N. et al. Natural killer cells migrate into and control simian immunodeficiency virus

replication in lymph node follicles in African green monkeys. Nat. Med. 23, 1277–1286 (2017).

250. Ayala, V. I. et al. CXCR5-Dependent Entry of CD8 T Cells into Rhesus Macaque B-Cell Follicles

167

Achieved through T-Cell Engineering. J. Virol. 91, e02507-16 (2017).

251. Gong, J. H., Maki, G. & Klingemann, H. G. Characterization of a human cell line (NK-92) with

phenotypical and functional characteristics of activated natural killer cells. Leukemia 8, 652–658

(1994).

252. Kitchens, W. H. et al. Interruption of OX40L signaling prevents costimulation blockade-resistant

allograft rejection. JCI insight 2, e90317 (2017).

253. Sakthiswary, R. & D’Cruz, D. Intravenous immunoglobulin in the therapeutic armamentarium of

systemic lupus erythematosus: a systematic review and meta-analysis. Medicine (Baltimore). 93,

e86 (2014).

254. Appelgren, D., Eriksson, P., Ernerudh, J. & Segelmark, M. Marginal-Zone B-Cells Are Main

Producers of IgM in Humans, and Are Reduced in Patients With Autoimmune Vasculitis. Front.

Immunol. 9, 2242 (2018).

255. Susanibar Adaniya, S. P., Cohen, A. D. & Garfall, A. L. Chimeric antigen receptor T cell

immunotherapy for multiple myeloma: A review of current data and potential clinical

applications. Am. J. Hematol. 94, S28–S33 (2019).

256. Manabu Fujisawa, Shigeru Chiba, M. S.-Y. Recent Progress in the Understanding of

Angioimmunoblastic T-cell Lymphoma. J. Clin. Exp. Hematop. 57, 109–119 (2017).

257. Sato, F. et al. Angioimmunoblastic T-cell lymphoma mice model. Leuk. Res. 37, 21–27 (2013).

258. Ehlers, M. et al. Immunoregulatory natural killer cells suppress autoimmunity by down-regulating

antigen-specific CD8 + T cells in mice. Endocrinology 153, 4367–4379 (2012).

259. Welcher, A. A. et al. Blockade of Interferon-γ Normalizes Interferon-Regulated Gene Expression

and Serum CXCL10 Levels in Patients With Systemic Lupus Erythematosus. Arthritis Rheumatol.

67, 2713–2722 (2015).

260. Wang, Z. et al. Diphtheria-toxin based anti-human CCR4 immunotoxin for targeting human

168

CCR4(+) cells in vivo. Mol. Oncol. 9, 1458–1470 (2015).

261. Sheppard, S. et al. The Murine Natural Cytotoxic Receptor NKp46/NCR1 Controls TRAIL Protein

Expression in NK Cells and ILC1s. Cell Rep. 22, 3385–3392 (2018).

262. Xu, H. C. et al. Type I Interferon Protects Antiviral CD8+ T Cells from NK Cell Cytotoxicity.

Immunity 40, 949–960 (2014).

263. Crouse, J. et al. Type I Interferons Protect T Cells against NK Cell Attack Mediated by the

Activating Receptor NCR1. Immunity 40, 961–973 (2014).

264. Knorr, D. A. et al. Clinical-scale derivation of natural killer cells from human pluripotent stem cells

for cancer therapy. Stem Cells Transl. Med. 2, 274–83 (2013).

265. Hermanson, D. L. et al. Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment

of Ovarian Cancer. Stem Cells 34, 93–101 (2016).

266. Wunderlich, M. et al. AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice

constitutively expressing human SCF, GM-CSF and IL-3. Leukemia 24, 1785–1788 (2010).

267. Sun, M. et al. Construction and evaluation of a novel humanized HER2-specific chimeric receptor.

Breast Cancer Res. 16, 61 (2014).

268. Han, J. et al. CAR-engineered NK cells targeting wild-type EGFR and EGFRvIII enhance killing of

glioblastoma and patient-derived glioblastoma stem cells. Sci. Rep. 5, 1–13 (2015).

269. Bock, A. M., Knorr, D. & Kaufman, D. S. Development, expansion, and in vivo monitoring of

human NK cells from human embryonic stem cells (hESCs) and and induced pluripotent stem cells

(iPSCs). J. Vis. Exp. e50337 (2013). doi:10.3791/50337

270. Okazaki, T. & Honjo, T. The PD-1-PD-L pathway in immunological tolerance. Trends Immunol. 27,

195–201 (2006).

271. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9

complex. Nature 517, 61422–61427 (2015).

169

272. Rezvani, K. & Rouce, R. H. The application of natural killer cell immunotherapy for the treatment

of cancer. Front. Immunol. 6, (2015).

273. Childs, R. W. & Carlsten, M. Therapeutic approaches to enhance natural killer cell cytotoxicity

against cancer: the force awakens. Nat. Rev. Drug Discov. 14, 487–498 (2015).

274. Ames, E. & Murphy, W. J. Advantages and clinical applications of natural killer cells in cancer

immunotherapy. Cancer Immunol. Immunother. 63, 21–8 (2014).

275. Meinhardt, K. et al. Identification and characterization of the specific murine NK cell subset

supporting graft--leukemia- and reducing graft--host-effects. Oncoimmunology 4, e981483

(2015).

170

Appendix – Treating a mouse model of lupus nephritis using PD-L1-expressing natural killer cells.

Summarized research proposal & findings of a Lupus Foundation of America Gina M. Finzi Student

Summer Fellowship (awarded in 2016)

Abstract

Despite standard care, 60% of Americans with systemic lupus erythematosus (SLE) develop lupus nephritis (LN). Current therapeutics for SLE and LN are broadly immunosuppressive: a high-risk strategy in the context of infection. Furthermore, 20% of LN leads to kidney failure, yet only one new treatment has been approved in over 50 years. There is a clear need for safe, effective, and innovative therapies for LN. SLE treatment largely fails to prevent LN potentially because it fails to specifically target the immune dysfunction that underlies disease. SLE is defined by rampant autoantibodies that cause widespread pathology and, when deposited in the kidneys, elicit LN. Autoantibodies arise from aberrant germinal centers (GCs) in which follicular helper T cells (Tfh) play a crucial role. Thus, pioneering approaches to restrain Tfh and GCs in SLE may be a potent tool to treat autoantibody-driven LN. We posit that inhibition of Tfh and GCs in SLE can be achieved by harnessing the regulatory role of natural killer (NK) cells. NK cells are gaining popularity as cell therapy for cancer. We find that NK cells suppress activated TFH during immunization, resulting in blunted GCs and reduced antiviral antibodies. We therefore propose that enhancing NK cell-mediated suppression of Tfh and GCs is an effective therapeutic strategy to ameliorate autoantibody-mediated LN. NK cells expressing programmed death ligand 1 (PDL1) killed autoreactive T cells expressing programmed cell death protein 1 (PD1) and alleviated disease in mouse models of autoimmune diabetes. Tfh express high levels of PD1, suggesting that they may also be amenable to NK cell-mediated killing in a context of LN. We will transfer PDL1- expressing NK cells into a mouse model of lupus-like disease and test their ability to subdue Tfh and GCs, thus alleviating nephropathy. Our innovative NK cell-based therapy can potentially reduce LN in a targeted manner while bypassing the high risks of broader immunosuppression.

171

Specific Aims

A promising new approach for targeted immunotherapy of lupus and lupus nephritis (LN) lies with natural killer (NK) cells – innate lymphocytes that are often diminished and dysfunctional in lupus patients120,121,125,135. NK cells are being successfully utilized as cancer-targeting therapeutic effector cells.

We discovered that NK cells also effectively suppress follicular helper T cells (TFH) and germinal center

(GC) B cell responses after immunization114. Moreover, the absence of NK cells in virus-infected mice led to increased antibody deposition in the glomeruli, a finding resembling the pathology of lupus nephritis

(unpublished data). Collectively, these data suggest that NK cells proficient in subduing TFH and GC reactions may be an effective therapy to restrain these aberrant processes that drive autoantibody- mediated nephropathy in lupus.

270 Since TFH express high levels of the inhibitory receptor programmed cell death protein 1 (PD-1) , we hypothesize that overexpression of the PD-1 ligand, programmed death ligand 1 (PD-L1), on NK cells represents an innovative means of therapeutically constraining TFH-mediated support of autoantibody production and alleviating LN disease pathogenesis. A similar administration of these “PD-L1high” NK cells in an autoimmune diabetes mouse model significantly reduced pathology by targeting PD-1 expressing T cells258. As part of a larger thesis project examining the capability of PD-L1high mouse and human NK cells to suppress TFH cells and ameliorate lupus-like disease, we propose to complete the following aims this summer:

high Aim 1. Determine the capacity of PD-L1 NK cells to suppress pathogenic TFH cells. Mouse NK cells stimulated with interleukin 18 (IL-18), an established method of increasing their expression of PD-L1258,

54 will be transferred into a TFH cell-driven mouse model of lupus-like disease to determine their ability to suppress early pathogenic TFH responses in these mice.

172

Aim 2. Ascertain the ability of PD-L1high NK cells to ameliorate autoantibody production and LN in a mouse model of lupus-like disease. After several weeks, the lupus-like mouse model used in Aim 1 results in autoantibody production and glomerulonephritis54. We will assess the ability of PD-L1high NK cells to inhibit lupus-like disease in this model.

Research Strategy

Background and Significance: Lupus is an autoimmune disease affecting 161,000 Americans in which rampant production of autoantibodies drives widespread immunopathology17,19,20. The high morbidity and mortality of lupus results from multi-organ involvement19, with LN as a predominant contributing factor to death68. LN is a renal disease triggered by lupus-associated autoantibody deposition in the glomeruli and subsequent autoimmune attack on renal tissues68. The current standard of care for lupus and LN is founded in the use of broadly immunosuppressive pharmacologic agents – a strategy that is inherently dangerous and largely ineffective at preventing disease sequelae17,19,20,65. Even with proper treatment, an estimated one in five patients with LN will progress to end-stage renal disease and kidney failure within ten years of diagnosis20,68. Importantly, nonspecific therapeutics for LN neglect the underlying production of autoantibodies that orchestrate the immune attack on renal tissue. A more

49–53 targeted approach to restrain aberrant TFH and GC responses would provide a powerful tool to prevent autoantibody-driven kidney disease for more than a million people worldwide who suffer this disease.

Innovation: We propose a highly innovative approach to treat LN by selectively subduing the specific immune processes that putatively precipitate LN pathogenesis. Importantly, our approach contrasts with conventional treatments that broadly suppress the immune system and undermine host defense against infection65. Our discovery that NK cells suppress many of the same immune phenomena

173 implicated in lupus pathogenesis prompted us to propose that harnessing this regulatory activity of NK cells can potentially prevent and treat the underlying immune dysfunction in LN. We intend to test NK cells as a prospective immunotherapeutic agent in an animal model of lupus and LN, a strategy that to our knowledge has not been explored. In corresponding experiments outside the scope of this summer fellowship, we will use cutting-edge strategies (e.g. CRISPRa) to prompt overexpression of the PD-L1 on human NK cells to enhance their capacity to suppress GC-like human T-B cell co-cultures237,271. Our experiments will stimulate further research and progress toward eventual clinical trials exploring this type of cellular therapy in LN. Our research has the potential to expand current uses of NK cell-mediated therapy272–274, and provide a powerful new tool for use in the fields of rheumatology and nephrology.

Approach: We hypothesize that PD-L1high NK cells will display enhanced immunosuppressive function. In the broader study, we will also test this using a spontaneous mouse model of lupus-like disease and in human in vitro assays as well. In this summer fellowship, we will focus on an inducible model of lupus- like disease that is used in our lab54. Splenocytes are first isolated from bm12 mice that are genetically identical to C57BL/6 mice other than 3 amino acid substitutions on MHC class II. Injection of these bm12 splenocytes into C57BL/6 mice induces alloactivation of bm12 CD4 T cells and a chronic graft-vs.-host disease (cGVHD) with disease manifestations closely resembling lupus54. Specifically, we observe an immense expansion of bm12 TFH cells and widespread GC responses within 2 weeks. Over the course of

2 months, mice show massive generation of ANAs (e.g. anti-dsDNA, anti-ssDNA, anti-chromatin antibodies), and accumulation of IgG deposits in the renal vasculature, interstitia, and glomeruli, resulting in glomerulonephritis and quantifiable proteinuria54.

high Aim 1. Determine the capacity of PD-L1 NK cells to suppress pathogenic TFH cells.

174

To test our hypothesis, NK cells isolated from C57BL/6 or PD-L1-/- mice will be induced to express high levels of PD-L1 via overnight culture with recombinant mouse IL-18258. Although our initial attempts to generate PD-L1high mouse NK cells via this method have shown success (unpublished data), wild-type donor NK cells will be assessed by flow cytometry to validate high expression of PD-L1. Groups of bm12- induced lupus-like mice will receive intravenous injections of IL-18-treated wild-type or PD-L1-deficient

NK cells every other day for 2 weeks. Mice will be euthanized at this time, and bm12 graft size and TFH differentiation will be measured by flow cytometry. Furthermore, GC B cell frequencies will be measured by flow cytometry and confocal microscopy. We expect injections of IL-18 stimulated wild-type (PD-

L1high) but not PD-L1-/- or untreated wild-type NK cells to reduce the bm12 graft expansion as well as the presence of TFH and GC B cells.

Aim 2. Ascertain the ability of PD-L1high NK cells to ameliorate autoantibody production and LN in a mouse model of lupus-like disease.

Lupus-like mice treated with NK cells for two weeks as described above will be monitored for 2 months via weekly collections of blood and urine to measure ANA titers and proteinuria (indicative of kidney dysfunction), respectively. At the 2 month time point, mice will be euthanized and kidney sections analyzed for presence of immunoglobulin, deposition of complement, and other pathological characteristics of glomerulonephritis (collaboration with Prasad Devarajan). We expect PD-L1high NK cells to reduce ANA production and ameliorate the development of lupus-like nephropathy as compared to mice treated with PD-L1-/- or unstimulated wild-type NK cells. In both Aims, a sample size of 4 mice/group will permit 80% power to detect reduced disease by chi-square analysis (p = 0.05).

Results

175

IL-18 optimization: Although our first attempt to generate PD-L1high mouse NK cells via overnight incubation with IL-18 were successful (data not shown), optimization of this approach was needed to ensure maximal PD-L1 expression before attempting our aims. Furthermore, concomitant examination of IL-18-induced phenotypic changes of other critical NK cell surface proteins may prove useful in evaluating our results. Thus, spleens from two male C57BL/6 mice were homogenized through a nylon mesh strainer, pooling splenocytes together. NK cells were then isolated from splenocytes via (negative) magnetic selection using an EasySep Mouse NK Cell Isolation Kit according to manufacturer’s instructions (Stemcell Technologies Inc.). NK cells were counted via a hemocytometer and resuspended in complete media at a concentration of 1 million cells per mL, then dispensed in 200µL aliquots into wells of a 96-well flat-bottom plate. All wells received 300U/mL of recombinant murine IL-2. Subsets of wells received 0, 25, 50, 100, or 200 ng/mL of recombinant murine IL-18, and the plate was incubated overnight (~19 hours) at 37°C. Aliquots were pooled according to condition, washed twice with buffer

(PBS supplemented with 5% FBS), and treated with an Fc receptor blocking agent (antiCD16/32). Cells were then stained with fluorescently-conjugated antibodies directed at the following surface proteins:

CD3, CD19, NK1.1, PD-L1, PD-1, DX5, NKG2D, TRAIL, CD27, CD11b, and C-kit. Analysis reveals that treating mouse NK cells with IL-18 (and IL-2) enhances their surface expression of PD-L1 vs. treatment with IL-2 alone, although using IL-18 concentrations >25ng/mL does not further enhance PD-L1 expression (Fig. 1a). Thus, we treated NK cells with 300U/mL IL-2 and 25ng/mL IL-18 in our experiment testing Aims 1 and 2. As an aside, IL-18 treatment does not appreciably alter the expression of the NK activation marker, NKG2D, or the expression of the apoptosis-inducing ligand TRAIL (data not shown).

Furthermore, the “maturity” of NK cells, based upon expression of CD27 and CD11b (Hayakawa et al.,

2006), is also not appreciably altered by IL-18 treatment (data not shown). Of interesting relevance, IL-

18 treatment does increase the proportion of NK cells expressing c-kit (Fig. 1b), a receptor for stem cell factor that is expressed by a regulatory-like subset of NK cells 258,275.

176

Aim 1: NK cells were isolated from the pooled splenocytes of six C57BL/6 female donor mice using an

EasySep Mouse NK Cell Enrichment Kit. Counted NK cells were treated overnight with IL-2 and IL-18, as previously optimized. The next day, half of these NK cells were treated with purified anti-PD-L1 blocking antibody (0.5µg per 106 cells) for 30 minutes at 4°C as described elsewhere 258 to block the effect of PD-

L1 expression. On this day (“Day 0”), 12 C57BL/6 female mice received an intraperitoneal injection of

~7.5×106 purified CD4 T cells magnetically isolated from five female Bm12 mice as described elsewhere

(Klarquist et al., 2015) to induce SLE-like disease, a one-time injection. These mice were then divided into three groups of four and injected i.v. with either: a) 5×105 IL-18-treated NK cells, b) 5×105 IL-18- treated, PD-L1-blocked NK cells, or c) saline alone. On Day 2, groups received a second i.v. injection of their respective treatments (a, b, or c, above). On Day 5, groups received a third and final i.v. injection of their respective treatments (a, b, or c, above), except that that roughly 1×106 NK cells were injected per mouse. Aliquots of each isolated NK cell population, as well as the CD4 T cell graft used to induce disease, were stained for identifying surface markers (e.g. CD3, CD4, NK1.1, NKp46) and analyzed via flow cytometry to determine cell purity (Table 1). Cell viability for each injection was also determined via trypan blue exclusion (Table 1). At Day 14, two mice from each treatment group were euthanized and spleens were collected and weighed. Following homogenization and lysis of red blood cells (via ACK lysis buffer), counted splenocytes were stained for flow cytometry using fluorescently-conjugated antibodies targeting the following surface markers: CD45.1, CD45.2, CXCR5, CD4, PD-1, Fas, GL7, CD19,

CD138, and B220, as well as Live/Dead Aqua (to exclude dead and dying cells). While a sample size of 2 mice per group was too low to establish statistical significance, some changes in mean values were observed. For example, although average spleen weight varies widely, mean splenocyte count is reduced by roughly 36% in mice receiving IL-18-treated NK cells over saline treated mice, and this decrease is fully reversed in mice treated with PD-L1-blocked NK cells (Fig. 2a). Mean Bm12 donor

177

(CD45.1+) CD4+ CXCR5+ PD-1+ T cell engraftment and host (CD45.1+ CD19+ B220+) Fas+ GL7+ GC B cell expansion does not appear to differ between treatment groups (Fig. 2b). Host CD138+ plasmablast populations could not be accurately determined (data not shown).

Aim 2: At Day 56 (~2 months), the remaining two mice from each treatment group were euthanized, cardiac-perfused with PBS, and kidneys were formalin-fixed. Left kidneys were placed in70% ethanol for future paraffin-embedding and H&E staining. Right kidneys were dehydrated in sucrose, frozen in O.C.T., sectioned using a cryostat (8µm), and acetone-fixed to slides for immunofluorescent (IF) staining.

Spleens were weighed and counted splenocytes were stained as on Day 14. Measures of splenomegaly

(Fig 3a) appear higher in mice receiving IL-18-treated NK cells, although a potential outlier in this group is likely skewing results. Although mean Bm12 donor T cell engraftment looks highest in mice receiving

PD-L1-blocked NK cells, mean splenic GC B cell expansion is reduced by roughly 62% in mice receiving IL-

18-treated NK cells over saline treated mice, and this decrease is mostly reversed in mice treated with

PD-L1-blocked NK cells (Fig. 3b). Prior to initiating disease (Day -1), and at weekly intervals until day 56, urine and serum were collected from these mice to monitor proteinuria and ANA titers, respectively.

Although ANA analysis is pending, urine protein (as measured by dipstick testing) suggests increased urine protein over time amongst all mice, but no noticeable difference between treatment groups (Fig.

4). Furthermore, IF staining for total IgG shows sparse and indistinct foci of IgG in kidney glomeruli amongst all treatment groups (Fig. 5), and enumeration of foci per glomerulus did not reveal a difference (data not shown). Pathology scores of H&E-stained kidney sections are pending.

178

Figures:

179

180

181