Moving in for the Kill: Natural Killer Cell Localization in Regulation of Humoral

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Moving in for the kill: Natural killer cell localization

in regulation of humoral immunity

A thesis submitted to the

Graduate School of the University of Cincinnati

In partial fulfillment of the requirements for the degree of

Master of Science

In the division of Immunology of the College of Medicine

By: Michael T. Moran B.S., University of Dayton, 2014

March 2016

Committee Chair: Stephen Waggoner, Ph.D Jonathan Katz, Ph.D J. Matthew Kofron, Ph.D Leah Kottyan, Ph.D

Abstract Modern medicine has advanced tremendously over the past century to develop vaccines that have successfully reduced the spread of, or eradicated, many infectious diseases including measles, smallpox and polio. While this progression has dramatically improved global health, the development of vaccination strategies for protection against deadly pathogens, such as human immunodeficiency virus (HIV) and hepatitis C virus, have been elusive. Further investigation of immune-regulatory mechanisms that are influential in the development of protective humoral immunity must be pursued for the generation of robust vaccinations to continue. Here, we present our findings on a regulatory role for natural killer (NK) cells in the generation of the humoral immune response to viral infection. Specifically, we have determined that in mice infected with the acute strain of lymphocytic choriomeningitis virus (LCMV), NK cells impair the development of T follicular helper cells and the germinal center response. We have determined that this suppressive function is carried out in a perforin-dependent manner at an early stage of infection. Importantly, during this early time frame, an anatomic redistribution of

NK cells occurs in the spleen to areas involved in generation of the germinal center responses, including the B cell follicle and T/B border of the white pulp. Mechanistically, we have identified a subset of these NK cells expressing the B-cell follicle homing receptor CXCR5 that are present in the spleen at these same early time points of infection. Taken together, these data support out hypothesis that this subset of white pulp localizing NK cells traffics to this region early in infection to facilitate direct contact with cells involved in the generation of T follicular helper cell and germinal center responses. While in contact with these precursors, we suspect

NK cells carry out perforin-dependent cytolysis of target cells to ultimately suppress the generation of humoral immune responses. These NK cells may represent an immunoregulatory subset and a possible new target of interventions to facilitate the generation of robust and protective vaccine strategies against pathogens.

Acknowledgements

I first and foremost would like to dedicate this thesis to my parents. Without their guidance and unwavering support, none of this would have been possible.

I would also like to thank my mentor Steve and all of the members of the Waggoner lab. Steve’s mentorship in developing me as a scientist has afforded me with skills that will continue to serve me in science and as a future physician.

The entirety of the lab’s ability to deal with my shenanigans, as well as add to them, has made the past two years beyond enjoyable. Thanks for that.

Table of Contents Abstract 2

Acknowledgements 3

Table of Contents 4

Background 6

I. Natural killer cells and innate immunity 6 II. Natural killer cell trafficking 8 III. The humoral immune response 9 IV. Natural killer cell regulation of adaptive and humoral immunity 10 Materials and methods 14

Results 17

I. Natural killer cells impair generation of the Tfh and GC response during viral infection 17 II. Natural killer cell suppression of humoral immunity is perforin dependent 18 III. Natural Killer cells localize to splenic white pulp 3 d.p.i. LCMV-Arm 19 IV. Use of intravascular staining method for analysis of natural killer cell localization to the white pulp 20 V. Natural killer cell localization to the white pulp is transient 21 VI. CXCR5 expression on natural killer cells correlates with white pulp localization 22 VII. White and red pulp localizing natural killer cells have different transcriptional profiles 23 Figures 26

Figure 1. NK cells suppress Tfh and GC response during LCMV-Arm infection 27

Figure 2. NK cell suppression of Tfh responses is perforin dependent 28

Figure 3. NK cells localize to the white pulp 3 d.p.i. 29

Figure 4. Intravascular staining method for analysis of white pulp localizing NK cells 30

Figure 5. NK cell localization to the white pulp is transient and NK cell suppression of humoral immunity occurs in the same time frame 31

Figure 6. NK cell expression of CXCR5 correlates with their localization to the white pulp 32

Figure 7. RNA-seq analysis of white and red pulp localizing NK cells 33

Discussion 34

I. NK cell localization to the white pulp and regulation of humoral immunity 35 II. Understanding a role for CXCR5 in NK cell localization and suppression of humoral immunity 37 III. Conclusions 39 References 41

Background

I. Natural killer cells and innate immunity Since the original description of NK cells in 1975 by Kiessling et al. and Herberman et al. as a

“previously undefined cytotoxic cell” with “naturally occurring killer activity”, the field has advanced tremendously to demonstrate the critical role of NK cells in tumor surveillance and the immune response to viral infection1–4. As alluded to by their name, NK cells are innate lymphocytes with a predetermined capacity to rapidly carry out cytotoxic effector functions against target cells, which does not require somatic antigen rearrangement, clonal expansion, or functional differentiation of these cells5. This innate killing capability is regulated by the net signaling input from an array of germline-encoded activating and inhibitory NK cell receptors

(NKRs)6,7.

Activating receptors, such as NKG2D, engage ligands associated with cell stress presented by cells that have undergone transformation to a cancerous state or become infected with a virus6,8. NK cell receptors and NK cell-sensitive pathogens appear to have co-evolved9. Most notably, the m157 viral peptide presented on cells infected by mouse cytomegalovirus (MCMV) engages Ly49H to induce NK cell killing of infected cells. This controls viral progression and promotes clearance of replicating virus in resistant strains of mice10–14.

Inhibitory NKRs provide the host with protection from NK cell-mediated tissue destruction through recognition of “self” ligands generally present on all healthy cells. The canonical “self” ligand in this context is major histocompatibility class I (MHC-I), which interacts with Ly49 receptors in mice, killer-cell immunoglobulin-like receptors (KIRs) in humans and NKG2A in both mice and humans6,7. Loss of MHC-I, which can occur in the context of cancer and viral infection, is often referred to as “missing self”, a phenomenon that relieves the inhibitory signal provided to the NK cells and thereby permitting lysis of the target. This interplay between NKR activating

7 and inhibitory signaling, known as the “dynamic equilibrium concept”, not only demonstrates the complexities involved in regulating NK cell cytotoxicity, but also the innate nature of these cells to respond to a stimulus without reliance on the protracted expansion of rare antigen-specific subsets of cells, as is the case for T and B cell responses5,15.

One of the primary mechanisms by which NK cells exert their killing effector functions is through the degranulation-mediated release of cytotoxic proteins, such as perforin and granzymes. The release of these two proteins work in tandem to induce cell death, where perforin polymerizes in the target cell membrane to form a pore to allow entry of granzymes that activate both caspase- dependent and independent apoptotic pathways. Although granzymes are the primary effector protein of cell death in this form of cytotoxicity, it has been shown perforin is by itself sufficient to induce killing16.

Although degranulation is induced through NKR signaling, subsets of NK cells possessing the

Fc receptor CD16 can be stimulated to degranulate by way of antibody-dependent cell cytotoxicity (ADCC) through interactions with antibodies that have opsonized a target cell. NK cells are also capable of killing independently of perforin and granzyme through expression of death receptor ligands, such as Fas ligand (FasL) and tumor necrosis factor-related apoptosis- inducing ligand (TRAIL), which interact with death receptors up-regulated on distressed cells to induce apoptosis.

Aside from killing, another major effector function of NK cells is the production of pro- inflammatory and immunosuppressive cytokines. This is largely influenced by stimulation from

IL-12, IL-15, IL-18 and type I interferons produced by dendritic cells (DCs)17–19. Production of cytokines such as tumor necrosis factor (TNF), transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) by NK cells is critical for shaping the immune response to infection by modulating both T and B cells as well as DCs19. The most notable cytokine produced by NK cells is interferon gamma (IFN-γ), produced during viral infection, which has been shown to be

8 crucial in supporting further cytokine production by antigen presenting cells (APCs), and in promoting T helper type 1 (Th1) polarization in the lymph node20,21.

II. Natural killer cell trafficking

NK cells are stationed throughout the body in both lymphoid and non-lymphoid tissues to facilitate their function as innate sensors of tumor growth and viral infection. At steady state, NK cells can be found predominantly in the blood as well as highly vascularized tissues such as the lung, liver and the red pulp of the spleen, while few are present in the lymph nodes22. Little is understood about the driving forces behind their anatomical localization in the periphery at steady state, but it is largely thought to be independent of chemokines since mice lacking various chemokine receptors normally found on NK cells show very few abnormalities in the distribution of these cells23. In contrast, NK cells possess a battery of chemokine receptors, such as CCR5, CXCR3, CXCR6, CX3CR1 and CCR2 that interact with chemokines produced by stromal cells and DCs during inflammation. This drives trafficking of NK cells to areas of inflammation and into lymphoid tissue, where they can modulate the immune response20,21,23–26

The anatomic localization of NK cells has been demonstrated in multiple contexts to be an important factor in their ability to modulate the immune response. Notably, Ly49H+ NK cell migration and localization to the splenic white pulp is essential for protection of this compartment from destruction by cytotoxic T cells during MCMV infection and is thought to occur in a CCR5 and CXCR3 dependent manner24,25. The receptor CXCR3, which responds to the inflammatory chemokines CXCL9, 10 and 11, has also been implicated in NK cell migration to draining lymph nodes where they provide essential IFN-γ to drive a Th1 polarized immune response 21,27. NK cell localization to the splenic white pulp has also been implicated in a similar role of providing IFN-γ for T cell priming and potentiating an inflammatory state20.

III. The humoral immune response The immune system has two primary arms, innate and adaptive immunity, which provide the host protection from pathogens and development of cancer. This system also plays an important role in maintaining homeostatic health, for example through immune-mediated pathways involved in development, wound healing, and maintenance of a beneficial microbiome. While the innate arm of the immune system is fast acting, the adaptive arm is somewhat slower as it requires rare, antigen-specific, subset of lymphocytes to proliferate, and often times remodel their receptors, to elicit a response. The deficits in speed of the adaptive response, which the innate system for the most part lacks, are offset by the development of memory and long-lived antigen-specific protection from secondary infection through the development of memory effector T cells.

In the case of humoral immunity, a branch of the adaptive immune response, this protective memory manifests through the production of neutralizing antibodies against the antigen by long- lived plasma cells generated in germinal center (GC) reactions in secondary lymphoid organs

(SLOs) throughout the body28,29. This GC reaction consists of foci made up of activated, antigen-specific B cells that cycle through rounds of rapid proliferation, somatic hyper-mutation and T-cell facilitated selection to form high-affinity B-cell clones that recognize the stimulatory antigen with greater avidity. This structure is also one site where B cells can undergo class switch recombination to change the isotype of antibody they are capable of producing depending on the inflammatory response driving this reaction29.

Lymph nodes and the splenic white pulp are made up of T-cell zones directly surrounded by B- cell follicles. This dichotomy is organized by constitutively expressed chemokine gradients of

CXCL19/21 (T cell zone) and CXCL13 (B cell zone), established by stromal cells located in each region. These chemokines either interact with CCR7 on T cells or CXCR5 on B cells in order to correctly localize these cells within the correct compartments30. In the context of

10 inflammation, this chemokine gradient is the driving force behind facilitating interactions between helper CD4+ T cells and activated follicular B cells in order to stimulate a humoral immune response31–33.

Specifically, when naive antigen-specific CD4+ T cells become activated in the T-cell zone, a subset of these cells differentiates towards a T follicular helper (Tfh) cell fate. These Tfh cells are the primary helper T cell subset in humoral immunity. Tfh cells are an essential source of stimulatory signaling for follicular B cells leading to their differentiation into GC B cells.

Importantly, these Tfh cells continue to support the GC throughout its functional life34. This initial stimulatory signaling is mediated through interactions between CD40 ligand on Tfh and CD40 on activated follicular B cells, as well as through other receptor pairings (e.g. ICOS) and specific cytokine (e.g. IL-21) signals35,36. As these two cell populations are anatomically separated from each other in the T- and B-cell zones prior to antigenic stimulation, a re-localization event must occur to facilitate direct cell-cell contact. For this, activated CD4+ T cells differentiating to a Tfh phenotype begin to express the B-cell follicle homing receptor CXCR5 while also becoming less responsive to CCL19/21 through lowered expression of CCR731. The dynamic interplay between the expression and functionality of these two receptors mediates the localization of these cells to the interface of the T-cell zone and B-cell follicle (T/B border) where stimulatory interactions between Tfh and activated follicular B cells can be carried out to initiate germinal center formation.

IV. Natural killer cell regulation of adaptive and humoral immunity NK cells are classically thought to function as innate “watch dogs” in surveillance of tumor development and viral infection. While these are undoubtedly essential roles for NK cells, there is a growing body of literature supporting their functions as regulatory cells of the adaptive immune responses during viral infection 37,38. NK cell killing of lymphocytes and production of cytokines are implicated in these regulatory processes. For example, NK cells can either

11 suppress or promote DC function and maturation following antigenic stimulation. In terms of suppression, this can occur either through the direct killing of immature DCs by NK cells or indirectly through control of antigen availability. An example of the latter is the direct NK cell- mediated cytotoxic control of MCMV replication that reduces viral titers in host tissues leading to less inflammation and attenuated activation of DCs39–41. In contrast, NK cell production of cytokines, such as tumor necrosis factor (TNF) and IFN-γ, can promote DC maturation and activation, which in turn produce IL-12 to further potentiate NK-cell effector functions. These NK-

DC interactions also have downstream implications in the overall outcome of T cell responses.

Notably, NK cell killing of target cells indirectly stimulates antigen-specific adaptive and humoral immune responses through robust priming of CD4+ and CD8+ T cells by cross-presentation of released antigens to these cells by DCs42. This NK-DC relationship also has negative consequences as well. For example, in vivo derived DCs isolated from lymphocytic choriomeningitis (LCMV) infected mice depleted of NK cells possess a greater capability to stimulate CD8+ T cells in vitro when compared to their NK cell replete counterparts43. Similarly, in the context of MCMV infection, Ly49H+ NK cells negatively influence T cell responses through lysis of virally infected DCs by limiting the number of antigen presenting cells available to activate DCs39.

NK cell regulation of adaptive immunity also occurs through shaping anti-viral T cell responses.

Many of the studies done in this area have been carried out using NK cell susceptible viruses, such as MCMV, which the clearance of is highly dependent on NK cells. Implications from these studies on the role of NK cell regulation of adaptive immunity are difficult to interpret as many are conducted in the context of NK cell depletion, which abrogates host immune control of virus replication. Overall, the findings are likely indirect results of viral titers and antigen availability, and not necessarily due to interactions with T cell and antigen presenting cells39,44,45. In response to this, various studies have been carried out using the non-cytolytic NK cell

“resistant” virus, LCMV, in order to shed light on NK cell interactions with T-cells during viral infection in shaping the immune response.

NK cell regulation of T-cell responses in the acute Armstrong (Arm) strain of LCMV was notably described by Su et al. utilizing infected β2-microglobulin deficient (β2-m-/-) mice46. These animals are deficient in MHC class I and have a subsequently deficient mature CD8+ T cell compartment, while possessing an otherwise normal NK cell compartment as this molecule is not necessary for NK cell education as it is for CD8+ T cells47. As LCMV-Arm viral titers are largely controlled by the CD8+ T cell response to infection, this model has provided valuable insight to the regulatory and effector roles for NK cells during this infection. Interestingly, in β2- m-/- mice depleted of NK cells and infected with LCMV-Arm, there were increased absolute numbers of CD4+ T cells in the spleen at the peak of the T cell response compared to NK cell replete control mice. Furthermore, CD4+ T cells were more proliferative, as defined by BrdU incorporation, and produced high quantities of IFN-γ in the absence of NK cells, indicating that

NK cells were carrying out a negative regulatory function towards CD4+ T cells responding to this infection47. This finding was further explored by our group in a model of chronic infection utilizing the clone 13 (Cl13) strain of LCMV where it was determined that NK cells were cytolytically attacking activated CD4+ T cells in a perforin-dependent manner 48.

We specifically found that this NK-cell suppression of CD4+ T cells had drastic implications in the outcome of pathology and control of infection due to regulation of CD8+ T cell responses in this model. Specifically, when C57Bl/6 mice are infected with a medium dose of LCMV-Cl13, NK cell mediated constraint of CD8+ T cells caused inefficient viral clearance. However, the remaining quantity of virus was insufficient to fully induce a state of T cell exhaustion, thereby permitting continued T cell functionality resulting in onset of severe disease pathology and mortality. The regulatory role of NK cells in this context is supported as NK cell depletion results in loss of this severe pathology due to a lack of constraint on CD4+ T cells, which in turn

13 promoted a greater CD8+ T cell response to effectively clear the virus before onset of pathology.

In contrast to the pathology associated with a medium dose of virus, a high infectious inoculum of LCMV-Cl13 caused a completely non-lethal relatively asymptomatic persistent infection associated with a severe state of T cell exhaustion. In this context, NK cell depletion resulted in severe disease pathology due to hyper-activation of virus-specific CD4+ and CD8+ T cell responses48,49. This study illustrates an important dichotomy between NK cells and T cells in the context of viral infection, where NK cells seem to function as “rheostats” in modulating T cell responses to influence the outcome of infection44,48.

The finding from this study, and others, have influenced further investigations in understanding how this NK cell control of T cells during viral infection has influenced the development of other aspects of the immune response38,50,51. Specifically, our group has demonstrated that NK cells suppress the generation of virus specific CD8+ and memory T cell responses in LCMV-Arm infection resulting in a reduction in viral clearance and a less robust recall response to secondary viral challenge51.

Furthermore, the finding that NK cells control CD4+ T cells in response to LCMV infection has lead us to investigate the role of this regulation in the generation of humoral immunity, which is the basis for the findings presented in this thesis51.

Materials and Methods

Mice

Male C57/Bl6 and perforin-deficient (prf1-/-) mice were purchased from The Jackson

Laboratories (Bar Harbor, ME), housed in specific pathogen free facilities, and utilized between

6-12 weeks of age. Studies were carried out under the ethical guidelines approved by the

Institutional Animal Care and Use Committee of Cincinnati Children’s Hospital Medical Center.

NK-cell depletion strategy

NK cells were selectively depleted from mice one day prior to infection by a single intraperitoneal (i.p.) injection of 25 µg anti-NK1.1 monoclonal antibody (PK136) or control treated with 25 µg mouse IgG2a antibody (C1.18.4; Bio X-Cell)48.

Histology and Fluorescence Microscopy

Tissues to be analyzed by fluorescence microscopy were harvested into and incubated in a solution of 4% formaldehyde (Thermo-Scientific) in PBS for 5 hours, followed by incubation in a

30% sucrose solution overnight at 4℃ for dehydration. Following dehydration, samples were embedded in optimal cutting temperature (OCT) media (Sakura) and frozen using a slurry of dry ice and 100% ethanol. Tissue was sectioned at a 12 µm thickness using a cryostat and affixed to positively charged slides (VWR). Prior to staining, slides were incubated in 100% acetone for

10 min at -20℃ followed by successive washes with PBS. Slides were then incubated with a cocktail of primary antibodies containing either goat anti mouse NKp46 (1:100, R&D Systems),

B220-AlexaFluor(AF)488 or AF647 (1:100), CD3-AF594 or AF647 (1:200, Biolegend). NKp46 primary antibody was revealed by application of donkey anti goat AF488 (1:800) or AF594

(1:200; Thermo). Tissues were mounted using prolong gold mounting media (Thermo-Scientific)

15 and covered with a coverslip. All imaging was performed using a Nikon A1 inverted confocal microscope in the Cincinnati Children’s Hospital Medical Center Confocal Imaging Core.

Formalin fixed and paraffin embedded tissues were prepared and stained in the CCHMC

Pathology Core. GCs were enumerated by immunohistochemistry using anti-B220 antibody

(1:50, BD Biosciences) and anti-PNA (1:50, Vector Labs). All imaging was performed using an

Aperio digital slide scanner at x20 magnification, and images were analyzed using ImageScope software (Leica Biosystems).

Flow Cytometry

Tissue to be analyzed by flow cytometry was harvested and converted into a single cell suspension by smashing tissue between two glass slides. Cells were then stained with a cocktail containing a mixture of any of the following antibodies: CD95/Fas (Jo2, 1:200), GL7

(1:200), CD44 (IM7, 1:200; BD Biosciences); CD19 (6D5, 1:200), NK1.1 (PK136, 1:200), NKp46

(29A1.4,1:50), CXCR5 (L138D7, 1:50), CD45R/B220 (RA3-6B2,1:200) CD4 (GK1.5, 1:400;

Biolegend). Cells were collected using a LSR Fortessa cytometer equipped with FACSDiva software (BD) present in the CHMC Research Flow Cytometry Core and analyzed using FlowJo software (TreeStar).

Intravascular Staining Method

To perform intravascular staining method, mice were injected via the tail vein with either 1.2 µg anti-NKp46 APC antibody (29A1.4; Biolegend) in 300 µL HBSS. Mice were routinely warmed using a heating lamp for a short period of time prior to injection as to promote vasodilation and aid injection. The injected antibody was allowed to circulate in the mouse for 3 minutes, after which the animal was euthanized by CO2 asphyxiation. Tissues to be analyzed were harvested into media and processed for analysis by flow cytometry or histology as previously described.

RNA-seq Analysis

Intravascular NKp46-APC staining method was performed on C57/Bl6 mice 3 d.p.i with LCMV-

Arm and splenocytes were then isolated and stained in preparation for flow cytometry based cell sorting. NK cells were sorted into tubes containing 100% fetal bovine serum (FBS) with red pulp and white pulp populations delineated by intravascular stain positive or negative. Following sorting, samples were submitted to University of Cincinnati Sequencing Core (supported by

NIEHS grant P30-ES006096) for PolyA-mRNA isolation, RNA-seq library preparation and HiSeq sequencing. Transcripts were aligned to the mouse genome, quantified, and normalized for downstream bioinformatics analysis using Tophat and Cufflinks protocols described in Trapnell et al., 201252. “Heatmaps” of resulting differentially regulated genes created using Genesis software produced by Sturn and Snajder of Graz University of Technology (Graz, Austria).

Results

I. Natural killer cells impair generation of the T follicular helper cell and germinal center response during viral infection

There is a growing body of literature demonstrating a role for NK cells in regulation of the adaptive immune response to infection. Our group has previously demonstrated that NK cells indirectly control virus specific CD8+ T cells during a model of chronic infection through the lysis of activated CD4+ T cells48. This finding lead our lab to hypothesize that NK cells could also impair the generation of T follicular helper (Tfh) cells in a model of acute viral infection to subsequently blunt the downstream development of humoral immunity51. To test this hypothesis,

NK cells were selectively depleted in C57/Bl6 mice by intraperitoneal (i.p.) administration of 25

µg anti-NK1.1 (clone PK136) monoclonal antibody one day prior to infection with 5x104 plaque forming units (p.f.u.) LCMV-Arm48. At the peak time point of the Tfh cell response to this infection, 6 days post infection (d.p.i.), a significant increase in the quantity and proportion of Tfh cells was measured in the spleen and inguinal lymph nodes when compared to NK cell-replete controls (Fig. 1A). Similarly, at this same time point we also measured an increase in proportion and quantity of germinal center (GC) B cells in these tissues (Fig. 1A, B). Suppression of the Tfh response by NK cells could have downstream implications in development of the GC.

We hypothesized that this overall increase in GC B cell numbers was due to an increase in the total number of discrete germinal centers in NK cell depleted mice or simply an increase in cellularity within a similar number of distinct germinal centers. To test this hypothesis, we quantified the number of B220+, peanut agglutinin+ (PNA+) GC structures in spleen sections at this same time point post-infection, we found no difference in the number of GCs between NK depleted and control groups, indicating this phenotype is likely manifested by an increase in cellularity of the GC structures themselves (Fig. 1C). These findings suggest a negative

18 regulatory function for NK cells in the development of humoral immunity through suppression of the Tfh response and GC reaction during acute viral infection.

II. Natural killer cell suppression of humoral immunity is perforin dependent

One of the primary effector functions of NK cells is the lysis of target cells through the release of cytotoxic proteins from its granules5,16. As previously mentioned, our group has demonstrated a role for NK cell mediated cytotoxicity in the killing of activated CD4+ T cells in a model of chronic infection48. We hypothesized that perforin-mediated NK cell cytotoxicity was responsible for Tfh and GC B cell suppression in our model of acute viral infection. To test this, we utilized mice deficient in perforin (prf1-/-), a pore forming protein present in the granules of cytotoxic lymphocytes that is critical to mediate this type of killing. These mice lack perforin in all cell types including NK cells, but also CD8+ T cells. Therefore, the CD8-dependent viral control is lost in these mice and they eventually succumb to immune pathology driven by overwhelming

IFN-γ production after about a week of infection53. However, Tfh cells begin to develop around 5 d.p.i., and we were able to take advantage of these differing kinetics to examine the effect of perforin deficiency in NK cells at early time points.

When C57Bl/6 mice were depleted of NK cells 1 day prior to infection with LCMV-Arm, an enhancement in both proportion and total number of Tfh cells was measured in the spleen and mediastinal lymph nodes (mdLN) at 5 d.p.i., a time point subsequent to peak NK cell activation

(3 d.p.i) and preceding the height of the CD8+ T cell response (8 d.p.i.)54. Additionally, when groups of prf1-/- mice were depleted of NK cells prior to infection, a similar enhancement of Tfh cells was observed in the spleen and mdLNs when compared to the wild type NK cell depleted animals at the same time point of infection. Interestingly, prf1-/- mice treated with isotype control antibody, and possessed an intact NK cell compartment, also showed enhanced proportions and total numbers of Tfh cells at 5 d.p.i. at comparable levels to both its NK depleted wild type

19 and prf1-/- counterparts (Fig. 2). Taken together, these findings provide evidence that perforin deficiency in NK cells is enough to recapitulate the phenotype of NK cell depletion in this model and that this suppression is perforin dependent.

III. Natural killer cells localize to the splenic white pulp 3 d.p.i. LCMV-Arm

Although we have shown NK cell-mediated cytotoxicity through a perforin-dependent mechanism plays a primary role in the ability of these cells to suppress the generation of a humoral immune response during infection, the exact cell type they act upon is not known (Fig.

1, 2). Understanding that perforin-mediated cytotoxicity requires close proximity or direct cellular contact with NK cells and their target cell16, we hypothesized that the anatomic localization of

NK cells in secondary lymphoid organs during infection could shed light on this question.

In the spleen of a naïve mouse, NK cells are found predominantly in the vascularized red pulp where they are anatomically separated from the B and T cell-rich white pulp22,55. Similarly, in the lymph nodes, NK cells mostly localize to the subcapsular and medullary sinuses facilitating easy transit through this organ and back to the lymphatics, while only transiently moving through the

T and B cell rich cortex and paracortex56. To understand if this distribution held true in the context of infection, we investigated spleen and lymph nodes of C57Bl/6 mice by confocal fluorescence microscopy at 3 d.p.i. with LCMV-Arm, the peak time point of NK cell responses to this infection54. While there seemed to be little change in the localization of NK cells within the various lymph nodes when compared to naïve mice, we observed a significant fraction of NK cells re-localized to the white pulp in the spleen (Fig. 3A, B). While the predominant fraction of these cells had a tendency to congregate in the T cell zone, a fraction was present at the T/B border, as well as deep in the B cell follicle (T zone = 769.22±84.50 NK cells/mm2 white pulp;

T/B border = 642.16±42.55 NK cells/mm2 white pulp; B follicle = 441.25±42.98 NK cells/mm2 white pulp, n = 19). This latter observation is interesting as these regions are critical areas

20 where activated T and follicular B cells interact to stimulate the development of Tfh cells to initiate formation of the GC reaction later in the immune response31,36. NK cell localization to this area, although preceding the peak of the humoral immune response, could allow for cytolytic interactions with precursors to Tfh and activated follicular B cells leading to downstream consequences of a dampened humoral immune response.

IV. Use of intravascular staining method for analysis of natural killer cell localization to the white pulp

In order to quantitate NK cell localization to the white pulp in a much faster manner, as well as isolate this group of cells through cell sorting, we sought a technique which would allow us to visualize white pulp-localizing NK cells by flow cytometry. This initially presented a challenge as flow cytometry requires the tissue to be generated into a single cell suspension, thus removing the spatial data that can be acquired from that tissue. To combat this, we employed an intravascular staining method consisting of intravenous (i.v.) administration of a labeled antibody via the tail vein of the mouse three minutes prior to sacrificing the animal (Fig. 4A)57. This method allows for the in vivo labeling of all antibody-reactive cells exposed to the vasculature, while leaving cells in less vascularized locations unlabeled. In the case of the spleen, this delineates cells present in the highly vascularized red pulp from those which localize to the less vascularized white pulp55. For our purposes, we chose to utilize a labeled antibody specific to

NKp46 (clone 29A1.4), a cell surface receptor present on all NK cells and few other cell types58.

To validate this method, we first sought to determine the efficacy of administering labeled anti-

NKp46 antibody through the vasculature of the mouse in order to stain NK cells in vivo. Three minutes following i.v. administration of 1.2µg labeled anti-NKp46-APC antibody to uninfected

C57Bl/6 mice, the animal was euthanized and whole blood and spleen were collected for analysis by flow cytometry. In the blood, which acts as a positive control for this method, we

21 observed that 99% of the NK cells present were efficiently labeled by the i.v. administered antibody (Fig. 4B). Furthermore, within the spleen of the same animal, we found that approximately 95% of splenic NK cells were positive for the i.v. administered antibody which is an expected result as the primary localization of NK cells in this tissue at steady state is the vascularized red pulp. Furthermore, we performed this technique using C57Bl/6 mice 3 d.p.i with LCMV-Arm and found that approximately 55% of splenic NK cells were not labeled by the i.v. administered antibody, implying these cells were not exposed to the antibody due to their localization in the weakly vascularized white pulp (Fig. 4B, C).

In order to ensure this finding was not due to the loss of NKp46 surface expression on splenic

NK cells, prior to analysis of these splenocytes by flow cytometry, we performed an additional ex vivo stain using an anti-NKp46 antibody conjugated to a different fluorophore (NKp46-PerCP-

Cy5.5) than the one utilized for the i.v. stain. From this, we are able to demonstrate that the i.v. anti-NKp46 negative NK cells do in fact express NKp46 on their surface to a similar extent as those which stain positive for the i.v. administered antibody, implying these i.v. negative cells are protected from staining due to their localization in the splenic white pulp and not downregulation of NKp46 (Fig. 4D).

V. Natural killer cell localization to the white pulp is transient

In order to confirm that 3 d.p.i. is the best time point to study NK cell localization in the white pulp, we employed our intravascular staining method over a range of time points early in infection. Utilizing groups of C57Bl/6 mice infected with LCMV-Arm we performed our anti-

NKp46 intravascular stain at one day increments ranging from 0 and 6 d.p.i. (Fig. 5 A). From this, we observed that peak NK cell localization to the white pulp over the first 6 days of infection occurs at 3 d.p.i, and is evident by both the proportion and number of intravascular stain-

22 negative NK cells in the spleen (Fig. 5 A, B). Furthermore, we can conclude this response is transient, with splenic NK cells predominantly localizing back in the red pulp by 6 d.p.i. (Fig. 5C).

To determine whether this anatomic redistribution of NK cells in the first three days of infection was crucial for suppression of Tfh and GC B cell responses, we employed a delayed NK cell depletion strategy. For this, groups of C57Bl/6 mice infected with LCMV-Arm on the same day were treated with 25µg anti-NK1.1 depleting antibody either 1 day prior to, or 1.5 or 3 days following infection, along with a control group. In mice depleted of NK cells one day prior to infection, we measured an enhanced quantity of GC B cells in the spleen 14 d.p.i. when compared to the control group (Fig. 5D). Interestingly, this enhancement of GC B cells was abrogated if NK depletion was performed 3 d.p.i. (Fig. 5D) This finding supports the assertion that the suppressive function of NK cells is mediated in the first few days following infection, and this correlates with the timing of NK cell infiltration into the white pulp (Fig. 5A). While this does not definitively prove this NK cell localization is necessary for suppression of Tfh and GC responses, it provides strong rational in support of this hypothesis.

VI. CXCR5 expression on NK cells correlates with white pulp localization

Lymphocytes are organized within, and move throughout, the splenic white pulp by responding to chemokines produced by stromal cells stationed throughout this 28,55. Taking this into consideration, along with our previous observation that NK cells localize to the T/B border and B cell follicle of the white pulp (Fig. 3A), we asked if NK cells could express the chemotactic B cell follicle-homing receptor, CXCR5. We hypothesized CXCR5 expression could direct NK cell interactions with cells involved in the Tfh and GC response which reside in these locations.

Prior to infection, NK cell expression of CXCR5 in wild-type mice was negligible and difficult to discern from background staining. Using C57Bl/6 mice at the peak of the NK cell response during LCMV-Arm infection, 3 d.p.i., we determined that a significant fraction of NK cells in the

23 spleen and multiple lymph nodes expressed enhanced surface expression of CXCR5 when compared to naïve controls (Fig. 6A, B). Furthermore, we determined that the frequency of

CXCR5+ NK cells in the spleen peaks during the first day of infection while holding constant, until 3 d.p.i. where it begins to return to steady state levels by 6 d.p.i. (Fig. 6). This is important as detectable CXCR5 expression is at its highest while the frequency of NK cells in the white pulp increases, then drops to pre-infection levels with similar kinetics as that of NK cells returning to a predominantly red pulp distribution by 6.d.p.i. (Fig. 6C) This suggests that CXCR5 expression on NK cells could act as a contributing factor to drive NK cell relocalization within the white pulp early in infection, but may also function to direct NK cells to specific areas, such as the B cell follicle31,36.

VII. White pulp and red pulp NK cells have different transcriptional profiles

Although previous studies have investigated some of the factors that drive NK cells to re- localize within lymphoid tissues following infection or immunization, little insight has been developed concerning the overall functional profile of this group of cells24,25. To shed light on possible effector functions these cells might carry out while in the white pulp, we investigated the transcriptional profile of white pulp localizing NK cells through RNA-seq analysis.

White and red pulp-localizing splenic NK cells, delineated by i.v. NKp46 staining, were isolated from C57Bl/6 mice 3 d.p.i with LCMV-Arm and were sorted by flow cytometry to be submitted for analysis by RNA-seq. Downstream pathway analysis of the output data set revealed a striking difference in the transcriptional profile between these two cell populations with increased levels of transcripts reported in white pulp over red pulp localizing NK cells in at least 28 genes involved in immune cell activation and effector functions (Fig. 7A). Furthermore, white pulp localizing NK cells had significantly increased expression of perforin as well as multiple subtypes of granzymes when compared to red pulp localizing NK cells (Fig. 7C). Additionally, we observed an increase in type one interferon signaling genes in white pulp localizing NK cells

24 when compared to their red pulp counterparts. This finding is significant since type I interferon signaling is known to be a vital component for early NK cell activation during viral infection (Fig.

7B)17. These results further suggest an increased state of activation and potential cytotoxic function in NK cells that have migrated to the white pulp during LCMV infection.

While a large number of genes in general signaling pathways were differentially regulated between red and white pulp localizing NK cells, we observed a fraction of up- or down-regulated genes that were not predicted from our a priori knowledge of NK cell activation during virus infection. This group of genes of interest includes: interleukin-10 (Il-10), lymphotoxin-β (Ltb),

CXCL10, and Cytotoxic T-lymphocyte-associated protein-4 (Ctla-4), all of which had reports of a significant increase in transcripts in white pulp localizing NK cells over red pulp NK cells (Fig.

7D). This is important as these all carry immune-regulatory implications that further support a regulatory role for this subset of NK cells.

Specifically, these set of genes are of particular interest in the context of this model of NK cell suppression of Tfh and GC responses. To speculate, both IL-10 and Ctla-4 have been implicated in immunosuppressive functions towards limiting the T cell response to infection, which is relevant to the highly T cell-dependent process of induction of humoral immunity59,60.

Furthermore, CXCL10, a chemokine which interacts with CXCR3 on activated T cells to act as a chemotactic factor could possibly have a “lassoing” effect in bringing T and NK cells in close contact with each other to possibly facilitate cytotoxic interactions27. Lastly, Ltb expression on

NK cells has not been fully described but this factor has been highly implicated in aiding the development and organization of secondary lymphoid tissue, specifically acting as a survival and differentiating factor for B cells61. This last point especially emphasizes the diverse role in the immune response of the differentially regulated genes which were reported from this data set in white pulp localizing NK cells. By a likely more relevant conclusion, these findings speak to the highly heterogeneous nature of this population of NK cells and suggest other immune

25 effector and regulatory roles these cells are likely carrying out in the context of infection in conjunction with a likely role in suppression of humoral immunity.

Figures

A 6 d.p.i; Spleen B + + B cells CD4 , CD44 T cells #TfhSpleen Spleen #TfhiLN iLN

24.3 s 5 s 4 11.2 1.2 l l l 410 l 510

e e P = 0.0064 58.7 5.78 c P = 0.0036 c

h h 4 f 5 f 410 T 310 T

+ +

1 1 - - 3104 Control D 5 D P 210 P

, , 4 + + 210

5 5

R 5 R C 110 C 4

X X 110

C C

. . o 0 o 0 N N Control NK Control NK

21.8 25.4 6.0 # GC Sp # GC iLN 6 4 42.0 10.8 s 2.010 s 510 l P = 0.054 l

l l

e e P = 0.067

c c

 4 B 6 B 4 10

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C C 4 ∆NK G G 310 + 6 + s 1.010 s a a 4

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CXCR5 GL7

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+ +

7 5.0105 7 L L 1104

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. .

o o

N 0 N 0 Control NK Control NK PD-1 Fas

C Control ∆NK

100

+ P = 0.982 A

PNA N 90

p 0 80

p .

G g 60

A B220 50 Control NK

Figure 1. NK cells suppress Tfh and GC response during LCMV-Arm infection: C57Bl/6 mice were treated by intraperitoneal (i.p.) injection with 25µg anti-NK1.1 depleting antibody (∆NK) or an isotype 4 control (Control) one day prior to infection with 5x10 p.f.u. LCMV-Arm A) Representative FACS plots + + + + of T follicular helper (Tfh) cells (CXCR5 , PD-1 , of CD4 , CD44 ; left), or germinal center (GC) B + + + + cells (Fas , GL7 of CD19 , B220 B cells) in the spleen from mice 6 d.p.i. B) Total number of Tfh cells (top) and GC B cells (bottom) in the spleen and inguinal lymph nodes (iLN) was quantified. Values were pooled from two independent replicate experiments, n=8 mice/group. C) Representative images of formalin fixed paraffin embedded serial spleen sections with GC structures marked by PNA (brown) and anti-B220 (pink) staining. Individual GC structures were quantified and averaged in

each whole spleen section (n = 4 mice/group). Scale bar = 500µm. Statistical significance determined by Student’s t-test and values are reported as mean28 ± s.e.m. B, C) Data presented here adapted from Rydyznski et al., 2015

Discussion

It is becoming increasingly appreciated that the role for NK cells as immune effector cells extends beyond tumor surveillance and lysis of virally infected cells, but also in regulating adaptive immune responses to infection. Our group has contributed significantly to this body of knowledge by describing a novel function for NK cells in impairing the generation of humoral immunity following viral infection15. The studies presented here contribute to this ongoing investigation by our lab through further characterization of the enhanced germinal center response observed in the absence of NK cells following viral infection, as well as by laying substantial groundwork for describing a possible cellular mechanism for this NK cell suppression of humoral immunity51.

Specifically, the work presented in this thesis demonstrates that 1) NK cell impairment of the germinal center reaction likely acts through restricting cellularity within GCs rather than limiting the total number of discrete GC reactions in secondary lymphoid organs, 2) that this suppression occurs in a perforin-dependent manner, and 3) that inhibition is most prominent during the first three days of infection. Furthermore, at time points post-infection correlating with this NK cell suppression, there is an 4) anatomic redistribution of NK cells in the spleen from the red to the white pulp where they 5) transiently congregate in the T cell zone and B cell follicle.

NK cell localization to the white pulp may act as a contributing mechanism to facilitate direct cell-mediated cytotoxicity of precursors to the Tfh and GC response which are present in this region. Within this same time frame, 6) a subset of NK cells in the spleen express the B cell follicle homing receptor CXCR5 which could aid in NK cell localization to areas within the white pulp critical in generation of humoral immune responses. Lastly, 7) the transcriptional profile of these white pulp-localizing NK cells suggests they are highly activated through induction of type

I IFN signaling and carry out perforin-mediated cytolysis as determined by significant increases in transcripts from these associated pathways when compared to red pulp localizing NK cells.

Furthermore, the wide array of differentially expressed genes in this population of NK cells, which have varying functions in the immune response to infection, speaks to the complexities of this system and the regulatory capacities this subset of NK cells holds in the context of humoral immunity and others.

I. NK cell localization to the white pulp and regulation of humoral immunity

The splenic white pulp acts as a hub for development of the humoral immune response to infection and is a primary site for activation of helper CD4+ T cells by resident dendritic cells in this tissue28. Subsequently, these helper T cells interact with activated follicular B cells to initiate

Tfh cell and GC formation in the spleen31. Here, we show that a significant proportion of NK cells in the spleen re-localize from a predominantly red pulp distribution, to one where ~55% of splenic NK cells are present in the white pulp (Fig. 3). Transcriptome analysis by RNA-seq suggests these white pulp-localizing NK cells are highly activated as indicated by induction of type I IFN signaling, and increased transcription of perforin and multiple subtypes of granzyme in these cells (Fig. 7)17. Our finding that activated NK cells localize to a region highly involved in the generation of humoral immunity led us to hypothesize that this event could be crucial for NK cell impairment in development of humoral immunity through direct, perforin-mediated, interactions with cells that reside there.

The anatomic redistribution of splenic NK cells to the white pulp has been reported in other murine models of inflammation such as MCMV infection and Poly (I:C) immunization24,25. In these models, NK cell localization to the white pulp was largely abrogated in mice deficient for the chemokine receptor CXCR3, indicating this receptor is a driving factor in facilitating NK cell redistribution to the splenic white pulp25. This localization is primarily driven by the DC and macrophage production of inflammatory CXCR3 chemokines ligands, including CXCL-9, 10, and 1127. The role of this localization by NK cells during inflammation has been associated with

36 various functions, such as protection of fibroblastic reticular cells from cytotoxic T cells during

MCMV infection, or through providing IFNγ to DCs in the lymph nodes to promote Th1 polarization following immunization21,24. Taking this body of knowledge into account, we felt manipulating the ability of NK cells to re-localize to the splenic white pulp through CXCR3 could provide evidence towards the role for NK cell localization to this region during LCMV-Arm infection in suppressing Tfh and GC responses.

While mice deficient for CXCR3 have been generated, a total knock-out of this protein could present further issues as this receptor is also utilized by T cells for trafficking to lymphoid tissues to facilitate an adaptive immune response27. This is problematic as a proper adaptive immune response is necessary for the development of humoral immunity through Tfh and GC responses. Blunting the humoral response to infection through a deficiency in T cells will likely present conflicting results in interpreting the outcomes from these experiments and will likely be difficult to distinguish between effects resulting from a lack of T cell mobilization or NK cell localization.

To address these discrepancies, we have chosen to take a mixed bone marrow chimera strategy utilizing donor bone marrow from rag-/-, cxcr3-/- mice at a 9:1 ratio with C57Bl/6 bone marrow. As rag-/- mice are deficient for B and T cells, but have an otherwise replete NK cell compartment, this will generate a mixed chimera where the vast majority of NK cells are deficient for cxcr3 (since the innate compartment from this background has a 9:1 numerical advantage) while the T and B cells, which can only arise from the C57Bl/6 bone marrow, will be wild-type. Although the possibility of wild-type NK cells developing from the C57Bl/6 bone marrow still exists in this model, we suspect it will occur to a minor degree and the resulting effects will be negligible. We can use congenically (Ly5.1) marked wild-type to formally discount the possibility that wild-type NK cells arise in this system.

Through this mixed chimera system, we expect that splenic NK cells will be unable to localize to the white pulp following infection with LCMV-Arm, and that an otherwise normal inflammatory response as seen in a C57Bl/6 mice will result due to an intact T and B cell compartment. We hypothesize that abrogating the ability of NK cells to localize to the splenic white pulp following

LCMV-Arm infection will recapitulate the enhancement of Tfh and GC responses seen during this infection in the context of NK cell depletion due to their inability to come in direct contact with precursors to this response and carry out perforin mediated cytotoxicity.

II. Understanding a role for CXCR5 in NK cell localization and suppression of humoral immunity

In this study, we describe our findings that a higher proportion of NK cells express the B cell follicle-homing chemokine receptor, CXCR5, on their surface at the peak of the NK cell response to LCMV-Arm infection (3 d.p.i.), and this occurs during the same time frame as peak

NK-cell localization to the white pulp (Fig. 6). The presence of CXCR5 on murine NK cells has not been previously described, so this finding sparked multiple lines of investigation by our group. Most notably, we hypothesize that this CXCR5+ NK cells might be a ‘follicular regulatory’ subset driving the suppression of humoral responses following LCMV Arm infection.

While understanding the functional consequences of CXCR5+ NK cells is our ultimate priority, as this receptor has yet to be described on murine NK cells, we wanted to determine if its presence was due to cxcr5 gene expression by NK cells. This question is especially relevant in understanding the repertoire of receptors present on NK cells as they have been shown to carry out an activity known as trogocytosis, which involves the “stealing” of pieces of another cell’s membrane, along with any associated surface proteins, to incorporate into their own62,63.

Before carrying out experiments to flow sort CXCR5+ NK cells and probe for mRNA, we took advantage of our previously completed RNA-seq analysis of splenic NK cells 3 d.p.i. with

LCMV-Arm to carry out an initial search for cxcr5 RNA transcripts. From this search, we found negligible quantities of transcripts for cxcr5 reported suggesting transcription of this gene occurs prior to 3 d.p.i. and is not detectable at that time point or, alternatively, that CXCR5 is possibly being acquired through trogocytosis.

Future experiments should sample RNA from splenic NK cells at multiple time points within the first 3 days of LCMV-Arm infection to probe for cxcr5 transcripts by quantitative PCR in order to determine the extent of cxcr5 expression by NK cells. While assays for trogocytosis have been described and implemented in other systems, it would require significant preparation and troubleshooting to carry out as a new technique for our group and will not be pursued until we are confident it is not acquired through classical protein expression62,63.

From a functional perspective, the presence of CXCR5 on the NK cell surface during infection is intriguing to us in the context of NK cell regulation of humoral immunity. CXCR5 interacts with the ligand CXCL13 produced by stromal cells which reside in the B cell follicle of the spleen and lymph nodes30. The gradient of this chemokine not only organizes B cells to form the B cell follicle, but is an important navigating factor to direct activated T and Tfh cells to this region, as well as the T/B border. Specifically, this receptor has been shown to be upregulated on activated CD4+ T cells in order to attract them to the T/B border of the white pulp to support stimulation of activated follicular B cells to differentiate into germinal center B cells and potentiate the humoral immune response31,33,34. We hypothesize that one way in which NK cells regulate humoral immunity is through trafficking to areas such as the T/B border in a CXCR5- dependent manner to suppress burgeoning GC responses

It would be surprising to find that CXCR5+ NK cells require this receptor for general entry into the white pulp as it has previously been demonstrated that white pulp localizing NK cells traffic to this region by CXCR3 and CCR5 these receptors are likely present on a predominant fraction

39 of this subset of NK cells as well. Furthermore, preliminary experiments have shown that the distribution of CXCR5+ NK cells between red and white pulp at 3 d.p.i. with LCMV-Arm, as delineated by i.v. NKp46 staining, is similar to that seen of the total splenic NK cell population at this time point (CXCR5+, i.v.- NK = 60.95±3.41% of total NK1.1+, CXCR5+ NK cells; NK1.1+, i.v.-

NK = 57.72±3.30% of total NK1.1+, CD3- NK cells, p = 0.519, n = 8, statistics by two-tailed student’s t-test). If CXCR5 is the primary factor driving these NK cells to the white pulp, it would be expected that a greater proportion of these cells would be present within the white pulp as compared to the total splenic NK cell localization at this time point suggesting the factors involved in driving this localization between both CXCR5 positive and negative NK cells are similar. We speculate that this receptor more likely allows for NK cell localization to areas such as the B cell follicle and T/B border as it has been shown that entry to these regions occurs largely in a CXCR5 dependent manner. To test our hypothesis that CXCR5 allows NK cells to localize to the white pulp, or to specific areas within it, we plan to adoptively transfer isolated splenic NK cells from either C57Bl/6 or cxcr5-/- mice into a C57Bl/6 recipient and analyze their localization in the spleen by histology 3 d.p.i. with LCMV-Arm.

Additionally, to further implicate a role for CXCR5+ NK cells in their ability to suppress the generation of humoral immunity, we plan to take a similar approach as described previously with the cxcr3-/- chimeras except utilizing cxcr5--/-, rag-/- mice as the predominant bone marrow donor and analyze them at 14 d.p.i. with LCMV-Arm for an enhancement of the germinal center response. Furthermore, these chimera mice can also provide insight into the acquisition of

CXCR5 on NK cells by determining if the cxcr5-/- NK cells become positive for the receptor after infection. If this is the case, then trogocytosis is the likely source of this chemokine receptor on

NK cells as they should be unable to produce the protein themselves.

III. Conclusions

High rates of morbidity and mortality caused by infectious pathogens is still a major global health issue and presents a need for the development of robust vaccines and therapeutics to combat these illnesses. Elucidating the nuances involved in the regulation and development of protective humoral immunity is a critical step in order for effective vaccination schemes against difficult pathogens, such as HIV and hepatitis, to be successful. Here, we build upon our previously described finding that NK cells play a regulatory role in the adaptive immune response through suppressing the generation of protective humoral immunity during viral infection. We have demonstrated a likely mechanism for this function through an anatomic redistribution of NK cells within the spleen during infection which we hypothesize allows for direct cytolytic interactions with cells involved in generating the humoral immune response in this organ. Furthermore, we have identified a possible subset of NK cells that express the chemokine receptor CXCR5 which could allow for NK cell localization to specific regions of the spleen, such as the T/B border and B cell follicle, where the initiation of the Tfh and GC reaction originate.

This novel group of NK cells offers a possible therapeutic target for the improvement of future vaccination strategies in order to boost protection from pathogens which have, so far, been difficult to provide immunity against. While further experimentation needs to be carried out to definitively implicate a role for NK cell localization in regulation of humoral immunity, the work presented here lays substantial groundwork for the continuation of these studies.

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