Regulation of Cd4 T Cell Responses to Il-7 by Ifn-Alpha and Tgf-Beta in Treated Hiv Disease

REGULATION OF CD4 T CELL RESPONSES TO IL-7 BY IFN-ALPHA AND TGF-BETA IN TREATED HIV DISEASE

By: THAO NGUYEN

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Advisor: Scott F. Sieg, PhD

Department of Pathology

Immunology Training Program

CASE WESTERN RESERVE UNIVERSITY

JANUARY 2017

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

THAO P NGUYEN

Candidate for the degree of Doctor of Philosophy*

Committee Chair

CLIFFORD HARDING, MD/PhD

Committee Member

SCOTT SIEG, PhD

Committee Member

MICHAEL LEDERMAN, MD

Committee Member

DONALD ANTHONY, MD/PhD

Committee Member

JOHN TILTON, MD

Committee Member

CLIVE HAMLIN, PhD

Date of Defense

November 21, 2016

*We also certify that written approval has been obtained for any proprietary material

contained therein.

I dedicate this dissertation to my sister and late mother.

iii

Table of contents

Page

Dedication iii

Table of contents iv

List of figures and tables viii

Acknowledgements x

List of abbreviations xi

Abstract 1

Chapter 1: Introduction 2

HIV pathogenesis 2

CD4 T cell count recovery in HIV infection following ART initiation 9

Homeostatic proliferation in response to interleukin-7 is a mediator of T cell 10 recovery

The role of IFN-I in treated HIV infection 14

The anti-viral and anti-proliferative functions of IFN-I 14

TGF-β and additional cytokines that may contribute to low CD4 T cell count 17 recovery in treated HIV infected patients

Chapter 2: Interferon-α inhibits CD4 T cells proliferation and signaling in 19 response to IL-7 and IL-2

Abstract 19

Introduction 19

Methods 21

Cells and cell culture

Flow cytometry

Statistical analyses

Results 24

IFN-α impairs IL-7-induced proliferation responses and diminishes 24 cellular function in CD4+ T cells

IFN-α diminishes T cell viability 29

IFN-α inhibits P-Akt but not P-STAT5 in IL-7-treated cells 31

IFN-α inhibits P-Akt signaling that is induced by IL-2 but not by SDF-1 33

Inhibition of P-Akt or P-STAT5 blocks cellular proliferation and 35 function

Discussion 37

Chapter 3: Diminished CD4 T cell responsiveness to IL-7 in immune 42 failure subjects is related to diminished CD127 expression and increased IFN-α expression

Abstract 42

Introduction 43

Methods 45

Patients

Peripheral blood mononuclear cell and T-cell isolation

mRNA analyses

Stimulation conditions

Flow cytometry

Statistical analyses

Results 48

IL-7 mediated proliferation and induction of CD25 are diminished in 48 CD4+ T cells from immune failure patients

CD4+ T cells from immune failure patients exhibit decreased CD127 53 expression and increased immune activation

Interferon-stimulated gene and IFN-α mRNA expression are increased in 53 CD3+T cells from immune failure patients

Diminished IL-7 responsiveness in immune failure patients is related to 55 expression of CD127 and IFN-α

IFN-α responsiveness is not diminished in CD4+ T cells from immune failure 57 patients

Discussion 59

Chapter 4: TGF-β inhibits IL-7 induced proliferation in memory but not 64 naive CD4 T cells

Abstract 64

Introduction 64

Methods 67

Peripheral blood mononuclear cell and CD4 T cell subset isolation

Stimulation conditions

Flow cytometry

Statistical analyses

Results 69

TGF-β inhibits memory but not naive CD4 T cell proliferation in response 69 to IL-7

TGF-β suppresses IL-7 mediated induction of c-myc expression in naive 72 and memory CD4 T cells

TGF-β does not inhibit IL-7 receptor signaling in memory CD4 T cells 74 and enhances S6 kinase signaling in naive CD4 T cells

GSK-3 inactivation partially overcomes TGF-β-mediated inhibition of 75 IL-7-induced proliferation and c-myc induction in memory CD4 T cells

Discussion 78

Chapter 5: Discussion and future directions 71

Diminished responsiveness to IL-7 may contribute to poor 81 = CD4 T cell count recovery in treated HIV infection

The role of IFN-I in poor CD4 T cell count recovery in treated 84 HIV infection

TGF-β selectively inhibits memory CD4 T cell proliferation in response to 86 IL-7

IFN-α and TGF-β utilize different mechanisms to inhibit IL-7-induced 87 proliferation in CD4 T cells

Literature cited 89

vii

List of Tables and Figures Page

Table 1. Clinical characteristics and immune phenotypes of subjects 46

Figure 1.1 CD4 T cell loss and viral load in HIV infection 2

Figure 1.2 Proposed contributors to HIV-associated immune activation 6

Figure 1.3. HIV life cycle showing sites of action of different classes of 8 antiretroviral drug

Figure 1.4 CD4 T cell recovery is highly variable and related to nadir 10 CD4 T cell count

Figure 1.5 IL-7 receptor signaling promotes cell survival and drives proliferation 11

Figure 2.1. IFN inhibits IL-7-induced proliferation 25

Figure 2.2. The magnitude of IFN-α mediated inhibition of IL-7 induced 26 proliferation is dose dependent

Figure 2.3 Pre-incubation of CD4+ T cells in IFN-α plus IL-7 reduces 27 T cell function compared with pre-incubation of cells in IL-7 alone

Figure 2.4 Gating strategy to assess viability 29

Figure 2.5 IFN-α causes cell death, primarily in nondividing T cells, 30 in IL-7-treated cell cultures

Figure 2.6 IFN-α impairs P-Akt but not P-STAT5 signaling in IL-7-treated cells 31

Figure 2.7 Impaired responses to IL-2 but not SDF-1 in CD4 cells exposed 34 to IFN-α

Figure 2.8 Inhibition of PI3K or STAT5 reduced cell proliferation, decreased 35 functionality, and enhanced cell death in cells treated with IL-7.

Figure 3.1 CD4+ T-cell responses to IL-7 are diminished in immune failure 49 patients

Figure 3.2 CD4- T cell proliferation in response to IL-7 is diminished in IF 51 subjects

Fig. 3.3 Induction of P-STAT5 and P-Akt in response to IL-7 52

Fig. 3.4 T cells from immune failure patients exhibit increased expression of 53

viii interferon stimulated genes and IFN-α

Fig. 3.5 IL-7 induced CD25 expression is related to expression of CD127 55

Fig. 3.6 CD4+ T- cell responses to IFN-α were not diminished in immune 57 failure patients.

Fig. 3.7 IFN-α mediated induction of cell death in CD4 T cell subsets 59

Figure 4.1 TGF-β differentially affects naive and memory CD4 T cell 69 proliferation in response to IL-7

Figure 4.2 TGF-β inhibits IL-7 induced % divided and proliferation index 71 in memory CD4 T cells

Figure 4.3 TGF-β inhibits IL-7 mediated induction of c-myc expression 72 in naive and memory CD4 T cells

Figure 4.4 TGF-β does not inhibit IL-7 receptor signaling in memory 74 CD4 T cells and enhances S6 phosphorylation in naïve CD4 T cells

Figure 4.5 TGF-β mediated inhibition of IL-7 induced proliferation and 76 c-myc expression in memory CD4 T cells is reversed by the inactivation of GSK-3

Figure 5.1 Differential mechanisms of inhibition of IL-7 induced 88 proliferation by TGF-β and IFN-α

Acknowledgements

I would like to thank my PI, Dr. Scott Sieg. During my time at Case, Scott provided a wonderful learning environment that allowed me develop as a scientist. He is encouraging, genuine, thoughtful and a great role model for me. I would also like to thank Dr. Michael Lederman for his guidance and feedback. I find Michael exceptional because his actions demonstrate his dedication to helping others develop their projects and careers on top of his various responsibilities.

I would like to thank Dr. Clifford Harding, Dr. Donald Anthony and Dr. John

(Chip) Tilton for their guidance and help over the years while serving on my thesis committee. Dr. Harding has done an excellent job in his role as the chair of my committee by always being clear and organized. I appreciate Dr. Don Anthony and Dr.

John Tilton for their guidance in helping me think about my project as well as my career goals.

Finally, I would like to thank my collaborators and the members of the Sieg and

Lederman labs. I want to thank Dr. Supriya Shukla and Dr. Robert Asaad for their contribution to my immune failure project. I would like to thank Doug Bazdar for teaching me various laboratory techniques as well as always being available to help me in the lab.

Overall, the contributions from all the mentioned people have resulted in a pleasant experience in my journey to obtaining my degree.

List of Abbreviations

AIDS -acquired immune deficiency syndrome

ART-antiretroviral therapy

CMV- Cytomegalovirus

CH-CHIR-99021 c-myc- myelocytomatosis

EBV- Epstein–Barr virus

GSK-3-glycogen synthase kinase 3

HC-healthy control

HIV-human immunodeficiency virus

IFI16-gamma interferon inducible protein 16

IgA-immunoglobulin A

IL-1β- interleukin-1beta

IL-2-interleukin-2

IL-6-interleukin-6

IL-7-interleukin-7

IL-10- interleukin 10

IF-immune failure

IS-immune success

IP-10-gamma interferon inducible protein 10

IRF-7- interferon regulatory factor 7

IRF-9- interferon regulatory factor 9

ISG-interferon stimulated genes

ISGF-3- interferon stimulated gene factor-3

ISRE- IFN stimulatory response elements

LAV-lymphadenopathy-associated virus

LN-lymph node

Mx-myxovirus

OAS- 2’, 5’-oligoadenylate synthetase

PBMCs-peripheral blood mononuclear cells pDCs-plasmacytoid dendritic cells

PKR-protein kinase R

SCID- severe combined immunodeficiency

SDF-1-stromal derived factor 1

SEB- Staphylococcal Enterotoxin B

STAT5- signal transducer and activator of transcription 5

TCR-t cell receptor

TGF-β- transforming growth factor-beta

TLR-toll like receptor

TRECs- T cell rearrangement excision circles

TYK2-tyrosine kinase 2

xii

The Regulation of Response to IL-7 by IFN-alpha and TGF-beta in Treated HIV Disease

Abstract

THAO NGUYEN

Antiretroviral therapy administration has nearly eliminated acquired immune deficiency syndrome (AIDS) -related mortality in human immunodeficiency virus (HIV) infected patients. Despite ART mediated reduction of viral replication, there is increased risk of non-AIDS related morbidities in person who fail to recover circulating CD4 T cell counts. These persons are referred to here as “immune failure” (IF) patients. IF subjects exhibit increased immune activation and expression of immune modulating cytokines, type 1 interferons (IFN-I), transforming growth factor-β (TGF-β), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Homeostatic proliferation in response to interleukin-7 (IL-7) is important to CD4 T cell recovery. Responsiveness to IL-7 may be diminished in IF patients. Diminished responsiveness to IL-7 may be partially related to increased CD4 T cell exposure to IFN-I in IF subjects. In chapter 2, we demonstrate that IFN-α, the primary IFN-I subtype in HIV-infection, inhibits IL-7-induced proliferation in CD4 T cells from HIV uninfected donors. In chapter 3, we show that responsiveness to IL-7 but not IFN-α is impaired in CD4 T cells from IF subjects. In chapter 4, we demonstrate that

TGF-β inhibits IL-7 induced proliferation in memory but not naïve CD4 T cells in HIV uninfected donors.

Chapter 1: Introduction

HIV pathogenesis

Discovery of HIV

In 1983, Françoise Barré-Sinoussi and colleagues isolated a retrovirus they referred to as lymphadenopathy-associated virus (LAV) from a patient exhibiting early symptoms of acquired immune deficiency syndrome (AIDS) (1). Subsequent work showed that LAV grew and increased in titers in CD4+ T cells by infecting and lysing these cells, in vitro (Fig. 1) (2). These observations provided important initial evidence suggesting that LAV may be the causative agent of AIDS, a syndrome characterized by

CD4 T cell loss. In 1986, LAV was given the name human immunodeficiency virus

(HIV) (3). Since then, productive collaborations between basic scientists and clinical researchers led to important insights about HIV, its interactions with the host and its pathogenesis.

Figure 1.1 CD4 T cell loss and viral load in HIV infection. This figure was reused from article titled "Pathogenesis of HIV Disease: Opportunities for New Prevention Interventions " authored by Anthony S. Fauci, published by Clinical Infectious Diseases. Permission to reuse this figure was obtained from licensed content publisher Oxford University Press on Nov 29, 2016.

HIV infections, when left untreated, eventually progress to CD4 T cell deficiency that is associated with AIDS related morbidities and death (Fig. 1.1) (4). These morbid events include infections with Pneumocystis jiroveci pneumonia (5, 6),

Mycobacterium tuberculosis, M. avium intracellular, Crytococcus neoformans,

Toxoplasma gondii cytomegalovirus, adenovirus, herpes simplex virus and Candida albicans (7, 8). HIV infected individuals are also at increased risk for developing Kaposi's sarcoma, non-Hodgkin lymphoma and other cancers (9). The course of HIV infection is comprised of three stages: primary infection, asymptomatic or clinical latency and AIDS defining illness (10) (Fig. 1.1). The rate in which disease progresses from primary infection to AIDS related morbidity is variable among HIV infected patients. For instance, there are typical progressors who develop AIDS defining illnesses in median time of 8-10 years, non-term nonprogressors who do not experience progression of disease for an extended period of time and rapid progressors who rapidly progress to

AIDS defining illnesses within 2-3 years following primary infection (10). The rate of disease progression can be evaluated by immunological and virologic markers. For example, peripheral blood CD4 T cell count is a well characterized predictor of disease progression (11, 12). Plasma HIV RNA level also serves as an important predictor of disease progression (13).

CD4 T cell decline begins in primary infection during the time of initial peak of plasma viraemia (Fig. 1.1). This decline in CD4 T cell count occurs in peripheral blood

(14) as well as the gut (15). Studies demonstrated that the loss occurs in the memory CD4

T cell subset, specifically in the CCR5+ expressing cells (16). The selective depletion of these cells is likely due to transmitted HIV being CCR5 tropic (17, 18). It is possible that multiple mechanisms are involved in the depletion of cells. For example, studies with

SIV infected macaques suggest that lamina propria CD4 T cell are lost due to Fas-Fas ligand mediated apoptosis of uninfected cells (19).

In the clinical latency phase, peripheral blood CD4 T cells count decline is slow and may be a result of indirect effects of HIV. Plasma HIV RNA level predicts disease progression (20) and reflects viral replication in lymph nodes (21). Infection of CD4 T cells by HIV is likely not the only mechanism of CD4 T cell loss since it is estimated that only 0.1-1% of CD4 T cells are infected in the blood and lymph nodes (16). To reconcile the proportion of cell death with viral replication in the lymph node, Finkel and colleagues demonstrate that apoptotic cell death in the lymphoid tissues mostly occurred in uninfected cells (bystander cell death) (22). More recently, studies from Doitsh and colleagues suggest that bystander apoptosis may only comprises of a small fraction of cell death. These scientists provided evidence to suggest that quiescent CD4 T cells in lymph nodes of HIV infected individuals die of caspase-1 induced pyroptosis (23). They further demonstrate that this manner of cell death is a result of abortive HIV infection induced by the DNA sensor IFI16 (24) and leads to the expression of the inflammatory cytokine, IL-1β (23). The conclusion as to whether this is a major mechanisms of cell death is controversial. Overall, these observations suggest that there are multiple mechanisms involving in CD4 T cell death during chronic HIV infection.

Dramatic and sustained immune activation and inflammation are important characteristics of HIV infection (25). Markers of immune activation in HIV infection include increased expression of activation markers (CD38 and HLA-DR) on CD4 and

CD8 T cells (26). Increased expression of inflammatory cytokines IL-1β, TNF-α, and IL-

6 in plasma and lymph nodes have been observed in the early stage of HIV infection (27-

29). Importantly, studies from Giorgi and colleagues demonstrated that the increased expression of CD38 on CD8 T cells is associated with disease progression (30).

Subsequently, Deeks and colleagues demonstrated that immune activation occurring in early HIV infection is predictive of progressive CD4 T cells loss in chronic HIV infection

(31). These observations suggest that immune activation contributes to CD4 T cell loss in chronic HIV infection.

Given the importance of immune activation to HIV disease progression, mechanisms driving these activation markers have been investigated. HIV replication can contribute to immune activation by eliciting CD8 T cell responses in acute stage of the infection. Due to continuous HIV replication in the chronic stage, CD8 T cells remain activated and become susceptible to apoptosis (32). In persons co-infected with CMV and/or EBV, reactivation of virus may be a result of suboptimal immune control as CD4

T cells are lost over time. It has been proposed that these viruses provide antigen stimulation to immune cells, thus contributing immune activation (26). Brenchley and colleagues demonstrated that levels of LPS in plasma, likely translocated from the damaged gut, correlates with immune activation in HIV infected individuals (33). In addition, others have demonstrated that LPS can activate TLRs on monocytes and result in elevated levels of sCD14, a marker of monocyte activation (34). Increased plasma

5 levels of sCD14 has shown to be predictive of mortality in treated HIV infection (35).

While viral replication and microbial translocation are likely to be important contributors to pathogenic immune activation in HIV, other drivers such as homeostatic T cell proliferation have been proposed (Fig. 1.2)

Figure 1.2 Potential contributors to HIV-associated immune activation This figure was reused from article titled "HIV-associated chronic immune activation " authored by Mirko Paiardini and Michaela Müller‐Trutwin, published by Immunology Reviews. Permission to reuse this figure was obtained from licensed content publisher John Wiley and Sons on Nov 29, 2016.

In addition to HIV related CD4 T cell loss and turnover, there is evidence of decreased CD4 T cell production and regeneration. There is observed diminished function of CD34+ lymphoid progenitor cells in the bone marrow of HIV infected individuals (36). In addition, there is evidence of diminished thymic function in HIV infections. Recent thymic emigrant express T cell receptor rearrangement excision circles

(TRECs), which are episomal DNA that result from T cell rearrangement. Since TRECs are not replicated in division, they are markers of recent thymic emigrants (37, 38).

Decreased TREC concentrations have been observed in HIV infected children and adults

(39, 40). Collagen deposition in lymph nodes during HIV infection can disrupt naïve

CD4 T cell access to homeostatic cytokine, IL-7 (41). These observations suggest that decreased CD4 T cell production and regeneration occurs in HIV infection and contributes to progression of disease.

Loss of T and B cell function is an important characteristic of HIV infection.

Specifically, reduced CD4 T cell function is commonly associated with development of

AIDS related morbidities (42). Specific loss of functions include the diminished production of IL-2 in CD4 T cells and decreased in vitro cytotoxic T cell generation in cells from HIV infected individuals (43). Defective B cell responses, as manifested as delayed neutralizing antibody response to HIV (44, 45), functional B cell exhaustion (46) and paucity of virus-specific IgAs at mucosal sites (47, 48) have been observed in HIV infected individuals. Collectively, these observations suggest that HIV infection results in dysfunction in multiple cell types.

In summary, HIV causes disease by contributing to the loss of CD4 T cell number and function. Antiretroviral treatment blocks HIV replication and often restores CD4 T cell number and function (49). Combination of three drugs targeting various phases of

HIV life cycle was shown to have sufficient antiviral potency to allow for durable control of HIV replication. These drugs include protease, nucleoside antiretroviral, reverse transcriptase inhibitor and were initiated in 1996 as a new standard of care for HIV- infected patients (50, 51). These drugs along with others can block HIV replication at

7 multiple steps (Fig. 1.3) and were found to markedly suppress HIV replication below detection limits (50).

Figure 1.3. HIV life cycle showing sites of action of different classes of antiretroviral drug. This figure was reused from article titled "HIV infection: epidemiology, pathogenesis, treatment, and prevention " authored by Gary Maartens, Connie Celum and Sharon R. Lewin published by The Lancet. Permission to reuse this figure was obtained from licensed content publisher Elsevier on Nov 29, 2016.

Longitudinal studies have shown that ART significantly reduced the risk of mortality and morbidity in HIV infected patients (52). Nonetheless, among individuals who achieve plasma HIV RNA levels below the limit of detection, there is often a lack of complete normalization of inflammation and immune activation (53). This abnormality is sometimes referred to as residual immune dysregulation syndrome in treated HIV infection (54). Treated patients with residual immune activation and inflammation also fail to normalize CD4 T cell counts, even after multiple years of ART (53). Given the

8 clinical significance of this incomplete immune recovery following antiretroviral therapy, there is great interest in determining the mechanisms that limit recovery.

CD4 T cell count recovery in HIV infection following ART initiation

ART mediated CD4 T cell count recovery is highly variable among patients as described in large longitudinal HIV cohort patient studies in Europe and the US (55, 56).

For example, some patients achieve CD4 count of >500 cell/µl of blood and experience morbidity risk comparable to that of HIV-uninfected persons (57) but a substantial proportion of patients fail to recover >350 cell/µL of blood despite control of viral replication with ART for >3 years (55). These patients are referred to here as immune failure (IF) patients. Persistently low CD4 T cell counts is predictive of risk for non-

AIDS related morbidities such as cardiovascular disease, fractures and infection-related malignancies when compared to immune success (IS) patients (58-61). CD4 T cell recovery occurs by a two phase process: lymphocyte redistribution from lymphoid organs as ART diminishes HIV replication followed by lymphocyte production by homeostatic proliferation in the periphery and thymic output. Lymphocyte redistribution occurs in weeks and homeostatic proliferation occurs in years following ART initiation (62).

Several risk factors are associated with poor CD4 T cell recovery including pre-ART

CD4 T cell count (referred to here as nadir CD4) (Fig. 1.5) and (53), older age (63) and possibly the presence of co-infection with hepatitis C (64). Among those factors, lower nadir CD4 is the most significant risk factor for development of poor CD4 T cell recovery as the lower nadir is associated with advanced disease progression and immune dysfunction (63). Older age is a risk factor that is explained by aging-related reduction in lymphocyte production in the thymus. Although gender did not predict CD4 T cell

9 recovery, ART induced CD4 T cell gains were greater in female patients than in male patients (55). Thus, IF patients are predominantly male, of older age and lower nadir CD4

T cells when compared to IS patients (53).

Figure 1.4 CD4 T cell recovery is highly variable and related to nadir CD4 T cell count. This figure was reused from article titled "HIV infection: Incomplete peripheral CD4+ T cell count restoration in HIV-infected patients receiving long term antiretroviral treatment " authored by Kelley CF and colleagues published by Clinical Infectious Diseases. Permission to reuse this figure was obtained from licensed content publisher Oxford University Press on Nov 29, 2016.

Homeostatic proliferation in response to interleukin-7 is a mediator of T cell recovery

Response to IL-7 is important for T cell homeostasis. IL-7 deficient mice are lymphopenic in the peripheral blood and lymphoid organs (65). Humans born with loss of function mutation in the gene encoding for IL-7Rα suffer from severe combined immunodeficiency (SCID) and an absence of T cells (66, 67). During lymphopenic conditions such as HIV infection, IL-7 concentration in the blood is increased and is inversely correlated with CD4 T cell counts (68). IL-7 is produced from stromal cells of the lymph nodes and bone marrow (69, 70). IL-7 then stimulates T cell proliferation until

T cell number increases to normal level. This is mediated by two mechanisms: thymic

10 output of naive CD4 T cells and homeostatic proliferation of all maturation subsets of

CD4 T cells in the peripheral blood (71). As thymic output diminishes as patients age, homeostatic proliferation in response to IL-7 may become a more important contributor to CD4 T cell recovery.

Due to its ability to promote survival and expansion of T cells, the effects of recombinant human IL-7 (rHIL-7) administration have been assessed in HIV infected patients. Based on the results of a phase 1 clinical trial in HIV infected patients, rIL-7 at dose range of 3-30µg/kg was well tolerated. In clinical trials involving HIV infected subjects, transient increases in plasma HIV RNA was observed along with increases in circulating T cell number, particularly in the central memory T cell subset (72, 73).

Although these results suggest that rhIL-7 administration may enhance CD4 T cell reconstitution in treated HIV infected patients, the long term clinical benefit of this approach is not yet known.

Figure 1.5 IL-7 receptor signaling promotes cell survival and drives proliferation. This figure was reused from article titled "Naive T cell homeostasis: from awareness of space to a sense of place" authored by Kensuke Takada and Stephen C. Jameson published by Nature Reviews Immunology. Permission to reuse this figure was obtained from licensed content publisher Nature Publishing Group on Nov 29, 2016.

The IL-7 receptor is composed of IL-7Rα (CD127) and γc (CD132). IL-7 delivers proliferation and survival signals to T cells following receptor binding. Cell survival is mediated by the phosphorylated STAT5 (P-STAT5) and proliferation is mediated by the phosphorylation and activation Akt (P-Akt) (74) (Fig.1.5). Studies have shown that IL-7 induced delayed but sustained Akt phosphorylation that was associated with proliferation

(75). P-Akt drives proliferation by inducing metabolic switch to aerobic glycolysis (76), increasing protein translation, and down-regulating cell cycle inhibitors (77).

Expression of myelocytomatosis (c-myc) oncogene product is important for T cell proliferation (78, 79). IL-7 induces c-myc transcription (80), and the inactivation of glycogen synthase kinase-3 (GSK-3), the negative regulator of c-myc expression (81). In resting cells, GSK-3 is constitutively active and promotes the degradation of c-myc (82).

As a result, c-myc expression is increased as GSK-3 is inactivated. Increased c-myc expression is associated with increased transcription of all the genes needed for proliferation. These genes encode nutrient uptake transporters as well as all the enzymes required for aerobic glycolysis. As part of aerobic glycolysis that is mediated c-myc, there is increased uptake of biosynthetic precursors required for cellular division.

Increased biosynthetic uptake is accomplished by c-myc induced expression of T cell specific glucose transporter Glut1 and amino acid transporter 4F2 cell-surface antigen heavy chain (CD98) (83).

P-Akt in response to IL-7 also up-regulates protein translation by signaling through the activation of mammalian target of rapamycin complex 1 (mTORC1) (84).

Activated mTORC1 phosphorylates and activates ribosomal S6 kinase which phosphorylates S6, a component of the translational machinery (77). Phosphorylated ribosomal S6 has increased translation activity in order to support cellular division (85).

IL-7 also promotes entry into the G1 phase of cell cycle through the destabilization of

P27kip1, a cyclin dependent kinase inhibitor in T cells. P-Akt phosphorylates P27kip1 leading to its degradation (77). FOXO3a can oppose this mechanism of driving proliferation by up-regulating P27kip1, IL-7 can also regulate FOXO3a expression by phosphorylation of FOXO3a leading to its exclusion from the nucleus, inhibiting its transcriptional control over pro-apoptotic genes and anti-proliferative genes and genes in involved in the regulation of glucose metabolism (86). Thus, IL-7 utilizes multiple mechanisms in order to drive cellular proliferation.

Responsiveness to IL-7 may be impaired in CD4 T cells from IF patients. Camargo and colleagues showed that P-STAT5 induction in response to IL-7 is positively correlated with CD4 T cell counts. They also observed a positive relationship between CD127 expression and P-STAT5 induction in response to IL-7 (87). Consistent with these observations, Tanaskovic and colleagues demonstrated that IL-7 induced P-STAT5 is diminished in CD4 T cells from IF subjects. They further show that P-STAT5 induction was inversely associated with percentage of CD4+CD57+ cells (senescent cells). In addition, they found that CD127 expression is diminished from naïve CD4 T cells from

IF subjects (88). These observations suggest that IL-7 responsiveness may be diminished in CD4 T cells from IF patients and this defect could be related to T cell senescence.

The role of IFN-I in treated HIV infection

In acute HIV infection, IFN-I blocks viral entry and replication by the actions of ISGs. In chronic HIV infection, sustained IFN-I production is a part of the systemic inflammatory response that is associated with disease progression and is not normalized by ART (89).

This is suggested by evidence of increased IFN-I exposure in IF patients based on interferon gene expression signature (90) as well as increased plasma levels of IP-10 (91).

In addition, IFN-I bioactivity is associated with low CD4 T cell count and high frequency of CD38 expressing CD8 T cells in HIV-infected patients (92). In chronic HIV infection, the source of IFN-I is controversial but studies involving acute SIV infection of monkeys suggest that plasmacytoid dendritic cells (pDCs) initially produce IFN-I and that IRF-7 mediated up-regulation of IFN-I leads to more production in other cell types (93, 94).

Also, microbial translocation from a damaged gut barrier, may activate TLR signaling and potentially contribute to IFN-I production (95). Overall, IFN-I expression is increased in IF patients and may adversely affect T cell homeostasis.

The anti-viral and anti-proliferative functions of IFN-I

IFN-I mediates antiviral activities and inhibits proliferation in a variety of cell types as part of its critical role in host defense (96). IFN-I is produced by immune cells following ligand engagement of innate receptor systems such as endosomal toll-like receptors

(TLR-3, TLR-7 and TLR-9) and cytosolic receptors (mda5 and RIG-1) (97). The IFN-I cytokine family consists of 12 subtypes of IFN-α, a single IFN-β and additional subtypes of IFN-ε, -κ, -ω. All IFN-I subtypes bind to the IFN-α receptor, which is comprised of

IFN-αR1 and IFN-αR2. Activation of IFN-α receptor triggers formation of transcription factors to drive interferon stimulated gene (ISG) expression (98). This process involves

14 the activation of receptor associated kinases, JAK1 and tyrosine kinase 2 (TYK2) to form

2 transcription factors: STAT1 homodimers and complex interferon stimulated gene factor-3 (ISGF-3). ISGF3 is composed of STAT1, STAT2 and interferon regulatory factor-9 (IRF-9). STAT1 homodimers direct the transcription of genes containing gamma-activated sequences (GAS) while ISGF-3 targets IFN stimulatory response elements (ISRE) and regulates >300 ISRE containing ISGs (96). Some ISGs are responsible for the inhibition viral infections by three mechanisms: the double stranded

RNA-dependent protein kinase R (PKR) pathway, the 2-5A system and the myxovirus resistance protein (Mx) pathway. PKR is a kinase with multiple functions including inhibition of protein synthesis following dsRNA binding and activation. While PKR exerts translational control to block viral replication, the 2-5A system mediates intracellular viral RNA cleavage by up-regulating 2’, 5’-oligoadenylate synthetase (OAS) expression, which generates the intermediate 2-5A from ATP. 2-5A activates RNase L, which cleaves single stranded RNA. As part of the Mx pathway, Mx proteins (MxA,

Mx1, etc) are GTPases in the dynamin family which interferes with viral replication at multiple steps of infection (98). Anti-viral responses can potentially cause inflammatory damage after viral clearance so regulation of ISG expression is mediated by a complex mechanism that involve interferon regulatory factor-7 (IRF-7) and FOXO3a. Since IRF-7 expression is controlled by an ISRE (99), this mechanism of regulation likely occurs as a result of IFN-I response. IFN-I induced PKR inhibits cell proliferation by down- regulating c-myc by a mechanism that is not dependent on activation by dsRNA (100,

101). However, other mechanisms of inhibition by IFN-I exists, including regulation of cell cycle control by increasing expression of retinoblastoma (102), suppression of cyclin

D3 (103), and increased expression of p27kip1 (104). Inhibition of cell growth by these

IFN-I inducible pathways inhibits entry into S phase (105). Consistent with the observation that IFN-I exert anti-proliferative effects, we have shown that IFN-α inhibits proliferation in CD4 T cells stimulated by IL-7 in chapter 2. Overall, IFN-I mediates potent anti-viral responses and anti-proliferative activity in T cells.

TGF-β and additional cytokines that may contribute to low CD4 T cell count recovery in treated HIV infected patients

Although IFN-I gene expression signatures are well characterized in cells from persons who experience poor CD4 T cell recovery during ART, other inflammatory cytokines and processes likely contribute to perturbations in T cell homeostasis (106). Systemic immune activation and inflammation that persists despite effective viral suppression may affect CD4 T cell turnover (53). Shive and colleagues determined that IL-6 and IL-1β down-regulate expression of CD127 and B-cell lymphoma 2 (BCL-2) in vitro (108). The down-regulation of CD127 expression by IL-6 and IL-1β could be important in CD4 T cell recovery following ART since IL-6 is elevated in plasma and IL-1β expression in lymph nodes remain elevated in treated HIV infected patients (106). Down-regulation of

BCL-2 by these cytokines may be important since BCL-2 is a prosurvival factor that is important for the maintenance of T cell homeostasis (107). Furthermore, by diminishing

CD127 and BCL-2 expressions, these cytokines have the potential to increase CD4 T cell turnover (53).

TGF-β is another cytokine that has complex biological activities and may affect

CD4 T cell count recovery. TGF-β produced by regulatory T cells stimulates the production of collagen leading to its deposition in lymph nodes of HIV infected individuals (41). LN fibrosis as a result of collagen deposition is characteristic of HIV

16 infection and is linked to naive CD4 T cell numbers in HIV infected patients (41). There is evidence to suggest that LN fibrosis is not corrected by ART, suggesting that TGF-β expression may remain elevated in LN of IF patients (109). In addition, TGF-β gene expression signature has been observed in T cells of immune failure patients (Sekaly, personal communication). These observations, along with the immunoregulatory effects of TGF-β (described below), suggest that TGF-β expression may play a role in poor CD4

T cell count recovery in treated HIV infection.

TGF-β inhibits proliferation in T cells as well as non-immune cells (110-112).

Additionally, TGF-β can be secreted from a variety of cell sources (113), including activated CD4 T cells (114). In epithelial cells, TGF-β arrests cell cycle by down- regulating c-myc expression (115) and up-regulating p27kip1, P15 (116) and P21 (117), inhibitors of cell cycle progression. In T cells, TGF-β inhibits TCR-activated proliferation by diminishing expression of metabolic genes and ribosomal S6 kinase phosphorylation and activation (118). Thus, TGF-β may inhibit proliferation in response to IL-7 since induction of metabolic gene expression, S6 kinase activity and cell cycle progression are promoted in response to IL-7.

The following chapter contains work published in the Journal of Leukocyte Biology in

June of 2015, volume 97, issue 6, pages 1139-1146. The authors are Thao P. Nguyen,

Doug Bazdar, Joseph C. Mudd, Michael M. Lederman, Clifford V. Harding, Gareth H.

Hardy and Scott F. Sieg. Copyright permission to use this work for this thesis was obtained from the Journal of Leukocyte Biology on November 28, 2016.

Chapter 2: Interferon-α inhibits CD4 T cell responses to interleukin-7 and interleukin-2 and selectively interferes with Akt signaling

Abstract: Persistent type I IFN production occurs during chronic viral infections, such as

HIV disease. As type I IFNs have antiproliferative activity, it is possible that chronic exposure to these cytokines could adversely affect T cell homeostasis. We investigated the capacity of IFN-α to impair T cell proliferation induced by the homeostatic cytokine,

IL-7, or another common γ-chain cytokine, IL-2, in cells from healthy human donors. We found that IL-7- or IL-2-induced proliferation of CD4+ T cells was partially inhibited in the presence of IFN-α. The CD4+ T cells that were exposed to IFN-α also displayed attenuated induction of IL-2 and CD40L following TCR stimulation. Analyses of signaling pathways indicated that IL-7 and IL-2 induced a delayed and sustained P-Akt signal that lasted for several days and was partially inhibited by IFN-α. In contrast, IL-7- induced P-STAT5 was not affected by IFN-α. Furthermore, IFN-α had no detectable effect on P-Akt that was induced by the chemokine SDF-1. Both inhibitors of P-Akt and

P-STAT5 blocked IL-7-induced T cell proliferation, confirming that both signaling pathways are important for IL-7-induced T cell proliferation. These results demonstrate that IFN-α can selectively inhibit cytokine-induced P-Akt as a potential mechanism to disrupt homeostasis of T lymphocytes.

Introduction

Type I IFNs represent a key innate defense mechanism to fight viral infection.

Among the various activities of type I IFNs, antiproliferative effects of these cytokines have been recognized as potentially important components of antiviral and -tumor

19 defenses (119-121). Although several molecular targets of cell-cycle regulation by type I

IFNs have been identified (100, 103, 122, 123), few studies have characterized these effects in primary T cells, and there is limited knowledge of the potential interactions of type I IFNs with homeostatic cytokines, such as IL-7 or IL-2, which are important for T cell survival and growth. Therefore, we performed studies to evaluate the effects of IFN-

α on T cell proliferation, T cell function, and T cell signaling in a model of IL-7-induced homeostatic proliferation.

IL-7 is an important cytokine that is critical for maintenance of T cell numbers.

Disruption of the IL-7/IL-7R axis results in severe lymphopenia (65, 124) and IL-7 is a critical factor in homeostatic T cell expansion that occurs in lymphopenic hosts (71, 125).

IL-7 administration in persons with HIV disease or other lymphopenic conditions results in T cell expansion and promotes T cell survival (73, 126-128). Thus, IL-7 is not only a key physiologic signal for T cell homeostasis but also, represents a developing tool for therapeutic interventions.

IL-7 mediates its effects by enhancing the expression of antiapoptotic molecules, such as B cell lymphoma 2 (71, 129, 130), and by inducing cellular proliferation through regulation of molecules that control cell-cycle progression, such as p27kip (131, 132). IL-7 binds to a heterodimeric receptor comprised of an α-chain (CD127) and the common γ- chain (CD132). IL-7R signaling includes JAK/STAT and PI3K/P-Akt activation, which affect cellular survival and proliferation (75, 133, 134).

IL-2 is also an important growth factor for T cells that induces proliferation and uses similar signaling machinery as IL-7. IL-2 activates T cells by signaling through the shared common γ-chain and through the β-chain (CD122), whereas the α-chain of the IL-

2R (CD25) provides for high-affinity interactions of this complex with the cytokine. IL-

2R activation, like IL-7R activation, leads to activation of JAK/STAT signaling along with P-Akt activation. Previous studies suggest that IFN-α may lead to impairments of

IL-2-induced STAT5 signaling that are demonstrable at the level of DNA binding (122).

Type I IFNs are produced at elevated levels in HIV disease, and although these cytokines play an important role in antiviral defenses, chronic exposure to these cytokines may have detrimental effects(92, 135, 136). For example, as a result of chronic exposure, it is thought that type I IFNs could contribute to T cell death by regulating various apoptotic pathways (137-139). An alternative, but not mutually exclusive, hypothesis is that type I IFNs could disrupt T cell homeostasis as a consequence of its antiproliferative effects. Here, we study the potential for IFN-α to inhibit T cell proliferation induced by the homeostatic cytokine, IL-7, and another T cell growth factor,

IL-2. Our studies uncover novel aspects of IL-7 signaling kinetics in primary T cells and suggest that IFN-α may mediate antiproliferative activity by selectively regulating P-Akt in T cells stimulated with these cytokines.

Methods

Cells and cell culture

Whole blood was collected from healthy adult volunteers who signed informed consent through a protocol approved by the University Hospitals of Cleveland

Institutional Review Board. PBMCs were isolated over a Ficoll-Hypaque cushion. In some assays, PBMCs were labeled with CFSE. PBMCs were incubated in 0.25 μM CFSE at 37°C for 10 min and washed with PBS, supplemented with 10% FCS. CFSE-labeled cells were resuspended in X-VIVO serum-free medium and incubated in 24-well plates at

21 a concentration of 2 million cells/ml. Cells were stimulated with rIL-7 (Cytheris; 5 ng/ml) to induce proliferation. IFN-α (PBL) was added at 500 U/ml or as otherwise indicated.

Cells were allowed to incubate for 7 days and were then, in some cultures, additionally stimulated with SEB (2 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 2 h, followed by a 4 h incubation with Golgi plug reagent. Intracellular flow analyses were performed to measure expression of CD40L, IL-2, and IFN-γ. Additional experiments involved purified CD4+ cells that were separated from PBMC by negative selection (magnetic bead separation; Miltenyi Biotec, San Diego, CA, USA). Cells were >97% pure, as determined by flow cytometric analyses. Purified CD4 cells were CFSE labeled and incubated with or without IL-7 (5 ng/ml) or IL-2 (50 ng/ml; BD PharMingen, San Diego,

CA, USA) ± IFN-α (500 U/ml or as indicated). After 3 or 7 days, cells were stimulated with CytoStim beads, which activate T cells by cross-linking TCRs (Miltenyi Biotec) for

2 h, followed by 3 h of Golgi plug (BD Biosciences, San Jose, CA, USA) treatment. Cells were assessed for CFSE dye dilution and for intracellular expression of CD40L. Some studies included IL-7-treated cells that were incubated additionally with wortmannin (500 nM; PI3K inhibitor; Sigma-Aldrich) or N′-[(4-Oxo-4H-chromen-3- yl)methylene]nicotinohydrazide (500 μM; P-STAT5 inhibitor; EMD Millipore, Billerica,

MA, USA).

Flow cytometry

SEB-stimulated cells and cells incubated without stimulation were assessed for expression of CD40L, IL-2, and IFN-γ. Cells were surface stained with antibodies reactive to CD4, and dead cells were excluded from analyses by staining with Live/Dead

Fixable Yellow Dead Cell Stain Kit (Invitrogen, Grand Island, NY, USA). Cells were

22 fixed and permeabilized with the Cytofix/Cytopermeabilization Kit (BD PharMingen) and then stained with fluorochrome-labeled antibodies reactive with CD40L (BD

PharMingen), IFN-γ (BioLegend, San Diego, CA, USA), IL-2 (BD Biosciences), or appropriate isotype controls. In some assays, cells were tested for expression of P-STAT5 and P-Akt by use of methods that we have described previously (140, 141). In brief, cells were incubated with or without IL-7 and IFN-α for 15 min overnight (1 day) or for 2 or 3 days. Cells were treated with 100 μl 16% ultrapure methanol-free formaldehyde

(Polysciences, Warrington, PA, USA) for 10 min at 37°C. Cells were then transferred to polystyrene tubes, washed with PBS, and resuspended in 500 μl cold 90% methanol for

30 min. Cells were washed and stained with anti-CD4 (BioLegend), anti-CD3

(BioLegend), anti-P-STAT5 (BD Biosciences; recognizing Tyr694), and anti-P-Akt antibodies (BD Biosciences; recognizing the Ser473 epitope) for 60 min on ice before analyses on a BD LSRII flow cytometer (BD Biosciences).

For experiments involving the assessment of apoptosis, CFSE-labeled PBMCs were incubated for 7 days under various conditions, washed, and surface stained with antibodies reactive to CD3 and CD4 (BioLegend) and additionally stained with

Live/Dead Fixable Yellow Dead Cell Stain Kit (Invitrogen). Cells were washed twice with 1 ml ice-cold PBS and then stained with Annexin V-PE (BD PharMingen) in the presence of 1× Annexin V binding buffer (BD PharMingen) for 15 min. Cells were examined by flow cytometry.

In some studies, PBMCs were preincubated with IFN-α (1000 U/ml) for 2 days, washed, resuspended in 300 μl X-VIVO medium, placed in polystyrene tubes, incubated in a 37°C water bath for 10 min, and then stimulated with SDF-1 (10 ng/ml; R&D

Systems, Minneapolis, MN, USA) for 1 min. Cells were treated with 400 μl BD Cytofix for 10 min, washed, and resuspended in 200 μl BD Phosflow Perm Buffer III. Cells were incubated on ice and in the dark for 30 min, washed, and stained with anti-CD3, anti-

CD4, and anti-P-Akt fluorochrome-conjugated antibodies as above.

Statistical analyses

SPSS software was used for statistical analyses. Nonparametric tests were used to assess differences between cells incubated in different conditions (Kruskal-Wallis multigroup comparison and Mann-Whitney U-tests). Nonparametric tests (Wilcoxon signed-rank tests and sign tests) were used to assess differences in paired data.

Results

IFN-α impairs IL-7-induced proliferation responses and diminishes cellular function in CD4+ T cells

To assess the effects of IFN-α on IL-7-induced CD4+ T cell proliferation, CFSE- labeled PBMCs or purified CD4+ T cells were incubated with IL-7 for 7 days in the presence or absence of IFN-α. The addition of IFN-α to IL-7-treated cells reduced proliferation (CFSE dye dilution) among CD4+ T cells within PBMCs and also in the purified CD4+ T cell populations (Fig. 2.1). The magnitude of inhibition by IFN-α was dose dependent and still detectable at concentrations as low as 30 U/ml in PBMC assays

(Fig.2.2). In contrast to the capacity of IFN-α to inhibit IL-7-induced T cell proliferation over 7 days, IFN-α had little effect on the induction of CD25 expression that was induced by IL-7 in CD4+ T cells after an overnight incubation (Fig. 2.1C). Thus, not all aspects of

T cell responses to IL-7 were impaired by IFN-α.

Figure 2.1 IFN inhibits IL-7-induced proliferation. PBMCs or negatively selected purified CD4+ T cells were CFSE labeled and incubated with IL-7(5 ng/ml) 6 IFN-a (500 U/ml). Flow cytometric analyses were performed after 7 days to assess cell proliferation. Cells were gated based on forward- and side-scatter characteristics to exclude debris and forward scatter height versus area to exclude doublets. Viability dye stain was used to exclude dead cells. Histograms represent CD4+ lymphocytes. (A) CFSE dye dilution of gated CD4+ cells at 7 days is shown on the x-axis for the various conditions. (B) Summary data of CFSE dye dilution from studies of PBMC gated for CD4+ T cells (left) and of purified CD4+ T cell cultures (right). Each pair of connected symbols represents data from a different donor. (C) Assessment of CD25 expression in CD4+ T cells incubated overnight in medium alone, IL-7 (5 ng/ml) or IL-7 plus IFN-a (500 U/ml). Cells incubated in IFN-a alone showed no change in CD25 expression (not shown). Connected symbols represent data from a single donor. Results are shown for 3 different donors.

Figure 2.2. The magnitude of IFN-α mediated inhibition of IL-7 induced proliferation is dose dependent.

To test the functionality of CD4+ T cells that had been preincubated for 7 days with IL-7 or IL-7 + IFN-α, we stimulated cells with SEB and measured expression of intracellular CD40L, IL-2, or IFN-γ. The cells that were preincubated with IL-7 plus IFN-

α were less able to express IL-2 and CD40L compared with cells incubated in IL-7 alone

(Fig. 2.3).

Figure 2.3 Pre-incubation of CD4+ T cells in IFN-α plus IL-7 reduces T cell function compared with pre-incubation of cells in IL-7 alone. PBMCs or purified CD4+ T cells were CFSE labeled and incubated with IL-7 (5 ng/ml) ± IFN-α (500 U/ml) for 7 days. Cells were then stimulated with SEB (PBMC) or CytoStim beads (purified CD4+ T cells) to induce intracellular cytokines or CD40L expression. Cells were gated based on forward- and side-scatter characteristics to exclude debris and forward-scatter height versus area to exclude doublets. Viability dye stain was used to exclude dead cells. CFSE dye dilution and intracellular CD40L expression among CD4+ cells were determined by flow cytometry. (A) Representative histograms depict IL-2, CD40L, and IFN-γ expression (y-axis), which were induced by SEB stimulation and CFSE dye dilution (x-axis). The CFSE dye dilution resulted from preincubation in IL-7 or IL-7 + IFN-α. The addition of SEB did not cause further proliferation, as the assay was performed over hours and the Golgi plug reagent had been added during the SEB incubation period (not shown). (B) Summary data of cytokine induction in PBMCs that had been incubated in IL-7 or IL-7 + IFN-α before SEB stimulation. (C) Summary data that use purified CD4+ T cells from 4 different donors, showing induction of CD40L expression in total and in CFSElow cells at day 7 following preincubation of cells in IL-7 or IL-7 + IFN-α (P = 0.068 in each experiment).

In contrast, the capacity of T cells to produce IFN-γ was not impaired significantly in T cells preincubated with IL-7 plus IFN-α. In 2 experiments, the cells were washed free of cytokine before SEB stimulation to determine if inhibition of SEB responsiveness was dependent on maintaining IFN-α in the cell culture. Even after washing the cells free of cytokine and replating before SEB stimulation, the cells that had been incubated previously with IFN-α + IL-7 demonstrated reduced SEB responses compared with cells incubated with IL-7 alone (percent inhibition of CD40L induction in

2 different donors = 56% and 50%, and percent inhibition of IL-2 induction = 30% and

61%, comparing cells preincubated with IL-7 + IFN-α with cells preincubated in IL-7 alone). Therefore, the magnitude of inhibition was similar whether IFN-α had been left in the culture for the duration of SEB stimulation or had been washed out of the culture before SEB stimulation.

To ascertain if the effects of IFN-α on T cell functionality could be mediated through direct effects in T cells, purified CD4+ T cells that had been preincubated for 7 days with IL-7 or IL-7 + IFN-α were stimulated with CytoStim beads, as examined for intracellular induction of CD40L. Similar to our observations in PBMCs, the addition of

IFN-α to IL-7 during the 7 day preincubation resulted in diminished induction of CD40L following TCR stimulation. Notably, the defects in CD40L induction among cells that were preincubated with IL-7 plus IFN-α were observed, even in cells that had proliferated during the preincubation period (Fig.2.3C).

Figure 2.4 Gating strategy to assess viability

IFN-α diminishes T cell viability

To assess the potential for IFN-α to affect cell viability, we incubated PBMCs from 14 healthy control donors in medium alone or medium supplemented with IFN-α,

IL-7, or both cytokines. After 7 days, CD4+ T cells were examined with viability dye stain to assess cell death (Fig. 2.4). Analyses indicated that there was an increase in cell death among CD4+ T cells cultured for 7 days with IFN-α (mean = 18.7%; n = 14) compared with death in cells incubated in medium alone (mean = 11%; P = 0.002). There was also an increase in cell death in cells incubated with IL-7 plus IFN-α (mean = 9.1%) compared with cells incubated in IL-7 alone (mean = 6.4%; P = 0.039). Thus, IFN diminished CD4+ T cell viability in 7 day cultures.

Figure 2.5 IFN-α causes cell death, primarily in nondividing T cells, in IL-7-treated cell cultures. PBMCs were CFSE labeled and incubated with IL-7 (5 ng/ml) ± IFN-α (500 U/ml) for 7 days. (A) Apoptosis was assessed by first gating out debris, then gating on CD4+ cells, and then CD3+ cells. SSC-A, Side-scatter-area; FSC-A, forward-scatter- area. (B) Histograms depict cell proliferation by CFSE dye dilution (x-axis) and apoptosis by Annexin V binding (y-axis) in IL-7 and IL-7 + IFN-α-treated cells. (C) Data summarize the percentages of apoptotic CFSElow cells among the CD4+CD3+ cells (left) and the percentage of CFSElow cells among only the apoptotic CD4+CD3+ cells (right).

Although this observation suggested an adverse effect of IFN-α on T cell viability, it did not address the possibility that cells treated with IL-7 + IFN-α might proliferate normally but then die after cell division. To address this possibility, we assessed cell viability in

PBMCs from 5 additional donors by use of Annexin V to identify early and late apoptotic cells (Fig. 2.5). Analyses of CD3+CD4+ cells demonstrated that among these 5 subjects,

30 the addition of IFN-α to IL-7-treated cells resulted in reduced frequencies of live and apoptotic CFSE dim cells (divided cells). Moreover, among the apoptotic cells, the proportion that had proliferated and then died during the culture period was actually reduced by IFN-α. These results are most consistent with a failure of cells to divide rather than division followed by death in cells exposed to IL-7 + IFN-α.

IFN-α inhibits P-Akt but not P-STAT5 in IL-7-treated cells

Figure 2.6 IFN-α impairs P-Akt but not P-STAT5 signaling in IL-7-treated cells. (A) PBMCs were incubated in medium alone, medium + IL-7 (5 ng/ml), or medium + SDF-1 (10 ng/ml) for various periods of time as indicated, and then cells were assessed for expression of P-Akt by intracellular flow cytometry. (B) Flow cytometry histograms (left) in CD3+CD4+ cells, showing induction of P-STAT5 (y-axis) and P-Akt (x-axis) at 3

31 days of incubation in PBMC treated with medium alone, medium + IL-7 (5 ng/ml), or medium + IL-7 and IFN-α (500 U/ml). No induction of P-Akt was observed in cells incubated with IFN-α alone (not shown). Summary data from 6 donors are provided (right). (C) PBMCs were incubated with IL-7 for 2 days, and then some cells were treated with an inhibitor of PI3K signaling (wortmannin). After an additional overnight incubation, cells were assessed for P-Akt expression. (D) PBMCs from 5 different donors were treated with IL-7 for 3 days and assessed for P-Akt expression by intracellular flow cytometry (labeled “IL-7”). In comparison, some cells were incubated with IL-7 for 3 h (IL-7, 3 h) or 1 day (IL-7, 1 day), washed, and then returned to culture in the absence of IL-7 until analysis for P-Akt at Day 3.

To understand the effects of IFN-α on cell signaling, we compared P-Akt with P-

STAT5 expression in PBMCs that had been incubated with IL-7 or with IL-7 plus IFN-α.

Cell signaling was assessed at various time-points. After IL-7 stimulation, a P-STAT5 signal was rapidly detected (1–15 min; not shown) and remained high through 3 days of cell culture (not shown). In contrast, P-Akt was not consistently detected at early time- points, ranging from minutes up through 1 day poststimulation, but became consistently discernible 2–3 days poststimulation with IL-7 and remained detectable over several days

(Fig. 2.6A). The delayed induction of P-Akt that was mediated by IL-7 was in contrast to a rapid and transient induction of P-Akt that was induced by the chemokine SDF-1 (Fig.

2.6A). Importantly, in the presence of IFN-α, P-Akt, which was induced by IL-7, was blunted, as assessed at day 2 (not shown) and day 3 poststimulation (Fig. 2.6B). In contrast, there was no appreciable effect of IFN-α on P-STAT5 expression (Fig. 2.6B).

To confirm the specificity of our P-Akt antibody, some cells were treated with the PI3K inhibitor wortmannin on day 2, and P-Akt was assessed on day 3. The blocking of PI3K resulted in blunted P-Akt expression but did not affect P-STAT5 (Fig. 2.6C).

To determine if the expression of P-Akt and P-STAT5, which we detected at day

3 poststimulation, was dependent on prolonged IL-7 exposure, we incubated cells with

IL-7 for 3 h or 1 day, washed the cells, and replated the cells in medium alone until day 3.

We compared the P-Akt expression and P-STAT5 signal at day 3 in these cells to signals detected in cells that had been left in IL-7 for 3 days continuously. The washing of IL-7 from the cell cultures after 3 h or 1 day of incubation in the absence of IFN-α resulted in diminished P-Akt and P-STAT5 detection on day 3 (Fig. 2.6D). These observations suggest that prolonged and continuous IL-7 exposure are required to observe optimal, late signaling events.

To ascertain if IFN-α might adversely affect expression of CD127, which provides a docking site for PI3K upstream of Akt activation (142), we incubated PBMCs with IFN-α for various periods of time (1–3 days). We found no evidence of diminished

CD127 expression after incubating CD4+ cells in IFN-α (not shown). This observation, together with the lack of an effect on P-STAT5 by IFN-α, suggests that the signaling deficiencies induced by IFN-α are more likely mediated by a postreceptor signaling mechanism rather than by modulation of CD127 expression.

IFN-α inhibits P-Akt signaling that is induced by IL-2 but not by SDF-1

We next considered the potential for IFN-α to inhibit P-Akt upon activation of T cells with other stimuli. PBMCs were stimulated with IL-2 or SDF-1 during or after exposure to IFN-α. For IL-2 studies, PBMCs were incubated with IL-2, plus or minus

IFN-α, for 3 days and assessed for P-STAT5 and P-Akt expression. Similar to the effects of IFN-α on IL-7 signaling, induction of P-Akt after 3 days of stimulation by IL-2 was

33 impaired by IFN-α, but there was little effect on P-STAT5 (Fig. 2.6A and B). To examine further the specificity of the IFN-α effect, PBMCs were preincubated with IFN-

α for 2 days and then stimulated with SDF-1, the chemokine ligand for CXCR4. In contrast to our observations with IL-7 or IL-2 stimulation, IFN-α had no detectable effect on P-Akt induction by SDF-1 (Fig. 2.6C). These data suggest that IFN-α may affect P-

Akt signaling from cytokine receptors more readily than from chemokine receptors.

Figure 2.7 Impaired responses to IL-2 but not SDF-1 in CD4 cells exposed to IFN-α. (A) PBMCs were CFSE labeled and incubated in medium alone, medium + IL-2 (50 ng/ml), or medium + IL-2 + IFN-α (500 U/ml). Histograms show expression of P-STAT5 (y-axis) and P-Akt (x-axis) after 3 days of incubation among CD3+CD4+ cells, and cells incubated in IFN-α looked similar to cells incubated in medium alone (not shown). (B) Results are shown for cells from 5 different donors comparing P-Akt and P-STAT5 induction in CD3+CD4+ cells incubated for 3 days in the presence of IL-2 or IL-2 + IFN-

α. (C) PBMCs were preincubated in medium alone or in medium + IFN-α (1000 U/ml) for 2 days before stimulating cells with SDF-1 (10 ng/ml) for 1 min. The percent of P- Akt+ cells is indicated for cells stimulated with SDF-1. (D) PBMCs were incubated for 7 days with IL-2 or IL-2 + IFN-α. The percentage of CFSElow cells was determined by flow cytometry (debris, doublets, and dead cells were removed from the analysis). Each pair of connected symbols represents cells from a different donor (n = 6).

As IFN-α impaired P-Akt expression in IL-2-stimulated cells, we also asked if proliferation of these cells was impaired at 7 days poststimulation. In 6 different donors, we found clear evidence that IFN-α reduced the proliferation of CD4+ T cells in response to IL-2 stimulation (Fig. 2.7D). These data indicate that IFN-α inhibited P-Akt induction and proliferation but only marginally reduced P-STAT5 induction in IL-2-stimulated T cells.

Figure 2.8 Inhibition of PI3K or STAT5 reduced cell proliferation, decreased functionality, and enhanced cell death in cells treated with IL-7.(A) CFSE-labeled

PBMCs were incubated in medium alone, medium + IL-7, medium + IL-7 + PI3K inhibitor, and IL-7 + STAT5 inhibitor and then assessed 7 days later for proliferation (CFSE dye dilution, x-axis) and response to SEB stimulation (CD40L induction, y-axis). Data are representative of results from 3 different donors. (B) Viability was assessed in the above cultures by gating on forward- and side-scatter to eliminate debris and monocytes, followed by forward-scatter-height versus forward-scatter-area to exclude doublets (not shown). Then, cells were gated on CD4 cells to assess viability dye stain. The percentage of dead CD4+ cells is shown on the y-axis under the various conditions (x-axis). Connected symbols represent data from a single donor. Data from 3 different donors are shown as different symbols.

Inhibition of P-Akt or P-STAT5 blocks cellular proliferation and function

To confirm that Akt and STAT5 signaling are important for cellular proliferation and to ascertain if disruption of either pathway results in functional impairments among cells that survive 7 day incubations, CFSE-labeled PBMCs were stimulated with IL-7 and incubated with or without a PI3K inhibitor (wortmannin) or a P-STAT5 inhibitor. After 7 days, CFSE dye dilution and responses to SEB stimulation were measured. Incubation of

IL-7-stimulated cells with an inhibitor of PI3K reduced cellular proliferation and diminished the ability of cells to respond to TCR activation. Similar to the effects of IFN-

α on T cell function, IL-2 production appeared to be more severely affected than IFN-γ production in surviving cells after TCR stimulation (Fig. 2.8A). The blocking of P-

STAT5 manifested in even more striking impairments that rendered cells completely dysfunctional. Inhibition of P-STAT5 also caused enhanced cell death in IL-7-stimulated cells after 7 days of culture (Fig. 2.8B). Therefore, T cells that have been stimulated with

IL-7 demonstrate impaired functional responses if signaling through P-STAT5 or P-Akt is blocked.

Discussion

Our studies demonstrate that IFN-α mediates inhibition of IL-7- or IL-2-induced

T cell proliferation. In IL-7-stimulated cells, we found evidence of enhanced cell death when IFN-α was also added to cultures, making it difficult to tease apart the potential role for cell death and the potential antimitotic effects of IFN-α. Analyses of apoptosis among cells that had divided during the 7 day incubation, however, suggested that IFN-α primarily caused cell death among cells that had failed to divide. Importantly, this observation does not exclude the possibility that the IL-7-treated cells, which died in the presence of IFN-α, may have entered cell-cycle originally but died before their first mitotic division.

Along with inhibition of T cell proliferation, our results indicate that the addition of IFN-α to IL-7-stimulated cells leads to decreased P-Akt but has no detectable effect on

P-STAT5 expression compared with cells stimulated with IL-7 alone. Although we measured P-STAT5 at a site that is important for DNA binding (Tyr694 (142)), our studies do not rule out the possibility that IFN-α may impair P-STAT5 DNA binding downstream of phosphorylation (122). Nonetheless, we found that IFN-α had little effect on the induction of CD25 expression by IL-7 during an overnight incubation. As the

CD25 gene promoter region has a well-defined STAT5 binding site, which promotes transcriptional activation (143, 144), this observation suggests that P-STAT5 signaling is not impaired downstream of phosphorylation when cells are treated with IFN-α, at least at the time-point and under the conditions used here. The sustained capacity of cells to signal via P-STAT5 in the presence of IFN-α, despite reductions in P-AKT signaling, is

37 also consistent with reports by others that IFN-α impairs IL-7-induced T cell proliferation but does not inhibit P-STAT5 signaling in primary T cells (145).

Our results indicate that P-Akt can be inhibited by IFN-α when cells are activated by IL-7 or IL-2; however, we did not find evidence that IFN-α could inhibit P-Akt induced by SDF-1. This raises the possibility that IFN-α mediates its effects on signaling machinery that is not shared between SDF-1 and common γ-chain cytokines, such as IL-7 or IL-2. Possibilities include PI3K subunits, adaptor molecules, or perhaps receptor components that may be differentially regulated by IFN-α. For the latter possibility, we found no evidence, at least for CD127, that cytokine receptor expression was disrupted by IFN-α (not shown). Recent studies by others also indicate that IFN-α does not impair

CD127 expression in T cells and may actually enhance expression in long-term cultures

(145). The delay in P-Akt that occurs after IL-7 stimulation is consistent with the possibility that IL-7 may induce a secondary factor that leads to enhancement of the signal. This raises the possibility that IFN-α could inhibit the induction of this unknown secondary signal. Further studies that address these possibilities will be needed to clarify the mechanism of P-Akt inhibition by IFN-α.

The kinetics of Akt activation that we have defined here add to our understanding of IL-7- mediated signaling. Previous studies indicate that PI3K signaling is rapid in thymocytes exposed to IL-7 but delayed in mature T cells (75, 146). Studies in primary bulk T cells suggest that IL-7-induced P-Akt is delayed but detectable within 3–6 h after

IL-7 exposure (75). Our results suggest that these signals are delayed for 1–2 days but then sustained for several days after IL-7 stimulation. Although we have focused our

38 analyses on CD4+ T cells, we noted somewhat stronger but transient P-Akt expression in

CD4−CD3+ cells (mostly CD8+ T cells) at earlier time-points (not shown). This may explain why we find a longer delay than the previous studies that used bulk T cells for analyses. Alternatively, the studies used different methods of detection (Western blot in previous studies and flow cytometry in our study) that could also contribute to these differences (75). In either case, both studies confirm that IL-7-induced Akt signaling is delayed in mature T cells.

Our experiments also demonstrate a requirement for prolonged IL-7 exposure for optimal induction of P-Akt (Fig. 2.6D). This observation may have important implications for mechanisms of homeostatic proliferation. Although it is difficult to extrapolate in vitro observations to in vivo conditions, our findings suggest that to induce homeostatic division, T cells likely need to experience sustained high levels of systemic

IL-7 exposure or dwell for prolonged periods close to an IL-7 source, such as the fibroblastic reticular network within lymph nodes or stromal cells of the bone marrow. It is interesting to speculate that T cells with the potential to arrest their migration within

IL-7-producing niches may be more likely to undergo homeostatic proliferation in vivo and that cells may alter their migratory properties in lymphopenic conditions to promote their exposure to IL-7.

Our observations also have implications for HIV disease where sustained IFN-α exposure has been implicated in pathogenesis. In particular, IFN-α is increased in plasmas of HIV-infected persons, and IFN-α levels are correlated directly with plasma viremia and inversely with CD4 cell counts (92). Furthermore, gene signatures indicative

39 of type I IFN activation are observed in CD4+ T cells from HIV patients who experience relatively poor CD4 T cell reconstitution during the administration of antiretroviral therapy (90). Thus, the antiproliferative activity of type I IFN and particularly, the impairment of IL-7 responsiveness could conceivably play an important role in limiting T cell reconstitution in HIV-infected persons receiving antiretroviral therapy or in persons with uncontrolled, chronic disease. Although strategies for neutralizing IFN are being considered to ameliorate the immunopathology of chronic, treated HIV infection, an alternative strategy may be to circumvent the antiproliferative activity of this cytokine while leaving antiviral and innate-immune activities intact.

The following chapter contains work published in the AIDS on August 24, 2016, volume

30, issue 13, pages 2033-2042. The authors were Thao P. Nguyen, Supriya Shukla,

Robert Asaad, Michael L. Freeman, Michael M. Lederman, Clifford V. Harding and

Scott F. Sieg. Copyright permission letter was not required to reuse this published work from AIDS.

Chapter 3: Responsiveness to IL-7 but not to IFN-α is diminished in CD4+ T cells from treated HIV infected patients who experience poor CD4+ T-cell recovery.

Abstract

Objective: To assess CD4 T-cell responsiveness to IL-7 and IFN-α in HIV-infected patients who experience poor recovery of CD4 T-cell counts during therapy (immune failure patients).

Design: Responses to IL-7 and IFN-α were compared between HIV-infected immune failure (CD4 cell counts <379 cells/μl) patients and immune success (CD4 cell counts

>500 cells/μl) as well as healthy control patients.

Methods: Flow cytometry was used to assess peripheral blood mononuclear cells for IL-

7-induced proliferation, CD25 expression, and signaling (signal transducer and activator of transcription 5 phosphorylation and Akt phosphorylation) in CD4 T cells. Freshly isolated cells were characterized by expression of IL-7Rα (CD127) among CD4 T-cell maturation subsets by flow cytometry and sorted CD3 T cells were assessed for expression of IFN-α and interferon stimulated genes (2'-5'-oligoadenylate synthetase-1 and myxovirus resistance A protein) by quantitative real-time PCR. Responses to IFN-α were assessed by induction of signal transducer and activator of transcription 1 phosphorylation and inhibition of IL-7-induced CD4 T-cell proliferation.

Results: IL-7-induced proliferation and CD25 expression were decreased in CD4 T cells from immune failure patients. CD127 expressing CD4 T cells were decreased, whereas expression of 2'-5'-oligoadenylate synthetase-1, myxovirus resistance A protein, and IFN-

α mRNA were increased in total CD3 T cells from immune failure patients. CD127 expression correlated with CD25 induction but not proliferation, whereas T-cell IFN-α mRNA was associated with reduced proliferation in CD4 T cells from immune failure patients. IFN-α-mediated induction of signal transducer and activator of transcription 1 phosphorylation and inhibition of proliferation were not diminished in CD4 T cells from immune failure patients.

Conclusion: IL-7 responsiveness is impaired in immune failure patients and may be related to expression of CD127 and IFN-α.

Introduction

Up to 30% of HIV-1-infected individuals receiving ART fail to recover CD4+

T cells counts to normal levels despite sustained HIV suppression (147). Such patients are referred to as immunologic nonresponders or immune failures. Persistently low CD4+

T-cell counts are associated with increased risk of non-AIDS-related morbidities (59, 60,

148). One mechanism that may contribute to poor CD4+ cell count recovery during ART is chronic immune activation as suggested by studies linking immune failure to increased

CD4+ T-cell activation and cycling as well increased plasma markers of inflammation

(sCD14 and IL-6) (53). Furthermore, increased expression of ISGs has been observed in

T cells from immune failure patients (90). Although chronic IFN-I expression has been implicated in models of HIV/simian immunodeficiency viruse (SIV) pathogenesis (92,

138, 149), it is not known whether IFN-I contributes to impaired CD4+ cell count recovery in treated patients.

Recovery of T cells in lymphopenic conditions is mediated by thymic output and homeostatic proliferation in response to IL-7 (71, 150). Ligation of the IL-7 receptor, which comprises CD127 and CD132, transmits signals that promote CD4+ T-cell survival and proliferation (71, 151, 152). IL-7-induced signal transducer and activator of transcription 5 phosphorylation (P-STAT5) results in the rapid induction of an antiapoptotic protein, bcl-2, and upregulation of IL-2Rα (CD25) (143). IL-7-induced Akt phosphorylation (P-Akt) is linked to cell cycle progression and T-cell proliferation (131,

133). Plasma IL-7 levels in immune failure patients are increased when compared with

IL-7 levels in patients who recovered CD4+ T cells (153). In contrast, the capacity of

CD4+ T cells to respond to IL-7 stimulation as measured by rapid induction of P-STAT5 is diminished in immune failure patients and associated with reduced expression of

CD127 (88). Thus, diminished IL-7 responsiveness may occur in CD4+ T cells of immune failure patients, impairing recovery. We consider the possibility that persistent

IFN-I expression could contribute to poor IL-7 responsiveness in immune failure patients.

We have demonstrated that P-Akt induction is delayed following exposure of peripheral

CD4+ T cells to IL-7 and that IFN-I impairs this signaling activity (154). Furthermore,

IFN-α inhibits CD4+ T-cell proliferation responses to IL-7 (145, 154). Thus, it is possible that IFN-[alpha] produced in HIV disease diminishes homeostatic proliferation in response to IL-7 and plays a role in poor CD4+ T-cell recovery.

We compared IL-7 responsiveness in CD4+ T cells from immune failure, immune success (persons who experience CD4+ cell count recovery over 500 cells/µl

44 during ART), and healthy control patients. We assessed CD127 protein expression as well as IFN-α mRNA and ISGs [2'–5'-oligoadenylate synthetase-1 (OAS1) and myxovirus resistance A protein (MxA)] mRNA expression to determine if IFN-I expression and exposure are related to IL-7 responsiveness. In addition, we evaluated the capacity of T cells from immune failure patients to respond to IFN-α stimulation as IFN-I tolerance has been reported in HIV disease (135). Our results suggest that IFN-α responsiveness is largely maintained, whereas IL-7 responsiveness is impaired in T cells from immune failure patients. Furthermore, our data suggest that IFN-α may play a role in mediating poor IL-7 responsiveness in T cells from immune failure patients.

Methods

Patients

The work was approved by the institutional review board at University Hospitals. After obtaining written informed consent, blood was drawn into heparin tubes. Immune failure patients were selected on the basis of past clinical history of CD4+ cell counts 350 cells/µl or less with treatment. As participation was based on past medical history, some patients had achieved slightly higher CD4+ cell counts at the time of blood draw, with no immune failure patients exceeding 378 cells/µl as indicated in Table 1. All immune success patients had achieved CD4+ cell counts more than 500 cells/µl leading up to the study. All HIV-infected donors had plasma HIV RNA below detection limits (typically

50 copies/ml) for at least 2 year leading up to the study. Both CD4+ cell count nadir and median age of the patients, which are recognized risk factors for immune failure, were not different between immune failure and immune success or healthy control (Table 1).

Peripheral blood mononuclear cell and T-cell isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by centrifugation over a Ficoll-Hypaque cushion. For T-cell isolation, the pan T-cell isolation kit (Miltenyi) was used resulting in a mean purity of 94%, as assessed by flow cytometric analysis. mRNA analyses

RNA was prepared from cell lysates using the RNeasy Plus Mini Kit (Qiagen,

Valencia, California, USA). Total RNA was reverse transcribed into cDNA using the

QuantiTect Reverse Transcription Kit (Qiagen). Equal quantities of cDNA for each experimental condition were amplified by real-time PCR using iQ SYBR Green

Supermix (Bio-Rad, Hercules, California, USA). Primer pairs used for genes encoding for GAPDH, OAS1, and MxA were previously published (135), including IFN-α (92).

Samples were amplified using a hot start at 95 °C for 3 min, followed by 50 cycles of 10 s at 95 °C, 10 s at 59 °C, and 30 s at 72 °C, and a postamplification melting curve ramping from 65 to 95 °C in increments of 0.5 °C per 5 s. Abundances of transcripts were calculated relative to Gapdh expression using the formula 2-[Ct(targetgene)-Ct(Gapdh)].

Stimulation conditions

To assess proliferation, PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE; 0.25 mmol/l at 37 °C for 10 min; Molecular Probes Invitrogen,

Grand Island, New York, USA). Staining was quenched with fetal bovine serum (FBS) for 5 min on ice, and then cells were washed with Roswell Park Memorial Institute 1640 medium (10% FBS). PBMCs were resuspended in an X-VIVO 15 serum-free medium supplemented with 1% penicillin streptomycin and incubated at a concentration of 1 million cells/ml ± rIL-7 (Cytheris; 5 ng/ml) ± IFN-[alpha] (PBL Assay Science,

Piscataway, New Jersey, USA; 500 U/ml).

Flow cytometry

Freshly isolated PBMCs were incubated with fluorochome-labeled monoclonal antibodies for 30 min: anti-CD3 peridinin chorophyll protein (PerCP), anti-CD45RA phycoerythrin-cyanine 7 (PE-Cy7), anti-CD8 allophycocyanin-cyanine (APC-Cy7), anti-

CD27 alexafluor-700 (AF-700), anti-CD127 fluorescein isothiocyanate (FITC), anti-

CD38 phycoerythrin-cyanine 5 (PE-Cy5) (all from BD Bioscience, San Jose, California,

USA), and anti-CD4 pacific blue (Biolegend, San Diego, California, USA). Cells were washed, fixed, and permeabilized with 2× perm/wash buffer (BD Bioscience) and incubated with anti-Ki-67 APC for 45 min. For proliferation assays, CFSE-stained

PBMCs were incubated with antibodies specific for CD3, CD4, and CD45RA following

47 incubation with Live/Dead Fixable Yellow Dead Cell dye (Invitrogen, Grand Island, New

York, USA). For apoptosis studies, cells were washed with cold PBS, resuspended in 1× binding buffer (BD Bioscience), and then incubated with annexin-V PE (BD Bioscience) at room temperature for 15 min. Intracellular detection of P-Akt and P-STAT5 was performed via the BD Phosflow protocol and incubated with the following fluorochome- labeled monoclonal antibodies for 30 min: anti-CD3, anti-CD4, anti-CD45RA, anti-

STAT5 (92) (pY649) APC, and anti-Akt (pS473) PE (BD Bioscience). Flow cytometric analyses were performed with FlowJo software (FlowJo LLC, Ashland, Oregon, USA).

Doublets, debris, and dead cells were excluded from analyses.

Statistical analyses

Prism 5 software (Graphpad, La Jolla, California, USA) was used to generate figures and perform statistical analyses. Nonparametric tests (Kruskal–Wallis and Dunn's multiple comparison post test) were used to assess differences between immune failure and immune success or healthy control. Wilcoxon signed rank test was used to assess differences in paired data. All the tests were 2-sided with a significance P value cutoff of

0.05.

Results

IL-7 mediated proliferation and induction of CD25 are diminished in CD4+ T cells from immune failure patients

Figure 3.1 CD4+ T-cell responses to IL-7 are diminished in immune failure patients. (a)–(d) Peripheral blood mononuclear cells were labeled with CFSE and incubated in the presence or absence of rIL-7. After 7 days, cells were assessed by flow cytometry. (a) The gating sequence is provided. Doublets were excluded from all analysis by the forward scatter area (FSC-A) and forward scatter height (FSC-H) gate and lymphocytes were identified by forward and side scatter. (b) Representative histograms showing

49 proliferation response by %CSFE low cell population in CD4+ cell counts. (c) Summary data of proliferation response in CD4+ cell counts (immune failure n=11, immune success n=10, and healthy control n=9) as well as CD45RA+ and CD45RA-CD4+ cell count subsets. (d) Summary data showing % divided and proliferation index in the CD45RA subset in cells from patients that were analyzed in part c. (e and f) Peripheral blood mononuclear cells were incubated in the presence or absence of IL-7. After 24h, cells were assessed for surface CD25 expression by flow cytometry. (e) Representative histograms showing CD25 expression in IL-7 treated CD4+T cells in comparison with untreated and isotype control. (f) Summary data of % IL-7 induced CD25 cells in CD4 T cells (immune failure n=11, immune success n=12, and healthy control n=9) and maturation subsets (naive defined by CD45RA+ CD27+, central memory defined by CD45RA-CD27+ and effector memory defined by CD45RA-CD27-). %IL-7 induced CD25 is the difference in CD25 expression between IL-7 treated and untreated. HC, healthy control; IF, immune failure; IS, immune success.

To test IL-7 responsiveness in CD4+ T cells, PBMCs were stimulated with rIL-7 and proliferation (CFSE dye dilution at day 7), cell signaling (P-STAT5 and P-Akt at day

3), and cell surface induction of CD25 expression (at day 1) were measured in CD4+ T cells by flow cytometry. Total CD4+ T cells from immune failure but not immune success patients exhibited diminished proliferation (%CFSE low) in response to rIL-7 compared with cells from healthy control (Fig. 3.1A-C). CD4+ T cells incubated in medium alone had median %CFSE low values of less than 1%, and these median values were not significantly different between the groups (data not shown). Furthermore, CD3+CD4- T cells from immune failure patients also displayed diminished proliferation (% CFSE low) in response to IL-7 stimulation when compared with cells from immune success and healthy control patients and diminished proliferation indices and % divided cells when compared with cells from healthy control (Fig. 3.2).

Figure 3.2 CD4- T cell proliferation in response to IL-7 is diminished in IF subjects. (A) %CFSE low (IF n=11, IS n=11, HC n=9). (B) %Divided (IF n=11; IS n=10; HC n=9). (C) Proliferation index (IF n=11; IS n=10; HC n=9). Data shown are CFSE dye dilution response to IL-7 when gating in CD4- cells under the experimental conditions described in Figure 14. Abbreviations: immune failure, IF; immune success, IS; healthy control, HC.

We also assessed proliferation responses among CD4+ T-cell maturation subsets.

Proliferation responses were diminished among the memory (CD45RA-) CD4+ T cells, and a similar trend that was not statistically significant was observed in naïve

(CD45RA+) CD4+ T cells (P = 0.059) from immune failure compared with cells from controls (Fig. 3.1D). To further understand the proliferation defect in the CD4+ cell counts, we calculated the average number of divisions per divided cell (proliferation index) and the proportion of precursor cells that divided at least once (% divided). Both indices were diminished in the memory (CD45RA-) subset of immune failure patients compared with healthy control (Fig. 3.1D). The proliferation index and % divided in the total CD4+ and CD45RA+ subset and were not statistically different between patient groups (data not shown). Consistent with the above findings, absolute numbers of CD4+

T cells at the end of the cell culture also suggested that T cells from control patients underwent greater expansion in response to IL-7 than cells from immune failure patients

(data not shown).

CD4+ T-cell proliferation in response to IL-7 stimulation is dependent on cell signaling mediated by P-STAT5 and P-Akt (155). Therefore, we assessed IL-7-induced P-STAT5 and P-Akt following 3 days of stimulation. P-STAT5 and P-Akt levels in CD4+ and CD4-

T cells following IL-7 stimulation were not different between immune failure and immune success or healthy control patients (Fig. 3.3).

Fig. 3.3 Induction of P-STAT5 and P-Akt in response to IL-7. PBMCs were incubated in the presence or absence of IL-7. After 3 days, cells were assessed for P-STAT5 and P- Akt by flow cytometry. (A) Graphs show responses in CD4+ (%P-STAT5+ IF n=12, IS n=12, HC n=9; %P-Akt+ IF n=13, IS n=12, HC n=9) and (B) shows CD4- T cells (%P- STAT5+ IF n=11, IS n=12, HC n=9; %P-Akt+ IF n=12, IS n=12, HC n=9). Abbreviations: immune failure, IF; immune success, IS; healthy control, HC.

IL-7-induced P-STAT5 results in up-regulation of CD25 expression (143). We compared IL-7 induced cell surface CD25 expression between immune failure, immune

52 success, and healthy control patients. PBMCs isolated from patients were incubated in the presence or absence of rIL-7 overnight. Surface expression of CD25 was measured by flow cytometry after gating on CD3+CD4+ T cells (Fig. 3.1E). CD25 induction in response to IL-7 was diminished in total CD4+ T cells and naive subset from immune failure patients. Among memory CD4+ T cells, induction of CD25 expression by IL-7 was reduced in immune failure patients, particularly among the effector memory subset

(Fig. 3.1F).

CD4+ T cells from immune failure patients exhibit decreased CD127 expression and increased immune activation

Fig. 3.4 T cells from immune failure patients exhibit increased expression of interferon stimulated genes and IFN-α. (a) Freshly isolated peripheral blood mononuclear cells were examined by flow cytometry for percentages of CD127+ cells in total, naïve, central memory and effector memory CD3+CD4+ T cells. (b) and (c) T cells were purified from freshly isolated peripheral blood mononuclear cells and gene expression of interferon stimulated genes and IFN-α was analyzed by quantitative real-time PCR. (b) mRNA

53 expression of 2'–5'-oligoadenylate synthetase-1 and myxovirus resistance A protein (immune failure n = 10, immune success n = 9, and healthy control n = 9) interferon stimulated genes (c) IFN-α mRNA analyses (immune failure n = 9, immune success n = 11, healthy control n = 7). HC, healthy control; IF, immune failure; IS, immune success; MxA, myxovirus resistance A protein; OAS-1, 2'–5'-oligoadenylate synthetase-1

To characterize freshly isolated CD4+ T cells from immune failure patients and investigate underlying mechanisms that may contribute to poor IL-7 responses in immune failure patients, we measured immune activation (CD38 expression), cell cycling (Ki-67 expression), and IL-7 receptor-α (CD127 expression) as well as proportions of maturation subsets among the CD4+ T cells (Table 1 and Fig. 3.4A). Among the CD4+ T cells, immune failure patients had decreased proportions of naïve cells but increased proportions of effector memory cells when compared with healthy control patients. CD4+

T cells from immune failure patients displayed diminished expression of CD127 within memory T-cell subsets as measured by either proportion of CD127+ cells (Fig. 3.4A) or

MFI of CD127 expression (not shown). Compared with immune success patients, immune failure patients had increased proportions of activated memory T cells (Table 1).

Furthermore, immune failure patients had increased proportions of cycling cells comparing immune failure with healthy control (naïve, central memory, and effector memory) and comparing immune failure with immune success (central memory). These data are consistent with previous observations and suggest that CD4+ T cells from immune failure patients are activated and cycling and may be less responsive to IL-7 stimulation due to lower CD127 expression (53).

Interferon-stimulated gene and IFN-α mRNA expression are increased in CD3+ T cells from immune failure patients

We have previously reported the inhibitory effect of IFN-α on IL-7-induced proliferation (154). To address the possibility that T cells from immune failure patients were exposed to IFN-I in vivo, we measured ISG expression in T cells from our patients and found that mRNA expression of OAS1 and MxA were increased in CD3+ T cells from immune failure patients (Fig. 3.4B). To determine if T cells could be a source of

IFN-I, expression of IFN-α mRNA was also assessed. We found evidence of increased mRNA expression of IFN-α in T cells from immune failure patients (Fig. 3.4C).

Fig. 3.5 IL-7 induced CD25 expression is related to expression of CD127.(a) Relationship between CD127 expression and induction of CD25 in response to overnight IL-7 treatment. (b) Relationship between CD127 expression and proliferation (percentage of carboxyfluorescein succinimidyl ester) in response to 7 days of IL-7 stimulation. Open symbols represent immune success patients and closed symbols represent immune failure patients. HC, healthy control; IF, immune failure; IS, immune success.

Diminished IL-7 responsiveness in immune failure patients is related to expression of CD127 and IFN-α

CD127 expression is a strong correlate of IL-7 responsiveness (P-STAT5 induction) in cells from immune failure patients (88). Therefore, we performed correlation analyses between CD127 expression and IL-7 responsiveness (CD25 induction and proliferation responses). We assessed these relationships using all HIV- infected patients to provide greater analytic power and then examined the relationships in immune failure and immune success patients separately. CD25 induction in response to

IL-7 directly correlated with CD127 expression in the HIV-infected patients. When assessed in the immune failure or immune success patients independently, the relationships remained positive and trending but not significant (Fig. 3.5A). In contrast, proliferation responses to IL-7 stimulation were poorly correlated with CD127 expression

(Fig. 3.5B) suggesting that diminished CD127 expression may not be a key determinant for diminished proliferation responses observed in CD4+ T cells from immune failure patients. We also considered the possibility that IFN-[alpha] or ISG mRNA expression might be related to proliferation responses to IL-7 stimulation. ISG expression did not correlate with either IL-7-induced CD25 expression or proliferation response (data not shown), whereas IFN-α mRNA expression in total T cells was inversely correlated with proliferation in the HIV-infected patients (Spearman r = -0.6379, P = 0.0033, n = 18).

The relationship was not significant although trending in the same direction among immune failure patients (Spearman r = -0.4667, P = 0.2125, n = 9).

Fig. 3.6 CD4+ T- cell responses to IFN-α were not diminished in immune failure patients. Peripheral blood mononuclear cells from patients were incubated in the presence or absence of IFN-α. All data shown are gated from CD3+CD4+ cells. (a) Signal transducer and activator of transcription 1 (STAT1) phosphorylation induction following 15min of IFN-α treatment (immune failure n=12, immune success n=12, and healthy control n=9). (b)-(d) Peripheral blood mononuclear cells were CFSE stained and assessed for proliferation and cell death in cells treated with IL-7 in the absence or presence of IFN-a for 7 days. (b) Compares percentages of CFSE low (immune failure n=10, immune success n=11, and healthy control n=9) and (c) are representative dot plots comparing cell death as measured by annexin-V binding CD4+ T cells that were not excluded by viability dye. (d) Summary data of cell death in divided cells (CFSE low) and undivided cells (CFSE high) (immune failure=10, immune success n=8, and healthy

57 control n=9). P values were obtained by Wilcoxon signed rank test. HC, healthy control; IF, immune failure; IS, immune success.

IFN-α responsiveness is not diminished CD4+ T cells from immune failure patients

Previous studies suggest that exposure to IFN-I in vivo during HIV or SIV infection may lead to tolerance and reduced IFN-I responsiveness (135, 149). To assess

IFN-α responsiveness in CD4+ T cells, we measured both the rapid induction of P-

STAT1 in freshly isolated PBMCs and the capacity of IFN-α to inhibit T-cell proliferation in cells from all patients. We found that IFN-α-induced P-STAT1 levels were not different between the patients (Fig. 3.6A). IFN-α significantly inhibited IL-7- induced proliferation in CD4+ T cells from all patient groups (Fig. 3.6B). The magnitude of IFN-α-mediated inhibition of IL-7-induced proliferation was also similar between groups (median % inhibition equal 55, 53, and 51 for immune failure, immune success, and healthy control, respectively). As IFN-Is have also been implicated in the induction of apoptosis in HIV disease (137), we also assessed cell death by annexin-V binding following IFN-α, IL-7, or IL-7 + IFN-α simulation. Cells incubated in IFN-α alone did not proliferate above background and did not reveal significant differences in cell death between the patient groups (median percentages of CD4+ T cells that were annexin V- bound was 7.3, 6.1, and 8.1 for immune failure, immune success, and healthy control patient groups, respectively; P = 0.79). Interestingly, when comparing cells incubated with IL-7 to cells incubated with IL-7 + IFN-α, we found an increase in cell death in the presence of IFN-α among divided (CFSE low) cells but not undivided (CFSE high) cells from immune failure but not immune success or healthy control patients (Fig. 3.6C and

D). Subset analyses of CD45RA+ and CD45RA- cells indicated that adding IFN-α to IL-

7-treated cells resulted in significant increases in cell death of divided cells within the naive-enriched (CD45RA+) T-cell subset of immune failure patients (Fig.3.7).

Fig. 3.7 IFN-α mediated induction of cell death in CD4 T cell subsets. Maturation subsets were based on (A) CD45RA+ (naive) and (B) CD45RA- (memory). Summary data of cell death in divided cells (CFSE low) and undivided cells (CFSE high) (IF n=10, IS n=8, HC n=9). P-values were obtained by Wilcoxon signed rank test. Abbreviations: immune failure, IF; immune success, IS; healthy control, HC.

Discussion

IL-7 responsiveness is important for the recovery of CD4+ T cells in ART-treated patients as suggested by evidence linking the rate of CD4+ recovery to CD127 polymorphisms (156). The importance of IL-7 responsiveness is also raised by studies

59 showing that CD4+ T cells from immune failure patients respond poorly to IL-7 stimulation as measured by rapid induction of P-STAT5 signaling (88). In addition to diminished IL-7 responsiveness, it is also possible that fibrosis of lymphoid tissues further contributes to poor CD4+ cell count recovery during ART by limiting access to

IL-7 within reticular networks (41). Thus, both limited access to IL-7 and limited capacity to respond to IL-7 stimulation may contribute to poor CD4+ cell count recovery in treated HIV.

Here, we describe additional impairments in IL-7 responsiveness in CD4+ T cells from immune failure patients that are characterized by diminished induction of proliferation over a 7-day stimulation and diminished induction of CD25 after overnight stimulation. Similar to the previous reports of diminished P-STAT5 signaling in T cells from immune failure patients (88), we found a direct relationship between induction of

CD25 and levels of CD127 expression among CD4+ T cells from HIV-infected patients.

These data and the observations that there are increased proportion of CD57+ ‘senescent’

CD4+ T cells in immune failure patients (88) point to a mechanism whereby the accumulation of cells poorly equipped to proliferate in response to IL-7 stimulation may contribute to poor CD4+ recovery in treated HIV disease. Unlike the previously reported observation of a defect in IL-7-induced P-STAT5 in CD4+ T cells from immune failure patients (88), we did not find evidence of diminished P-STAT5 signaling in CD4+ T cells from immune failure patients. This may be related to differences in approach as we assessed P-STAT5 following 3 days of IL-7 stimulation compared with 15 min in the previous work. We examined the later time point to allow a comparison between P-

STAT5 signaling and Akt signaling since Akt signaling is consistently measurable after 3

60 days but not after 15 min of IL-7 stimulation (154). It is possible that early P-STAT5 signaling defects in immune failure are transient, or alternatively, cells that fail to induce

P-STAT5 signals early are lost over a few days of culture.

Consistent with previous observations (88), we found that CD127 expression was diminished in T cells from immune failure patients, and other studies indicate that IL-7 is increased in plasma of immune failure patients (153). It is possible, therefore, that reduced CD127 expression is a consequence of increased chronic exposure of T cells to high concentrations of IL-7 in vivo leading to IL-7 hyporesponsiveness. Although CD127 expression correlated with CD25 induction mediated by IL-7, it was a poor predictor of

T-cell proliferation responses to IL-7 stimulation. This suggests that the mechanism of impaired IL-7-induced proliferation in CD4+ T cells from immune failure patients is likely to be complex and not simply a reflection of reduced CD127 expression.

We also confirm a previous report that T cells from immune failure patients display increased ISG mRNA expression (90) and add that these cells also express increased IFN-α mRNA. Notably, the analyses of ISGs and IFN-α mRNA were limited to total CD3+ T cells. Thus, determining the contribution of CD4+ and CD8+ T cells as well as the contribution of memory and naïve cell subsets to IFN-α mRNA will be important in future studies. Although T cells are not necessarily recognized as a predominant producer of IFN-I, previous studies in HIV+ patients indicate that various cell types, including T cells, can contribute to IFN-α production (157). It is possible that a small contaminating population of cells may have accounted for the IFN-α mRNA signal in our assays; however, we did not observe a relationship between the purity of the sample and the expression levels of IFN-α mRNA (data not shown). Although limited by a small

61 sample size, the increased IFN-α mRNA signal observed in T cells from immune failure patients and the trending inverse relationship with T-cell proliferation responses to IL-7 leads us to speculate that autocrine IFN-α production could contribute to poor IL-7 responsiveness in T cells from immune failure patients. Interestingly, this relationship may reflect an indirect mechanism because there was no discernible relationship between

ISG expression and IL-7-induced proliferation. Further studies will be important to confirm and expand on these observations.

Increased levels of ISG and IFN-α mRNA expression in T cells from immune failure patients suggest that IFN-I exposure may be sustained in immune failure. In certain cell types, chronic exposure to IFN-I in vivo can lead to IFN-I tolerance. For example, monocytes from untreated HIV infected but not healthy control patients are refractory to IFN-α2a-mediated induction of OAS and MxA presumably due to chronic

IFN-I exposure during HIV infection (135). Diminished expression of ISGs also has been observed following IFN-α2a prolonged administration in rhesus macaques (149). Our data suggest that IFN-α responsiveness as measured by P-STAT1 or inhibition of IL-7- induced proliferation was not impaired in CD4+ T cells from immune failure patients.

Furthermore, CD4+ T cells from immune failure patients that have divided in response to

IL-7 appear more susceptible to IFN-α-enhanced cell death, revealing a potential mechanism that could limit homeostatic recovery of CD4+ T cells in these patients.

The following chapter contains unpublished work and copyright is owned by the journal

and licensed content publisher that will publish this work in the future.

Chapter 4: TGF-β inhibits IL-7-induced proliferation in memory but not naive CD4 T cells

Abstract

TGF-β is a potent suppressor of T cell activation and expansion. Although the anti-proliferative effects of TGF-β are well-characterized in TCR-activated cells, the effects of TGF-β on T cell proliferation driven by homeostatic cytokines such as IL-7 are poorly defined. Here, we find that TGF-β inhibits IL-7-induced proliferation in memory, but not naïve, CD4+ T cells. TGF-β caused an impairment in c-myc induction that was relatively less well sustained in naïve than in memory CD4+ T cells. TGF-β had no discernible effect on IL-7R signaling (P-STAT5, P-Akt or P-S6) in memory T cells but selectively enhanced P-S6 signaling in naïve T cells. The inhibitory effects of TGF-β on memory T cell proliferation were partially overcome by chemical inhibition of GSK-3, which also led to reversal of c-myc expression inhibition in these cells. These data suggest that TGF-β could play an important role in limiting homeostatic proliferation of memory T cells while sustaining homeostatic proliferation of naïve T cells. Our observations also point toward a novel strategy to subvert TGF-β-mediated inhibition of memory T cells by targeting GSK-3 for inhibition.

Introduction

TGF-β1 is a cytokine that affects various cellular activities including proliferation, differentiation and apoptosis (158). TGF-β has well-defined immunosuppressive effects on the immune system (114, 159-163) and is therefore important in the protection from immunopathology. Deficiencies in TGF-β receptor expression can lead to T cell hyper-activation (161, 164), while inhibition of TGF-β

64 during parasitic infections may lead to poorly controlled immunopathology (165).

Overexpression of TGF-β can also be detrimental in certain circumstances, as TGF-β expression has been linked to susceptibility to certain parasites (166) and to impaired immune responses in the tumor microenvironment (167). Thus, an appropriate balance in

T cell exposure to TGF-β is important for efficient immune responses and homeostasis.

In order to mediate complex biological effects, TGF-β signals through its receptor to activate Smad 2 and 3. Activated Smad2/3 binds to Smad4 and this complex translocates to the nucleus to regulate gene transcription (168, 169). Cellular responses to TGF-β are complex and may be determined by expression of cell type-specific co-factors (170-

172), TGF-β receptors, signaling pathway regulators, and by epigenetic mechanisms

(173). While there are multiple levels of complexity by which TGF-β affects cell responses, various studies have defined TGF-β signaling through smad3 as a potent suppressor of proliferation in epithelial cells, keratinocytes and TCR-activated T cells

(118, 174, 175). In epithelial cells and keratinocytes, TGF-β arrests cell cycle at the G1 to

S phase transition by down-regulating c-myc expression (176) which is a key determinant in cell cycle progression (78, 79). In TCR-activated T cells, TGF-β suppresses the expression of metabolic genes and diminished mTORC1 mediated S6 kinase activity and also may mediate smad-independent mechanisms to impair proliferation (118). Early studies also suggested that TGF-β could impair Jak/STAT signaling in T cells stimulated with IL-2 (177); however, this observation was later disputed (178). Collectively, these studies suggest that TGF-β inhibits the induction of c-myc expression and the cellular metabolic program to impair cellular proliferation.

Despite our understanding of the activity of TGF-β in TCR-activated cells, the potential importance of this cytokine in T cells responding to homoeostatic cytokines such as IL-7 is poorly defined. Interestingly, recent studies in mice with deficiencies in

TGF-βR demonstrated that these mice suffered from a loss of naïve T cells and a corresponding decrease in sensitivity to IL-7. This observation raises the possibility that

TGF-β may have differential effects on naïve and memory T cells, particularly in the context of IL-7 stimulation. Since IL-7 is a critical mediator of T cell restoration in lymphopenic conditions and has a non-redundant role in sustaining T cell numbers in the periphery (71), the potential interactions of TGF-β with IL-7 represent an important consideration in T cell homeostasis.

IL-7 signals through the IL-7 receptor, comprised of IL-7 receptor α (IL-7Rα or

CD127) and the common gamma chain (γc or CD132) (151). IL-7 receptor engagement activates Jak1 and 3, leading to STAT5 phosphorylation and subsequent expression of genes promoting cell survival (179). IL-7 induces additional signaling through PI3K that is associated with delayed phosphorylation of Akt (133, 154) and S6 (77), the 40s ribosomal subunit. Phosphorylation of S6 is associated with increased protein translation

(180) while P-Akt inactivates the negative regulator of proliferation, glycogen synthase kinase-3 (GSK-3) (81). In resting cells, GSK-3 is a constitutively active kinase that interacts with many kinases (181) and promotes the degradation of c-myc (182). Thus,

IL-7 promotes c-myc expression and global protein translation to drive cellular proliferation.

In this study, we have examined the effects of TGF-β on human naïve and memory T cell proliferation that is induced by IL-7. We find that TGF-β inhibits IL-7-

66 induced memory, but not naïve T cell proliferation. We also uncover differential effects of TGF-β on the duration of c-myc inhibition and S6 signaling in IL-7-stimulated memory and naïve T cells. Furthermore, we find that chemical inhibition of GSK-3 at least partially overcomes TGF-β-mediated inhibition of c-myc expression and proliferation in IL-7-stimulated memory T cells. Overall, our studies highlight striking differences in the effects of TGF-β on IL-7 responses in naïve and memory T cells, suggesting that TGF-β may play a differential role in the homeostasis of these cellular subsets.

Methods

Peripheral blood mononuclear cell and CD4 T cell subset isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by centrifugation over a Ficoll-Hypague cushion. For naive CD4 T cell isolation, naive

CD4+ T cell isolation kit II (Miltenyi Biotec, San Diego, CA, US), human was used. For memory CD4 T cell isolation, the central memory CD4+ T cell isolation kit was used for magnetic labeling and separation of non-CD4+ T cells and naive CD4+ T cells. Purity of the sorted naive and memory CD4 T cells was assessed by flow cytometry.

Stimulation conditions

To assess proliferation, PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE; 0.25mmol/l for 37 C for 10 min; Molecular Probes Invitrogen,

Grand Island, New York, USA). Staining was quenched with fetal bovine serum (FBS) for 5 min on ice, and then cells were washed with Roswell Park Memorial Institute 1640

(RPMI 1640). PBMCs were resuspended in an X-VIVO 15 serum-free medium (Lonza,

Basel, Switzerland) supplemented with 1% penicillin streptomycin and incubated at a concentration of 1 million cells/mL +/- rIL-7 (5ng/mL; Cytheris) +/- rTGF-β1 (5ng/mL;

R&D systems). In experiments testing inactivation of GSK-3, small molecule inhibitor,

CH 99021 (Axon Medchem) were stimulated with rIL-7+/-rTGF-β1 at described concentrations in the Results section.

Flow Cytometry

Freshly isolated peripheral blood mononuclear cells were sorted for naive and memory CD4 T cells, which were incubated with the following fluorochrome labeled monoclonal antibodies for 30 minutes: anti-CD3 peridinin chorophyll protein (PerCP), anti-CD45RA phycoerythrin-cyanine 7 (PE-Cy7), anti-CD27 alexafluor-700 (AF-700)

(all from BD Bioscience, San Jose, California) and anti-CD4 pacific blue (PB) (from

Biolegend).

To measure proliferation and c-myc expression, CFSE-labeled PBMCs were incubated with fluorochome labeled monoclonal antibodies anti-CD3 brilliant violet 711

(BV 711), CD45RA PE-Cy7, CD27 PerCP (all from BD Bioscience) and CD4 PB (from

Biolegend). Cells were washed, fixed, and permeabilized with 2x perm/wash buffer (from

BD Bioscience) and incubated with anti-hc-Myc AF-700 (from R&D Systems, Inc,

Minneapolis, MN, US) overnight. Following intracellular staining, cells were washed with 2x perm/wash buffer and assessed by flow cytometry. To assess P-S6, P-Akt and P-

STAT5, cells were fixed with fixation buffer at 37 degrees C for 10 min and permeabilized with perm III buffer at 4 degrees C for 30 min before labeling with anti-

CD3 PerCP, anti-CD45RA PE-Cy7, anti-CD27 FITC, anti-Akt (pS473), anti-STAT5

(pY694) (all from BD Bioscience), anti-CD4 pacific blue (Biolegend), anti-S6 pS240-

APC (Miltenyi Biotec, San Diego, CA, US) for 30 min at 25 degrees C.

Statistical analyses

Prism 5 software (Graphpad, La Jolla, California, USA) was used to generate figures and perform statistical analyses. The nonparametric paired test, Wilcoxon matched-pairs signed rank test was used to compare responses to IL-7 in the presence or absence of TGF-β. To compare % TGF-β mediated inhibition of c-myc expression and proliferation in response to IL-7 among the CD4 T cell maturation subsets, the Kruskal-

Wallis T test was used and followed by the post test, Dunn's multiple comparison test. All tests were 2-sided with a significant P value cutoff of 0.05.

Results

Figure 4.1 TGF-β differentially affects naive and memory CD4 T cell proliferation in response to IL-7. (A-C) Peripheral blood mononuclear cells were labeled with CFSE and incubated with rIL-7 (5ng/mL) in the presence or absence of rTGF-β1 (5ng/mL). After 7 days, cells were analyzed by flow cytometry for proliferation or CFSE dilution as measured by %CFSE low. (A) The gating sequence is provided. Doublets were excluded from analysis by the forward scatter area (FSC-A) and forward scatter height (FSC-H) gate and lymphocytes were identified by forward and side scatter. (B) The representative data shows %CFSE low in CD4 T cell maturation subsets (naive; central memory, CM; effector memory, EM). (C) Summary data show % CFSE low in CD4 T cell maturation subsets (n=8). Additional PBMCs were used to isolate naive and memory CD4 T cells. Purified naive or memory CD4 T cells were labeled with CFSE and stimulated with rIL-7 (10ng/mL) in the presence or absence of rTGF-β1 (5ng/mL). After 9 days, cells were analyzed by flow cytometry for %CFSE low. (D) Shows the summary data (n=3 for all subsets)in purified naive and memory CD4 T cells. CD4 maturation subsets were defined by expression of CD45RA and CD27 (naive defined by CD45RA+CD27+, CM defined by CD45RA-CD27+ and EM defined by CD45RA-CD27-). Significant difference between % CFSE low response in rIL-7 in the presence or absence of rTGF-β1 was assessed by Wilcoxon matched-paired signed rank test.

TGF-β inhibits memory but not naive CD4 T cell proliferation in response to IL-7

To characterize the effects of TGF-β on naive and memory CD4 T cell proliferation in response to IL-7, we labeled PBMC with CFSE tracking dye and stimulated these cells with rIL-7 in the presence or absence of TGF-β. We used flow cytometric analysis to gate on cells with naïve (CD45RA+CD27+), central memory (CM;

CD45RA-CD27+) and effector memory (EM; CD45RA-CD27-) phenotype 7 days post

IL-7 stimulation and assessed the percentage of each population that had diluted CFSE tracking dye as a measure of cellular proliferation (Fig. 4.1). Median % TGF-β-mediated inhibition of proliferation was 50% and 62% in CM and EM CD4 T cells, respectively. In contrast, TGF-β did not inhibit naïve T cell proliferation. Expression of CD45RA and

CD27 on CD4 T cell subsets did not appear to be changing during these cultures. To further characterize the inhibitory effect of TGF-β on memory CD4 T cell proliferation, we used Flow Jo analytical software to calculate the proportion of precursor cells that divided at least once (% divided) and the average number of cell divisions among the

70 divided cells (proliferation index). TGF-β inhibited IL-7 induced % divided in the CM and EM subsets and significantly reduced the proliferation index in the EM CD4 T cell subset, which was also the subset where cells tended to undergo several rounds of replication (Fig. 4.1). These data suggest that TGF-β limits both efficiency of initial cell cycle progression and the capacity of proliferating cells to undergo multiple rounds of division in memory CD4+ T cells.

Figure 4.2 TGF-β inhibits proliferation in memory CD4 T cells. (A) Graphs show % divided and proliferation index data analyzed from figure 4.1C. Significant differences in % divided and proliferation index between rIL-7 and rIL-7+rTGF-β1 were assessed by Wilcoxon matched-paired signed rank test.

To determine if the inhibitory effect of TGF-β on IL-7-driven memory CD4 T cell proliferation was due to a direct effect on T cells, we assessed the effects of TGF-β on IL-

7 induced proliferation using negatively selected, purified naive or memory CD4 T cells.

Cell purity reached a minimum of 98.5% and 99.1% for naïve (CD4+CD45RA+CD27+) and memory (CD4+CD45RA-) T cells, respectively. We found that TGF-β inhibited proliferation in purified CD45RA- memory T cells that were further defined by CD27 expression (for example, CD45RA-CD27+ CM and CD45RA-CD27-EM cells) (Fig.

4.1D). In contrast, TGF-β did not inhibit and even had a tendency to enhance proliferation of purified naïve T cells stimulated with rIL-7.

Figure 4.3 TGF-β inhibits IL-7 mediated induction of c-myc expression in naive and memory CD4 T cells. Peripheral blood mononuclear cells were incubated with rIL-7 (5ng/mL) in the presence or absence of rTGF-β1(5ng/mL). After 3 or 5 days, cells were analyzed by flow cytometry for intracellular c-myc expression. (A) Representative graph shows relative cell size as measured by forward scatter (FSC) versus c-myc expression in CD4 maturation subsets at day 5. (B) Summary data show c-myc expression of CD4 T cell maturation subsets after 3 and 5 days of stimulation. (C) Summary data of TGF-β mediated inhibition of IL-7 induced c-myc expression in CD4 T cell maturation subsets after 3 and 5 days following stimulation. % Inhibition of IL-7 induced c-myc was calculated as the difference between c-myc expression in IL-7 stimulated cells and IL- 7+TGF-β stimulated cells, divided by c-myc expression in IL-7 stimulated cells,

72 multiplied by 100. Significant difference between % c-myc in rIL-7 in the presence or absence of rTGF-β1 was assessed by Wilcoxon matched-paired signed rank test. Significant differences in % TGF-β mediated inhibition of IL-7 induced c-myc expression between CD4 T cells subsets were assessed by Dunn's multiple comparison test.

TGF-β suppresses IL-7 mediated induction of c-myc expression in naive and memory CD4 T cells

In keratinocytes and epithelial cells, TGF-β has been found to inhibit cell proliferation in a smad3-dependent manner by suppressing c-myc transcription (175). To test whether IL-7 induced c-myc expression and if TGF-β mediated inhibition of c-myc induction in T cells, we stimulated PBMC with IL-7 ± TGF-β and measured intracellular c-myc protein by flow cytometry. In preliminary studies, we found evidence that IL-7 mediated up-regulation of c-myc expression in CD4 T cells within 3-5 days of stimulation (not shown). At day 5 post-stimulation, IL-7 treatment caused some cells to increase in size (defined by increased forward scatter by flow cytometry) in the CD4 T cell maturation subsets and this corresponded to increased expression of c-myc. Both IL-

7 mediated c-myc up-regulation and increased cell size were diminished by TGF-β treatment in naive and memory CD4 T cell subsets (Fig. 4.3A&B). The magnitude of c- myc inhibition was compared among the T cell subsets at 3 and 5 days post stimulation with IL-7 + TGF-β. Whereas similar levels of inhibition were noted at 3 days post stimulation among the T cell subsets, naïve T cells tended to display a lesser degree of

TGF-β-mediated inhibition at 5 days post stimulation compared to memory cells and particularly compared to EM cells. This suggested that the relative inhibitory effect of

TGF-β on c-myc expression diminished more substantially over time in the naïve T cell subset (Fig. 4.3C). Nonetheless, TGF-β inhibited induction of IL-7-induced c-myc expression in all T cell subsets.

Figure 4.4 TGF-β does not inhibit IL-7 receptor signaling in memory CD4 T cells and enhances S6 phosphorylation in naïve CD4 T cells. PBMCs were incubated with rIL-7 (5ng/mL) in the presence or absence of rTGF-β1 (5ng/mL), rTGF-β1(5ng/mL) alone or medium alone. After 3 days, cells were analyzed by flow cytometry for P-STAT5, P-Akt and P-S6. (A) Representative graphs show induction of P-STAT5 and P-S6 in naive, central memory (CM) and effector memory (EM) CD4 T cells in the absence of rIL-7 or rTGF-β1 (medium), stimulated with rIL-7 or stimulated with rTGF-β1 or rIL-7 and rTGF-β1 following 3 days of stimulation. (B) The summary data comparing P-Akt and P- S6 induction in response to rIL-7 alone or with rTGF-β1 after 3 days of stimulation. Significant difference in P-S6 between rIL-7 in the presence or absence of rTGF-β1 was assessed by Wilcoxon matched-paired signed rank test.

TGF-β does not inhibit IL-7 receptor signaling in memory CD4 T cells and enhances S6 kinase signaling in naïve CD4 T cells

We and others have shown that P-Akt signaling in response to IL-7 is delayed and important to proliferation (133, 154). Phosphorylation of S6 is downstream of P-Akt and promotes proliferation by increasing protein translation (77). TGF-β has been shown to inhibit S6 kinase phosphorylation and activity in TCR-activated CD4 T cells (118). To

74 further examine the mechanism for the differential effects of TGF-β on proliferation in response to IL-7 between the CD4 T cell maturation subsets, we assessed the effects of

TGF-β on the induction of phosphorylated STAT5 (P-STAT5) and S6 (P-S6) in response to IL-7 after 3 days following stimulation (Fig. 4.4). This time point was chosen based on our previous experience with IL-7-induced P-Akt signaling that becomes detectable by flow cytometry 2-3 days post IL-7 stimulation (154). TGF-β did not have a consistent effect on P-STAT5 and P-S6 in memory CD4 T cells but surprisingly, caused a significant enhancement of P-S6 expression in naive T cells stimulated by IL-7. The effect of TGF-β on S6 signaling may have been independent of Akt, as we did not see a consistent effect of TGF-β on Akt signaling in any T cell subset (Fig. 4.4B). Overall,

TGF-β had no discernible effects in memory T cell subsets but enhanced P-S6 signaling in naïve T cells.

GSK-3 inactivation partially overcomes TGF-β-mediated inhibition of IL-7-induced proliferation and c-myc induction in memory CD4 T cells

P-Akt phosphorylates GSK-3, resulting in its inactivation in response to IL-7 stimulation. Since GSK-3 constitutively promotes c-myc degradation, inactivated GSK-3 results in increased c-myc expression (81). Therefore, we asked if a chemical inhibitor of

GSK-3 might overcome TGF-β-mediated inhibition of T cell proliferation and c-myc suppression in IL-7 stimulated memory T cells. CHIR 99021 (CH) is a highly specific small molecular inhibitor of GSK-3α and β isoforms, which inactivates GSK-3 by competitive ATP binding. We assessed T cell proliferation (% CFSE low cells) in response to IL-7 ± TGF-β in the presence or absence of CH. The addition of CH to

PBMC resulted in modest increases in memory T cell proliferation compared to cells

75 incubated in medium alone and CH also increased proliferation in cells stimulated with

IL-7 (Fig. 4.5A).

Figure 4.5 TGF-β mediated inhibition of IL-7 induced proliferation and c-myc expression in memory CD4 T cells is reversed by the inactivation of GSK-3. PBMCs were labeled with CFSE and were not stimulated or stimulated with rIL-7 alone or rIL- 7(5ng/mL) and rTGF-β1 (5ng/mL) in the presence or absence of CHIR 99021 (1µM). After 7 days, cells were analyzed by flow cytometry. (A) Representative graphs and (B) summary data show %CFSE low in CD4 T cell maturation subsets following stimulation. Additional PBMCs were stimulated in the same conditions and intracellular c-myc expression was assessed by flow cytometry. (C) Graphs show expression of c-myc after days 3 and 5 following stimulation. Significant differences in % CFSE low response

76 between rIL-7+rTGF-β1 and IL-7; rIL-7+rTGF-β1 in the presence or absence of CH; rIL-7 and rIL-7+rTGF-β1+CH were assessed by Wilcoxon matched-paired signed rank test.

The addition of CH to cells treated with IL-7 + TGF-β resulted in proliferation responses that were greater than those observed in cells treated with IL-7 + TGF-β alone and in the case of CM cells, similar to the responses observed in T cells stimulated with

IL-7 alone (Fig.4.5A and B). Thus, CH largely overcame the TGF-β mediated inhibition of IL-7 induced proliferation in the CM CD4 T cell subset and partially overcame the inhibition of proliferation in the EM CD4 T cell subset (Fig. 4.5A and B). Similarly, CH reversed the TGF-β mediated inhibition of c-myc expression in CM CD4 T cells and partially reversed inhibition EM CD4 T cells at days 3 and 5 following IL-7 stimulation

(Fig. 4.5C). Notably, proliferation of T cells stimulated with CH + TGF-β + IL-7 was consistently lower than proliferation of cells stimulated with CH + IL-7 (Fig. 4.5A and data not shown), suggesting that some anti-proliferative effects of TGF-β still remained in the presence of CH. This might be expected given evidence from other studies that

TGF-β can mediate inhibition of T cell proliferation by smad-independent mechanisms.

Although the purpose of the above experiments was to determine the effect of the

GSK-3 inhibitor on memory T cells, we also found striking effects in naïve T cells. For example, at the concentration of inhibitor (1 µM) used to overcome TGF-β inhibition in memory T cells, we found that the inhibitor alone markedly induced naïve T cell proliferation (median % CFSE low cells = 34% in CH treated PBMC after 7 days; n=6).

Moreover, the addition of CH to IL-7 stimulated PBMC induced marked proliferation in naïve T cells that far exceeded the proliferation observed in cells stimulated with IL-7 alone (medium % CFSE low = 18% and 86%, for naïve T cells stimulated with IL-7 or

IL-7+ CH, respectively, n= 6; p = 0.0313). These observations suggest that GSK-3 plays a more critical role in regulating proliferation in naive T cells than memory T cells.

Discussion

Differential effects of TGF-β on naive and memory CD4 T cell proliferation has been described previously in T cells activated by anti-CD3 plus IL-2 by de Jong and colleagues (183). These scientists showed that TGF-β inhibited memory, but enhanced naive TCR-driven proliferation, in the presence of IL-2 but not in the presence of IL-7.

The TGF-β mediated enhancement of proliferation in naive CD4 T cells was associated with increased IL-2Rα or CD25 expression (183). Our data also point to differences in the effects of TGF-β on proliferation in naïve and memory T cells. Our studies did not include TCR stimulation and centered instead on the direct effects of TGF-β on T cell proliferation in response to the homeostatic cytokine, IL-7. We find that TGF-β impairs memory but not naïve CD4+ T cell responses to IL-7 stimulation.

Since TGF-β inhibits c-myc expression in epithelial cells, we speculated that a similar effect might occur in T cells. We further hypothesized that inhibition of T cell proliferation would correspond to inhibition of c-myc expression and therefore might be more pronounced in memory CD4+ T cells than in naïve CD4+ T cells. Our data demonstrate that the TGF-β mediates inhibition of c-myc expression in both memory and naive T cells; however, it only impairs proliferation of memory CD4 T cells. The reason behind this is observation is unclear but may be related to the TGF-β mediated increase in

P-S6 induction in response to IL-7 in naive but not memory CD4 T cells (Fig. 4.4).

Enhancement of IL-7 induced P-S6 by TGF-β in naïve T cells may enhance global protein translation, perhaps compensating in part for lower c-myc expression. This might also contribute to what appears to be a waning effect of TGF-β on inhibition of c-myc in

78 naïve T cells that becomes distinguishable from the relative levels of inhibition in memory T cells over 5 d of incubation (Fig. 4.3C). Overall, these observations suggest that TGF-β may exert stimulatory and inhibitory effects on naive CD4 T cells while primarily delivering inhibitory signals to memory CD4 T cells.

In contrast to enhancing P-S6 signaling in naïve T cells stimulated with IL-7,

TGF-β did not discernible effect on IL-7 receptor signaling measured by P-STAT5, P-

Akt or P-S6 in memory T cell subsets and no stimulatory effects on P-Akt or P-STAT5 in naïve T cells. Our findings are consistent with reports that TGF-β does not impair

Jak/STAT signaling (178). The mechanism behind the enhancement of P-S6 in naïve T cells that was mediated by TGF-β is uncertain but may be related to the role of TGF-β in enhancing CD127 expression in these cells (184). Nonetheless, in preliminary experiments, we did not find consistent evidence of higher CD127 expression in naïve T cells incubated with TGF-β compared to unstimulated cells or higher CD127 expression in cells incubated with TGF-β + IL-7 compared to cells incubated with IL-7 alone (not shown). Also, we did not find a consistent enhancement of P-Akt signaling or P-STAT5 signaling in naïve T cells exposed to IL-7 + TGF-β compared to IL-7 stimulated cells alone. These observations suggest that the effects of TGF-β on naïve T cells may occur independently of effects on IL-7R expression. Further studies with more detailed kinetic and dose response analyses may ultimately help to discern the role of CD127 expression in this effect. Alternatively, we propose that TGF-β may enhance P-S6 activation independent of IL-7R signaling, potentially via other pathways such as MAP kinases, that are induced by TGF-β (185, 186) and that may also modulate P-S6 signaling.

The importance of IL-7 and TGF-β in maintaining T cell homeostasis have been highlighted in mouse models, where IL-7 deficiency results in T cell lymphopenia (71) while loss of TGF-βR results in T cell hyper-activation (164, 187). Moreover, in mice,

TGF-β plays an important role in enhancing naïve T cell survival by sustaining CD127 expression. This effect involved suppression of gfi-1, a regulatory factor that reduces

CD127 transcription (184). Thus, although recognized for its immunosuppressive activities, there is a growing appreciation that TGF-β plays an important role in sustaining naïve T cell numbers in vivo.

The interplay between IL-7 and TGF-β may also be important in inflamed lymph node microenvironments such as those observed in HIV disease. Memory and naïve CD4

T cells are exposed to IL-7 expressed by stromal cells in reticular networks within the lymph node and (41, 188, 189) TGF-β may be secreted from activated or regulatory T cells during HIV infection (41, 114, 190). Thus, memory CD4 T cells that rely on IL-7 to proliferate and maintain homeostasis could be inhibited by TGF-β exposure in T cell zones within lymph nodes. Similar interactions may occur in mucosal microenvironments where IL-7 is produced by epithelial cells (191) and TGF-β may be produced by various cells (192). It is likely that TGF-β and IL-7 interactions influence homeostasis of other cell types that rely on IL-7, such as innate lymphoid cells or gamma/delta T cells in the mucosa and that these interactions could also be modified in the context of inflammatory conditions.

We show here that chemical inhibition of GSK-3 can at least partially overcomes

TGF-β-mediated inhibition of IL-7-induced memory T cell proliferation. The effects of the inhibitor are more pronounced in CM than EM cells, suggesting that GSK-3 may play

80 less of a role in regulating T cell proliferation in cells that are further along the differentiation pathway (EM cells). This observation and our finding that the GSK-3 inhibitor, CH alone provided a sufficient signal to induce proliferation of naïve T cells is consistent with previous studies indicating that CD28-mediated control of GSK-3 is more important for naïve than it is for memory T cell proliferation responses during TCR triggering (193). Our studies also raise the intriguing possibility that targeting GSK-3 for inhibition in vivo may be a strategy to circumvent TGF-β-mediated inhibition of memory

T cells that have the potential to respond to tumors or pathogens. This strategy may be limited, however, by the potential for TGF-β to enhance production of other immunosuppressive cytokines such as IL-10 in memory T cells.

Overall, our studies uncover novel interactions of TGF-β and IL-7 that have distinct implications for naïve and memory T cell proliferation and homeostasis. Our studies are consistent with studies in mice, suggesting that TGF-β may play an important role in maintaining naïve T cell homeostasis while limiting memory T cell expansion under steady state conditions. Understanding the molecular mechanisms that account for these differential outcomes will ultimately be important for uncovering fundamental mechanisms that support T cell reconstitution while regulating T cell hyper-activation in vivo. Chapter 5: Discussion and future directions

Diminished responsiveness to IL-7 may contribute to poor CD4 T cell count recovery in treated HIV infection

ART administration has nearly eliminated AIDS related mortality in HIV infected patients (54). Following the initiation of ART, there is an immediate and sustained reduction of detectable plasma HIV RNA in most patients. Following the reduction of

81 detectable plasma HIV RNA, recovery of CD4 T cell count is gradual and highly variable among the patients (55). Studies suggest that nadir CD4 T cell count is a consistent predictor of impaired CD4 T cell recovery following ART administration (56). Multiple immunological mechanisms have been proposed to explain incomplete recovery of CD4

T cell number after years of ART administration. These mechanisms include increased

CD4 T cell turnover and senescence (53, 88), limited access to IL-7 in lymphoid tissue

(194) as well as decreased IL-7 responsiveness (88), which has been reported by

Tanaskovic and colleagues as well as described in this thesis. These defects are related to the residual immune activation and inflammation. For example, work from the Schacker and colleagues showed that lymphatic tissue collagen predicts the magnitude of CD4 T cell recovery in persons on ART (195). There is evidence of increased IL-6 and IL-1β in treated HIV infected patients who fail to recover CD4 T cell counts. The expression of these cytokines has been linked to increased T cell turnover and survival since they have the potential to reduce IL-7 responsiveness by reducing of CD127 expression in vitro

(108).

We observed diminished ex vivo CD4 T cell responsiveness to IL-7 in IF patients when compared to immune success patients and healthy control donors. Defects in IL-7 responsiveness, as measured by proliferation and CD25 induction were observed predominantly in memory CD4 T cells. In support of the hypothesis that CD127 expression is a contributor to these defects, diminished CD127 expression was observed in central memory and effector memory CD4 T cell subsets. Since CD127 expression did not correlate with proliferation in response to IL-7 in CD4 T cells from IF subjects,

CD127 may not be the only contributor to impaired proliferation in response to IL-7.

The diminished CD127 expression of freshly isolated memory CD4 T cells from

IF subjects when compared to controls may be explained by increased IL-6 and IL-1beta expression as well as elevated plasma IL-7 levels. Shive and colleagues showed that IL-6 and IL-1beta diminishes CD127 mRNA levels as well as induction of BCL-2 expression, a response that is downstream of IL-7 receptor activation. CD127 mRNA expression is also down-regulated in response to IL-7 signaling. Collectively, any or all of these mechanisms may contribute to diminished CD127 expression in IF subjects. Future therapy approaches such as blocking IL-6 activity may help determine the importance of these cytokines in regulating CD127 expression in IF subjects.

Another mechanism that may contribute to diminished proliferation in response to

IL-7 in IF patients may be FOXO3a expression. As discussed in chapter 1, FOXO3a belongs to a family of transcription factors that resides in the nucleus to up-regulate anti- proliferative genes such as p27kip1 and p21in the absence of cytokine stimulation.

P27kip1 and p12 expression are associated with arrested cell cycling. To determine whether FOXO3a expression is increased in T cells from IF subjects, we assessed

FOXO3a mRNA expression in purified T cells that were freshly isolated from the subjects discussed in chapter 3. We found that FOXO3a mRNA expression was increased from T cells from IF subjects when compared to HC subjects (unpublished data). Consistent with this observation, FOXO3a mRNA expression has also been observed in transcriptomic analysis of memory CD4 T cells from IF subjects (Sekaly, unpublished).

IL-7 activates P-Akt signaling, which inactivates FOXO3a. Inactivated FOXO3a does not translocate to the nucleus to up-regulate expression of genes that promote cell

83 cycle arrest. While FOXO3a mRNA expression is increased in T cells from IF subjects, it is not clear whether this abnormally high expression of FOXO3a in some IF subjects represents a mechanism by which proliferation in response to IL-7 is diminished in these subjects. One way to test the hypothesis that FOXO3a expression can contribute to impaired proliferation in response to IL-7 in IF patients is through determining whether chemical inhibition of FOXO3a will enhance proliferation in CD4 T cells from IF subjects.

The role of IFN-I in poor CD4 T cell count recovery in treated HIV infection

The IFN-I gene expression signature in T cells from IF subjects and the inhibition of IL-7 induced proliferation in CD4 T cells by IFN-a suggest that IFN-I play a role in impairing CD4 T cell count recovery in treated HIV infected patients. Since ISG expression in T cells did not correlate with proliferation in response to IL-7 in CD4 T cells from IF subjects, we did not find the mechanism by which IFN-I may contribute to the proliferation defect in IF subjects. In chapter 2, we found that IFN-α can inhibit IL-7 induced proliferation as well as the delayed receptor (P-Akt) signaling that is associated with proliferation. We did not observe impaired P-Akt signaling at day 3 stimulation with

IL-7 in CD4 T cells from IF subjects. These observations suggest that either in vivo exposure to type 1 interferon does not impair ex vivo IL-7 induced P-Akt or the mechanism underlying impaired ex vivo IL-7 responsiveness is not related to in vivo type

1 interferon exposure. The studies described in chapter 3 had limitations in the measurement of IL-7 receptor signaling. For example, we assessed IL-7 induced P-

STAT5 and P-Akt but not P-S6, a marker associated with protein synthesis as a mechanism of proliferation. In addition, the measurement of P-S6 is helpful in

84 determining whether signaling downstream of P-Akt is intact in CD4 T cells from IF patients. A further limitation of our assessment of our assessment of IL-7 receptor signaling was that only one signaling time point was assessed. In chapter 2, we demonstrated that a sustained P-Akt signaling is important for proliferation in response to

IL-7. Since IL-7 induced P-Akt was not measured beyond day 3 following IL-7 stimulation, it is not clear whether there is a defect in the sustained IL-7 induced P-Akt.

In future studies, the multiple time point (days 3 and 5) assessment of P-Akt and P-S6 induction in response to IL-7 in CD4 T cells from IF subjects may determine whether IL-

7 signaling is impaired. If this is the case, then type 1 interferon exposure may play a role in diminishing IL-7 induced proliferation.

Future studies could further describe the role of type 1 interferon in the mechanism involved in the impaired CD4 T cell proliferation in response to IL-7. To determine if IFN-α mRNA expression from T cells from IF subjects inhibit proliferation in response to IL-7, in vitro IFN-α or IFN-αR blocking studies would be needed. While

ISG expression in T cells from IF subjects has been consistently observed. It is not known if there is increased ISG protein expression among the CD4 T cell subsets from IF patients. We have validated a flow cytometry technique to analyze ISG (ISG15, IRF-7 and tetherin) expression in CD4 T cell maturation subsets from IF patients. Additional studies include assessment of ISG expression in cells dividing in response to IL-7 to determine whether diminished proliferation occurs in cells with increased expression of

ISGs as a result of in vivo IFN-I exposure.

As suggested by figure 3.6, the role of type 1 interferon in mediating poor CD4 T cell recovery may be related to its anti-proliferative function as well as its pro-apoptotic

85 function. It was surprising to observe intact responses to IFN-α in IF patients since interferon tolerance is a well described outcome of chronic type 1 interferon exposure. T cells from IF patients had increased exposure to type 1 interferon, suggesting that these cells may function in the context of chronic type 1 interferon exposure. It was not clear whether cells are tolerant to the anti-viral or anti-proliferative activities of interferon or both since the interferon tolerance has been described by the diminished anti-viral response. Therefore, it is possible that T cells from IF patients experience interferon tolerance in respect to the induction ISGs but still maintain anti-proliferative and pro- apoptotic activity.

TGF-β selectively inhibits memory CD4 T cell proliferation in response to IL-7

TGF-β may play a role in inhibiting T cell proliferation in treated HIV infection.

Plasma TGF-β levels positively correlate with IL-7 levels in treated HIV infected patients

(196). More recently, TGF-β gene expression signatures are observed to be increased in T cells from IF patients (Sekaly, personal communications). Consistent with this observation, studies have found that there an inverse relationship between pre-ART TGF-

β expression from duodenal bioposies and CD4 T cell recovery (197). Collectively, these observations suggest that CD4 T cell exposure to TGF-β may contribute to poor CD4 T cell recovery in treated HIV infection.

While TGF-β is a pleitropic cytokine, its anti-proliferative effects are consistently observed in human T cells. For example, TGF-β has been shown to inhibit IL-2 induced proliferation in memory CD4 T cells (183). Consistent with this observation, we demonstrated in chapter 4 that TGF-β inhibits memory CD4 T cell proliferation in response to IL-7. Our mechanistic studies revealed that TGF-β inhibits c-myc induction

86 in response to IL-7 in naive and memory CD4 T cell subsets. Elevated c-myc protein levels are important for driving cell cycling. C-myc has been shown to promote the transcription of a large subset of genes that drive aerobic glycolysis, including CD98 as well as mediate the increase in glucose uptake (83). TGF-β mediated inhibition of c-myc expression in response to IL-7 would suggest that genes involved in aerobic glycolysis,

CD98 expression and glucose uptake, particularly in memory CD4 T cells are inhibited by TGF-β. In preliminary studies, we found that CD98 expression in response to IL-7 were not inhibited by TGF-β in similar magnitude as inhibition of c-myc induction

(unpublished data).

We found that TGF-β did not inhibit naive CD4 T cell proliferation in response to

IL-7. In purified naïve CD4 T cells, TGF-β enhanced proliferation in response to IL-7. In contrast, TGF-β inhibits proliferation in response to IL-7 in memory CD4 T cells. These observations suggest that TGF-β selectively inhibits memory CD4 T proliferation in response to IL-7. An open question is what may allow TGF-β to distinguish between naïve and memory CD4 T cells in order to selectively inhibit proliferation.

IFN-α and TGF-β utilize different mechanisms to inhibit IL-7-induced proliferation in CD4 T cells

Exposure to TGF-β and IFN-I have a greater inhibitory effect on CD4 T cell recovery than exposure with TGF-β or IFN-α alone. In data that was not submitted to peer-reviewed journals, stimulation with TGF-β and IFN-α inhibited proliferation

(%CFSE low) that was greater magnitude than with stimulation with TGF-β or IFN-α alone (data not shown). This would suggest that TGF-β and IFN-α may target different pathways by which IL-7 induces proliferation.

Our data suggest that IFN-α and TGF-β may target different mechanisms by which IL-7 induces proliferation in CD4 T cells (Fig.5.1). In chapter 2, we showed that

IFN-α inhibits induction of P-Akt in response to IL-7. In contrast, we showed that TGF-β did not inhibit IL-7 signaling, as measured by P-S6 and P-STAT5 as well as P-Akt (data not shown) as shown in chapter 4. We found that TGF-β inhibits c-myc expression in response to IL-7. Induction of c-myc expression in response to IL-7 is driven by IL-7 receptor signaling through P-Akt as well as c-myc transcription, possibly through P-

STAT5 binding of the c-myc promotor. Since we did not find that TGF-β inhibited IL-7 induced P-Akt or P-STAT5, TGF-β may inhibit c-myc transcription in response to IL-7.

To address this possibility, memory CD4 T cells purified from PBMCs from healthy donors can be assessed for c-myc mRNA following stimulation with IL-7 and TGF-β in comparison with memory CD4 T cells stimulated with IL-7 or TGF-β alone.

Figure 5.1. Differential mechanisms of inhibition of IL-7 induced proliferation by TGF-β and IFN-α

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