Immune Evasion by Mycobacterium Tuberculosis: Mannose

IMMUNE EVASION BY MYCOBACTERIUM TUBERCULOSIS: MANNOSE-

CAPPED LIPOARABINOMANNAN INDUCES GRAIL AND CD4+ T CELL

ANERGY

OBONDO JAMES SANDE

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Advisor: Dr. W. Henry Boom

Department of Pathology: Immunology Training Program

CASE WESTERN RESERVE UNIVERSITY

May 2016

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of Obondo James Sande, candidate for the

Ph.D. degree*

(Signed)

Alan D. Levine (Chair of the committee)

Clive Hamlin

Clifford V. Harding

Roxana E. Rojas

W. Henry Boom

(Date) 18 January 2016

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

Dedication

I dedicate this work to my parents Pantaleo O. Wantono (RIP) and Loyce Nandera and to my wife, Hellen Beatrice Anyait. They have provided nothing but support before and throughout my doctoral training.

iii

Acknowledgements

I would first and foremost like to thank Henry, Roxana and Cliff. Their tireless guidance has not only shaped my skills in the laboratory but they have taught me how to effectively communicate and share my ideas, both in text and through regular presentations, which I have come to understand is instrumental in becoming a successful scientist. I would also like to thank all the members of the Boom/Rojas laboratory (Scott

Reba, Qing Li, Xuedong Ding, Ahmad Faisal Karim, Sophia Onwuzulike and Keith

Chervenak), all have made my graduate student career enjoyable and as fruitful as possible. I would also like to thank all the members of the Harding laboratory (Jaffre

Athman, Nancy Nagy, Sukula Supriya, Claire Mazahery and Pamela Wearsch) for their help along the way. Last but not least I would like to thank Robert N. Mahon for initiating this work. Lastly I would like to thank Grace M. Svilar and Marla Manning, the administrators of the Fogarty Scholarship for their kind guidance. I am grateful for your timely assistance throughout my graduate student studies.

Table of contents Page Dedication iii Acknowledgements iv Table of Contents v List of Figures viii List of Abbreviations x Abstract xi

Chapter 1: Introduction Tuberculosis overview 2 Mycobacteria 3 The course of M. tuberculosis infection 4 M. tuberculosis evasion of CD4+ T cells 6 M. tuberculosis cell wall glycolipids 9 Trafficking of M. tuberculosis molecules outside infected cells 11 Mannose-capped Lipoarabinomannan (LAM) and immune regulation 12 LAM and T cell receptor signaling and regulation of T cell activation 14 T cell anergy 17 Models of T cell anergy induction 17 Mechanisms of anergy induction 18 GRAIL (Gene Related to Anergy in Lymphocytes) 19 Other factors that confound anergy 21 Pathogens and anergy induction 22 Reversal of T cell anergy 22

Chapter 2: Mycobacterium tuberculosis ManLAM inhibits T-cell-receptor signaling by interference with Zap70, Lck and LAT phosphorylation

Abstract 24 Introduction 25 Materials and Methods 27 Results 33 Discussion 45

Chapter 3: Mannose-Capped Lipoarabinomannan from Mycobacterium tuberculosis

Induces CD4+ T cell Anergy via GRAIL

Abstract 50 Introduction 51 Materials and Methods 54 Results 63 Discussion 86

Chapter 4: Discussion and future direction LAM trafficks outside M.tuberculosis-infected macrophages, inhibits T cells 93 LAM and inhibition of TCR signaling 94 A Model of anergy induction by LAM 101 Role and induction of GRAIL by LAM 107 Reversal of LAM-induced anergy by IL-2 111 GRAIL and anergy induction by other pathogens 114 Conclusion and future directions 115

Appendix 1: Correlation of figures with experiments 122 Appendix 2: List of Publications and abstracts 123 Works cited 125

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List of Figures

Figure Page

Figure 1.1 Potential outcomes of exposure to M. tuberculosis 7

Figure 1.2 Mycobacterial cell envelope 10

Figure 1.3 Proposed structures of LAM, LM, PIM 11

Figure 2.1 ManLAM inhibits antigen-specific CD4+ T cell activation 34

Figure 2.2 ManLAM inhibits the activation of human T cells 36

Figure 2.3 ManLAM inhibits Lck and LAT phosphorylation 38

Figure 2.4 ManLAM does not activate the cAMP/PKA pathway or

inhibit Lck-Tyr505 pphosphorylation 40

Figure 2.5 Inhibition of TCR signaling by ManLAM is temperature sensitive 42

Figure 2.6 ManLAM is found in T cell membranes and does not

affect localization of Lck in lipid rafts of activated Tcells 44

Figure 3.1 Viability and function of CD4+ T cells with IL-7 treatment 65

Figure 3.2 Presence of LAM on CD4+ T cell membranes is required

for inhibition of CD4+ T cell activation after primary stimulation 66

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Figure 3.3 LAM induces anergy in P25 CD4+ T cells 68

Figure 3.4 LAM-induced P25 CD4+ T cell anergy occurs over a range

of Ag85B peptide and LAM concentrations 70

Figure 3.5 LAM does not induce FoxP3-positive regulatory T cells

and/or increase activation-Induced cell death (apoptosis) 72

Figure 3.6 LAM-induced CD4+ T cell anergy is not due to cell death 73

Figure 3.7 LAM does not affect receptor expression on LAM-anergized

P25 TCR-Tg CD4+ T cells 76

Figure 3.8 LAM induces increased GRAIL protein expression both

during priming and upon re-stimulation 78

Figure 3.9 Knockdown of GRAIL expression by siRNA prevents

inhibition of CD4+ T cell activation by LAM 81

Figure 3.10 Exogenous IL-2 down-regulates GRAIL expression and

restores T cell proliferation in LAM-anergized CD4+ T cells 83

Figure 3.11 LAM associates with lipid rafts and CD3 on human CD4+ T

cells, and up-regulates GRAIL upon activation with anti-CD3/CD28 85

Figure 4.1 Model of anergy induction by LAM 102

List of Abbreviations

Ag85B, 85B antigen from M. tuberculosis APC, antigen presenting cell BCG, Bacille Calmette Guerin BMM, bone marrow derived macrophages Cbl-b, Castas b-lineage lymphoma proto-oncogene b Csk, C-terminal Src kinase CTLA-4, cytotoxic T lymphocyte antigen-4 Erk, extracellular regulated kinase FITC, fluorescein isothiocyanate Foxp3, forkhead box P3-expressing GRAIL, gene related to anergy in lymphocytes ITAM, Immunoreceptor tyrosine-base activation motif Lag-3, lymphocyte activation gene 3 LAM, Lipoarabinomannan LAT, linker for the activation of T cells Lck, lymphocyte-specific protein tyrosine kinase LM, lipomannan MAPK, mitogen activated protein kinase MHC, Major Histocompatibility Complex PBMC, peripheral blood mononuclear cell PD-1, programmed death-1 PIM, phosphatidylinositol mannosides SLP-76, SH2 domain-containing leuckocyte phosphoprotein of 76 kDa Tim-3, T cell immunoglobulin and mucin domain-containing protein 3 ZAP-70, Zeta-associated protein of 70 kDa

Immune Evasion by Mycobacterium tuberculosis: Mannose-Capped Lipoarabinomannan

Induces GRAIL and CD4+ T cell anergy

OBONDO JAMES SANDE

Abstract

Mycobacterium tuberculosis (Mtb) persists and survives in the host in the face of many T cells subsets recognizing a range of Mtb antigens due to its ability to evade innate and adaptive immune responses. CD4+ T cells and infected antigen presenting cells

(APC) are central for control of Mtb but also targets of its immune evasion strategies.

The major Mtb cell wall glycolipid, mannose-capped lipoarabinomannan (LAM) is one of the molecules involved in immune evasion. Previous studies determined that LAM can inhibit phagosome maturation and antigen processing in macrophages and thus indirectly affect memory and effector CD4+ T cell function. LAM is trafficked from Mtb-infected macrophages via bacterial vesicles into the microenvironment of the infected site, where it can bind to uninfected APC and T cells. We proposed that this provides a mechanism for direct delivery of LAM to surrounding T cells, thereby further regulating their function. Earlier studies determined that LAM directly inhibits polyclonal murine CD4+

T cell activation by blocking ZAP-70 phosphorylation.

In the first part of this thesis we extended our observation of direct inhibition of T cell activation by LAM in two directions. First we determined if LAM inhibition of murine primary CD4+ T cells could be extended to antigen-specific CD4+ T cell

xi activation by antigen presenting cells and whether human CD4+ T cells were similarly inhibited. Second, we determined the mechanism of LAM-mediated inhibition of TCR signaling in terms of its effect on Lck and LAT phosphorylation and lipid raft integrity.

We found that LAM inhibited antigen-specific murine CD4+ T cells and primary human

T cells as well. In addition to ZAP-70, LAM inhibited phosphorylation of Lck and LAT.

Inhibition of proximal TCR signaling was temperature sensitive, suggesting that LAM insertion into T cell membranes was required. We established that direct interaction of

LAM with T cells inhibits antigen-specific CD4+ T cell activation by interfering with very early events in TCR signaling through LAM’s insertion in T cell membranes.

Previous studies show that inhibition of proximal TCR signaling is associated with induction of T cell anergy.

In the second focus of this thesis, we tested if LAM-induced inhibition of CD4+ T cell activation results in T cell anergy. We found that LAM induces anergy in P25 TCR transgenic CD4+ T cells (specific for P25 peptide of the 38 kDa antigen 85B of Mtb).

Anergy induction required the presence of LAM in the T cell membrane during primary

T cell activation. Once anergy was induced, LAM was no longer required in the T cell membrane, as removal of LAM did not affect the T cell anergic state. The induction of anergy was due to up-regulation of GRAIL (Gene Related to Anergy In Lymphocytes) protein in both murine and human CD4+ T cells. We further determined that exogenous

IL-2 reversed LAM-induced anergy by downregulating GRAIL expression. We propose that LAM inhibits CD4+ T cell activation and up-regulates GRAIL expression to induce anergy in Mtb-reactive CD4+ T cells. Together, the studies described in this thesis determined that induction of CD4+ T cell anergy by LAM may represent one mechanism

xii by which Mtb evades T cell recognition.

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Chapter 1: Introduction

Tuberculosis overview

Mycobacterium tuberculosis (Mtb) and other members of the Mycobacterium tuberculosis complex cause tuberculosis (TB). TB is a chronic infectious disease mainly transmitted by the aerosol route in humans that remains a major global health problem despite the availability of BCG vaccine and drug treatment (1-4). The success of Mtb as a pathogen is due to its efficiency of transmission, the ability to resist the available drugs and its ability to evade elimination by both innate and adaptive immune responses (5, 6).

There are extensive public health measures in place including improvement of the healthcare infrastructure, contact case tracing to reduce transmission, but these efforts need to be complemented by other preventive strategies including effective drug therapy for active TB cases and an effective vaccine. However, these measures cannot be fully achieved because of various challenges. Mtb is susceptible to few antibiotics due to its less permeable cell wall envelope, complicated metabolism and (3). Moreover, Mtb has developed resistance to both the available first line drugs (isoniazid, ethambutol, pyrazinamide and rifampicin) and second line drugs (multi-drug and extensively drug resistance (7-9). The host immune response that would be expected to help clear the bacilli is often rendered ineffective by Mtb immune evasion mechanisms. The only currently available vaccine for Mtb infection is the M. bovis bacille Calmette-Guerin

(BCG). BCG is used in newborns mainly to prevent disseminated disease in young children. However, BCG is not effective at preventing reactivation disease in adolescents and adults, which is the disease manifestation thought to be responsible for the majority of TB in the world (10). And to date, it is not clear if this vaccine prevents reinfection

(new infection). In addition Mtb/HIV-1 coinfection has complicated the

2 immunopathogenesis and control of TB. All together, these challenges have increased the public health impact of TB worldwide.

Because of these challenges, there are now many initiatives including TB

Alliance, FIND, the Bill and Melinda Gates Foundation, AERAS and the Global Fund for

AIDS, TB and malaria (11) to improve diagnostics and detection of cases, discover new chemotherapeutic agents, immunotherapy and develop effective vaccines. Despite these efforts, the major barrier to development of improved or new prevention strategies is incomplete understanding of the biology of mycobacteria, the pathogen and the host- pathogen interaction during Mtb infection. This thesis focuses on the host-pathogen interaction and Mtb is evasion of the host’s immune response.

Mycobacteria

Mycobacteria are gram-positive bacteria, and members of the phylum

Actinobacteriae and family actinomycetale (12, 13). Within actinomycetes, mycobacteria belong to the Corynebacterium-Mycobacterium-Norcadia branch. There are over seventy species of mycobacteria, most of which are nonpathogenic saprophytes (14). M. smegmatis, for example, is an exclusively saprophytic species referenced in some of the work in this thesis. There are few species that are pathogens of humans and other animals: M. avium complex and M. kansii are not common pathogens, but cause TB-like

(atypical) disease in immunocompromised humans (15-17). M. ulcerans and M. leprae cause the human diseases Buruli ulcers and leprosy, respectively. M. tuberculosis (Mtb) complex which consists of six closely related species are the commonest cause of TB, including M. tuberculosis (the most predominant cause of human TB), M. africanum (an

3 agent of human TB mostly in West Africa), M. bovis (the agent of TB in cattle, humans and few other mammals), M. canettii (rarely seen in humans), M. microti (agent of TB in voles), and M. marinum (agent of TB in zebra fish) (18). The work in this thesis focuses on M. tuberculosis (Mtb) the main species of Mtb complex, and its laboratory strains Mtb

H37Rv (the virulent strain) and Mtb H37Ra (the avirulent strain).

The course of M. tuberculosis infection

Mycobacterium tuberculosis is an intracellular pathogen well adapted to living in man (19, 20). Mtb infects phagocytic antigen presenting cells (APCs) in the lungs, including alveolar macrophages, dendritic cells (DCs) and lung macrophages (21-23).

Virtually all Mtb infections occur by airborne transmission of droplet nuclei containing a few viable organisms, and spread from person to person by inhalation of aerosolized bacilli from active TB patients during expectoration. Once the Mtb bacilli enter the alveolar space, they are phagocytosed by antigen presenting cells, including alveolar macrophages, lung macrophages, monocytes and dendritic cells (20, 21).

In these cells, M. tuberculosis survives in phagosomes and uses a wide range of evasion mechanisms to first evade innate immunity and indirectly evade adaptive host immune response, including inhibition of phagosome maturation, resistance to microbicidal killing by reactive oxygen species, prevention of major histocompatibility complex class-II (MHC class II) antigen processing and presentation by APCs (21, 23-

27). Mtb components, including glycolipids (eg LM, LAM) and lipoproteins, are recognized in APCs by Toll-like receptors (TLRs), mannose receptors (10), resulting in the secretion of chemokines and inflammatory cytokines. Infected APCs migrate to

4 regional lymphoid tissues, where adaptive immunity develops through antigen presentation to naïve T cells. APCs process Mtb antigens by intravacuolar proteolysis to produce peptides that bind to MHC class II molecules, which then translocate to the cell surface and present Mtb peptides to CD4+ T cells. Mtb peptides are also presented by

MHC class I molecules to CD8+ T cells. Memory and effector T cells migrate back to sites of infection and help APCs control Mtb growth through secretion of effector cytokines (IFN-γ, TNFα, IL-2 and other effector cytokines), which facilitate the maturation and killing capacity of APCs. In this process, granulomas develop through the secretion of the effector cytokines (10). Granulomas consist of a central area of necrosis surrounded by macrophages and giant cells containing bacilli, T cells, B cells, and fibroblasts. If APCs do not destroy Mtb, the bacilli multiply logarithmically within in the lung, and the logarithmic growth can be arrested by the development of cell-mediated immunity. CD4+ T cells are central to controlling Mtb infection (24, 27).

In mice, between 1 and 3 weeks after infection, Mtb-specific T cells appear in the lungs, IFN-γ, secreted by mainly CD4+ T cells, helps APCs control the bacterial load. In addition CD4+ T cells can kill Mtb-infected target cells and provide help for other T cell subsets such as CD8+ T cells and gamma-delta T cells which may be important for control of Mtb infection (21, 28).

In humans, 90% of health individuals control Mtb in the lungs when infected with the bacteria (Figure 1.1). However, the pathogen is usually not eliminated and a few mycobacteria can persist for years (latent infection), residing inside macrophages in granulomas and evading elimination by the host immune response. This persistence may allow progression to active TB, either as progressive primary disease (5-10%) or, years

5 later, as reactivation TB when T cell immunity fails. This is often exacerbated in immunosuppressive conditions such as HIV-1 infection, malnutrition, newborns, elderly and likely some genetic factors (10). The most common manifestation of TB is pulmonary TB, often characterized by cough for more than 3 weeks, loss of weight, evening fevers, Mtb in lung cavities in some patients who then become the source of further transmission.

Therefore, CD4+ T cells and infected APCs are central for control of Mtb.

Suboptimal function of CD4+ T cells during Mtb infection could play a significant role in failure of CD4+ T cell to optimally recognize and help infected APCs to eliminate the bacilli.

M. tuberculosis evasion of CD4+ T cells

Mtb is an intracellular pathogen that infects mainly alveolar macrophages, and as such the first target in regulation are the Mtb-infected macrophages. However, effective immune response to Mtb depends on T lymphocytes, as T cell-deficient mice, nonhuman primates and humans are susceptible to rapidly progressive disease (29). Among T lymphocytes, CD4+ T cells are essential for protective immunity to TB: in HIV-infected humans, deficiency or loss of CD4+ T cells significantly increases susceptibility to TB, and reconstitution of CD4+ T cells by antiretroviral therapy reduces susceptibility to TB

(29).

Figure 1.1. Summary of the potential outcomes of M. tuberculosis infection. Most M. tuberculosis exposures (70%) are cleared and do not result in bacilli deep into the alveoli.

Infections that occur are usually controlled but not eradicated and lead to latent Mtb infection with up to 10% life time risk of reactivation. Progressive primary TB develops in some cases, especially children and immunosuppressed individuals where the immune system fails to control initial infection. This figure was generated from (10, 30, 31)

Other T cell subsets including CD8+ T cells, γδ T cells are also generated in response to Mtb antigens during infection, but the contribution of these T cell subsets to overall protection is less clear (32-34).

Despite robust development of Mtb antigen-specific CD4+ T cell responses, the bacteria can persist for the life of an individual, allowing for reactivation, progression, disease and transmission of Mtb. Pathogens that evade adaptive immunity typically exhibit antigenic variation. By contrast, Mtb T cell epitopes are hyperconserved, implying that antigenic variation may be minimal and an unlikely mechanism of evasion of CD4+ T cell recognition by Mtb. Therefore the mechanisms that inhibit CD4+ T cell function and promote persistence, reactivation or progression of Mtb infection are still incompletely understood, but they include inhibition of Mtb antigen processing and inefficient MHC II antigen presentation (6, 10, 35, 36). By inhibiting the antigen processing and presentation function of APCs, the bacteria indirectly interfere with CD4+

T cell recognition of Mtb- infected APCs, and thus downregulate CD4+ T cell activation and effector functions, allowing Mtb to survive long term in the infected APCs.

However, besides the above known indirect regulation of CD4+ T cells by infected APCs, recent reports show that Mtb-infected APCs can secrete Mtb molecules including lipoproteins and glycolipids (eg LAM) via exosomes and bacterial vesicles into the microenvironment of infected cells. These molecules have been observed bound to uninfected cells in the infection site, suggesting that they could directly modulate the function of these cells (37, 38). Based on this hypothesis, Athman et al. recently showed that Mtb vesicles laden with glycolipids can directly inhibit macrophage function (37).

Mahon et al. demonstrated that Mtb glycolipids could directly inhibit CD4+ T cell activation. Chapters 2 and 3 of this thesis build on Mahon’s work to explore Mtb evasion of CD4+ T cell recognition via direct interaction of CD4+ T cells with inhibitory glycolipids.

M. tuberculosis cell wall glycolipids

The mycobacterial cell wall (Figure 1.2) is rich in mannosylated glycolipids, and they include Lipoarabinomannan (LAM), Lipomannan (LM) and phosphatidyl-myo- innositol mannosides (PIM) (39, 40). PIM is further classified into PIM2, PIM6, etc.

These glycolipids share a common glycophosphatidyl-myo-inositol (GPI) anchor, but differ in their glycosylation, with PIM consisting of a small number of mannosyl units,

LM having a long mannan core and LAM consisting of the most complex polysaccharide structure of branched arabinomannosyl units (39, 40).

Therefore these Mtb glycolipids are related structurally (Figure 1.3), however, in spite of their structural relatedness, LM, PIM and LAM induce different effects on the immune system. Whereas LM is a TLR2 agonist and induces pro-inflammatory effects on macrophages, LAM on the other hand induces inhibitory effects on macrophages (41).

PIM6 but not LAM or LM, can induce T cell adhesion to fibronectin via VLA-5 (42).

However, previous studies focused mainly on glycolipid effects on macrophages and dendritic cells, and little has been done on the direct effects of these glycolipids on T cells. Work in this thesis addresses this gap by focusing mainly on the direct effects of

LAM on CD4+ T cells.

Figure 1.2. Mycobacterial cell envelope. This is the current proposed structure of M. tuberculosis cell envelope. Glycolipids including LAM, LM and PIM are located in the plasma membrane (PM) and the outer membrane (OM). Adapted from (43).

Figure 1.3. Proposed structures of mycobacterial LAM, LM and PIM. These glycolipids share a common GPI anchor which is composed of D-myo-inositol, Palmitic acid and tuberculostearic acid, but differ in their glycosylation, with PIM consisting of a small number of mannopyranose units, LM having a long mannan core and LAM consisting of the most complex polysaccharide structure of branched arabinofuranose units. Adapted from (44).

Trafficking of M. tuberculosis molecules outside infected cells

Several intracellular bacterial pathogens use bacterial vesicles (BVs) as an alternative way to deliver ligands that can be recognized by host cells (45, 46). Some of the membrane vesicles carry bacterial components with virulence factors such as toxins, adhesins, or immunomodulatory compounds that are important for pathogenesis (45-47).

This vesicular transport and delivery system has been described in many intracellular pathogenic bacteria, including Mycobacteria species (48).

Mtb cell wall molecules glycolipids (eg, LAM, PIM, LM) and lipoproteins (eg, LprG,

LpqH) are released from Mtb in phagosomes of the infected macrophages, first into the intracellular environment where they are packaged in membrane vesicles such as BVs

11 and exosomes and trafficked to the extracellular environment (37, 49, 50). These membrane vesicles enable mycobacterial glycolipids, protein antigens and lipoproteins to be found both associated with the macrophage membrane and in supernatants of infected macrophages (50). Although dead mycobacteria in the granulomas may act as source of

Mtb molecules, recent studies demonstrate that viable mycobacteria in infected APCs are the major source of intra-and extracellular Mtb molecules (50). Moreover these bacterial vesicles showed immunomodulatory effects on macrophages both in vitro and in vivo

(50). The adaptive immune response including the effects on T cells was not covered in the study. We proposed that released Mtb molecules from Mtb infected macrophages may act as immunomodulators of nearby T cells in the infected site (granuloma), and work in this thesis sought to use purified LAM to elucidate LAM’s direct effects on

CD4+ T cell function.

Mannose-capped Lipoarabinomannan (LAM) and immune regulation

LAM is a major glycolipid in the Mtb cell wall. LAM is mainly made up of three components: the membrane anchor (mannosyl-phosphatidyl-myo-inositol), backbone

(mannopyranose and arabinofuranose homopolysaccharides) and the capping motif (39,

40). Three types of LAM have been described based on the presence and structure of the motifs capping the non-reducing termini of the arabinan domain: mannose-capped LAM

(ManLAM) in Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium leprae; phospho-myo-inositol (PI)-capped LAM (PILAM) in Mycobacterium smegmatis; and non-capped LAM (AraLAM) in Mycobacterium chelonae (51). In addition variations in structure of ManLAM from Mtb clinical isolates have been identified in some studies.

Mtb strains, HN885 and HN1554 contain truncated and more branched forms of

ManLAM, whereas Erdman, the laboratory strain used to study the pathogenesis of TB, and H37Rv have fully branched mannose and can bind mannose receptors on macrophages (52-54).

The effects of these types of LAM on APCs have been well known: ManLAM inhibits phagosome maturation and pro-inflammatory cytokine production, while PILAM and

AraLAM appear to upregulate pro-inflammatory cytokine production by APCs (51, 54).

Further evidence shows that in addition to insertion in lipid rafts in the macrophage membrane, ManLAM interacts with the mannose receptor on macrophages and DC-

SIGN on dendritic cells, inhibiting IL-12, TNF-alpha and nitric oxide production (51,

20). Moreover clinical isolates from Mycobacterium tuberculosis with truncated

ManLAM structures have a low association with mannose receptor and altered immunological properties, ie, they are defectively phagocytosed by THP-1 cells and primary human macrophages (55). Therefore, since M. tuberculosis, M. bovis and

M.leprae are pathogenic, while M. smegmatis and M. chelonae are non-pathogenic, studies on APCs have tended to implicate the mannose cap for the anti-inflammatory effects seen with ManLAM. T cells lack mannose receptors, but LAM, via its GPI anchors (acyl fatty tails) can associate with T cell membranes (56, 57). This thesis focuses on CD4+ T cells. Although we did not design studies to rule out the role of mannose cap in modulation of T cells, studies described in both chapter 2 and 3 of the thesis also examine the requirement of LAM insertion into lipid rafts in modulation of

CD4+ T cell activation.

LAM and T cell receptor signaling and regulation of T cell activation

The T cell receptor (TCR) was first described in the early 1980s (58, 59). TCR engagement promotes multiple signaling cascades that ultimately determine cell fate through regulating cytokine production, proliferation, cell survival and differentiation

(60). The engagement of TCR/CD3 complex on naïve T cells by MHC-peptide complex initiates a complex process that leads to the induction of cytokines, cell surface receptors, and proliferation and differentiation into effector and memory T cells (61, 62). The signaling cascades produced by the binding of antigen to the TCR have been worked out over the past few years (63-66). Upon TCR/CD3 complex engagement by MHC-peptide complex, Lck, a member of Src family kinases phosphorylates immunoreceptor tyrosine- based activation motifs (ITAMs) on CD3-zeta on the cytosolic side of the TCR/CD3 complex. Once phosphorylated, ITAMs become recruitment sites for molecules containing Src-homology domain-2 (SH-2) including Zeta chain-associated protein kinase of kilodalton 70 (ZAP-70), a member of Syk family of kinases. ZAP-70 gets phosphorylated at amino acid residues including tyrosine 319 by Lck (67-69). Activated

ZAP-70 then promotes recruitment and phosphorylation of downstream adaptor or scaffold proteins including linker for activation of T cells (LAT), SH-2 domain- containing leukocyte phosphoprotein of 76 KDa (SLP76), and phospholipase C. These adaptor proteins branch the signal into multiple pathways that each can activate a unique transcription factor. The most well characterized pathways are Ca2+ /calcineurin signaling, PKC pathway and the Ras/MAPK/ERK pathways (63, 70). These pathways activate a group of transcription factors, including NFAT, NF-kB and AP-1 that promote

IL-2 gene expression and T cell activation (71). Signaling through the TCR is not

14 sufficient for full T cell activation, and a second signal, co-stimulation is required via the binding of CD28 to CD80/CD86 (72-74). CD28 is constitutively expressed on T cells while CD80 and CD86 are inducible and expressed in mature, activated APCs. This pathway leads to activation of the transcription factor AP-1, which, together with partner,

NFAT promotes transcription of the IL-2 gene and IL-2 production. In addition lipid rafts are important for the initiation of effective TCR signaling and activation.

Lipid rafts are referred to as specialized microdomains within the plane of the plasma membrane with a lipid composition different from the glycerophospholipid bilayer of the surrounding membrane. They are made up of glycosphingolipids, sphingomyelin and cholesterol (62, 75, 76). The proteins referred to as

Glycosylphosphatidylinositol (GPI)-anchored proteins and the ganglioside GM1, preferably localize in lipid rafts. These proteins are used as markers for membrane rafts, and GM1 is the target of cholera toxin B subunit (77). Lipid rafts act as organizing centers for membrane assembly of signaling complexes important in initiation of signaling and T cell activation. The early TCR signaling molecules, Lck, and adaptor protein, LAT reside in lipid raft domains (77). For productive activation, the rafts have to be mobile and upon engagement of TCR with its ligand, allow clustering of TCR, CD3 and other key proximal TCR signaling molecules to initiate effective signaling.

Interference with any of the components of this signaling cascade may result in inhibition of T cell activation: a previous study, for example, showed that T cells deficient in ZAP-

70 have greatly decreased TCR-induced tyrosine phosphorylation for downstream signaling molecules (78). Other studies reveal that some virulent bacteria including

Helicobacter pylori, Yersinia pestis and Salmonella typhimiurium release protein

15 molecules that interfere with TCR signaling to escape host immunity or cause significant immunopathology (79, 80). Mahon et al. demonstrated that LAM from Mtb directly inhibit CD4+ T cell activation by down-regulating ZAP-70 phosphorylation (81). How

LAM down-regulates ZAP-70 phosphorylation was not addressed. Studies in chapter 2 focus on the mechanisms underlying LAM-induced inhibition of CD4+ T cell activation and lipid raft integrity.

T cell activation is tightly regulated to maintain immune homeostasis and prevent autoimmunity while helping to eliminate the pathogen. Studies show that multiple complex pathways, with competing feedback mechanisms operate in the regulation of T cell activation. These pathways are active during TCR signaling and aid the TCR to not only discriminate between strong (agonists) and weak ligands (antagonists), but also the environment in which the antigen is encountered (82, 83). Regulation can occur at any part of the TCR signaling cascade, both proximal or distal. The most studied positive pathways are the Ca2+ arm of the TCR signal transduction pathway and the

PKC/IKK/Ras/MAPK arm that are predominant when strongly binding ligands and full co-stimulation (CD28 signaling) stimulate the TCR /CD3 complex, involving Lck modification by ERK, resulting in long lasting signaling for gene expression and productive T cell activation (61, 83). In contrast stimulation of the TCR with weakly binding ligand predominantly activate negative feedback loops leading to rapid gene expression and recruitment of proteins that down-regulate TCR signaling and IL-2 gene transcription, resulting in partial/suboptimal T cell activation (84, 85). Suboptimal T cell activation is well known first step in the induction of T cell anergy (86, 87).

T cell anergy

The TCR like the B cell receptor recognize not only antigens derived from organisms and pathogenic cells, but also self-antigens expressed on the body’s own tissues and nonpathogenic antigens (eg allergens) responsible for allergic reactions. In healthy individuals, self-antigens are not supposed to elicit a significant immune response. This is because self-reactive T cells are clonally eliminated by negative selection in the thymus during development (central tolerance), and cells that survive this process are rendered tolerant to self-antigens in the periphery (88). The main mechanism for inducing peripheral T cell tolerance is anergy induction, an intracellular process in which antigen receptors (TCR) become uncoupled from their downstream signaling pathways (Ca2+ /calcineurin signaling, PKC and the Ras/MAPK/ERK pathways) (86, 89).

Schwartz et al referred to T cell anergy as “a hypo- or nonresponsive state, in which a T cell is intrinsically functionally inactivated following an antigen encounter, but remains alive for a long time in a hyporesponsive state”. Functionally, anergy is characterized by defective proliferation and IL-2 production by previously primed T cells upon re- stimulation. Anergy induction appears to be a negative feedback process in the regulation of T cell activation.

Models of T cell anergy induction

The first and most studied in vitro anergy induction model to be characterized is

TCR engagement with its cognate antigen in the absence of effective costimulation (89).

The other models used to induce in vitro T cell anergy are still being characterized, but include pretreatment of T cells with calcium ionophore ionomycin, anergizing cytokines

17 including IL-10, TGF-β, and suppression via T regulatory cells (89, 90). These models, although still being elucidated, induce anergy when the TCR is engaged with strongly binding ligand in the presence of full costimulation, and mimic TCR stimulation without full costimulation, especially in respect to inducing upregulation of anergy-inducing proteins (90). The other means of inducing anergy involves engagement of the TCR with weakly binding ligands (self or altered peptides, high concentrations of soluble peptides) in presence of full costimulation (91).

In vivo anergy is induced in TCR transgenic mice by administration of cognate protein antigens via intravenous or oral route (91, 92). The studies in chapter 3 focus on in vitro anergy induction by the mycobacterial glycolipid LAM.

Mechanisms of anergy induction

T cell anergy induction occurs at priming, and is a rapid and active process that is closely linked to inhibition of T cell activation (86). The observed defects in most models are the absence of CD28 signaling or the inability to provide strong TCR signaling through the calcium/calcineurin and/or PKC/IKK/Ras/MAPK pathways to synergize with an intact

CD28 signaling pathway to produce sufficient IL-2 to block the induction of inhibitors i.e. anergy-inducing proteins. According to several studies the following defects in the

TCR signaling pathway and its downstream biochemical pathways underlie T cell anergy induction: partial or blocked proximal TCR signaling, increased Ca2+/calcineurin or decreased ras/MAP kinase arms of the TCR signaling pathway and blocked IL-2R signaling (92-94). Studies demonstrate that engagement of the TCR in the presence of anergizing stimuli such as ionomycin, promotes dominant Ca2+ signaling that activates

NFAT, promoting rapid expression of genes and production of anergizing proteins that block proximal TCR signaling molecules or cytokine production. NFAT belongs to a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. When cells are activated, NFAT is dephosphorylated by the Ca2+/calmodulin-dependent phosphatase calcineurin, translocates to the nucleus and becomes transcriptionally active.

In the nucleus, it cooperates with the transcription factor AP-1 (Fos/Jun), to induce expression of cytokine genes including the IL-2 genes that are central for productive T cell activation. In the absence of costimulation, only NFAT is activated, and this induces expression of anergy-associated genes. The most studied of these are the E3 ubiquitin ligases GRAIL, Cbl-b and Itch (61, 62, 95-100, 101-103). Work in chapter 3 of the thesis focuses on GRAIL.

GRAIL (Genes Related to Anergy In Lymphocytes)

GRAIL is a 428 amino acid type 1 transmembrane single subunit ubiquitin ligase protein with a cytosolic Zinc-binding RING finger domain and a luminal or extracellular protease-associated (PA) domain. GRAIL is localized to the transferrin-recycling endocytic pathway, and targets proteins for proteosomal degradation (94, 104, 105). Its

RING finger domain functions as an E3 ubiquitin ligase, while the PA domain captures transmembrane protein targets for ubiquitination. GRAIL is constitutively expressed in the brain, liver and kidneys, but exclusively inducible only in T cells. Its role in the brain and kidneys is still not clear, but thought to be related to regulation of autoimmunity and cell survival (92). In the liver, GRAIL plays a role in maintenance of glucose metabolism

(106). GRAIL is also implicated in the modulation of cell cycle progression in cancers

(107) and Crohn’s disease (108). However, in T cells, GRAIL expression has been highly

19 implicated in negative regulation of T cell activation (109-123). Recent studies, for example, show that GRAIL deficiency in Th2 cells is associated with allergies including asthma (124). GRAIL mRNA and protein are present at basal levels in naïve, effector and memory T cells (90), suggesting a ready role to counter weak TCR stimulation upon T cell activation. GRAIL levels increase but quickly return to baseline to allow productive

T cell activation to proceed. This is thought to be due to overwhelming TCR signal strength with positive regulators of T cell activation that down-regulate or degrade

GRAIL. However, TCR stimulation, under anergizing conditions, promotes sustained expression of GRAIL.

GRAIL is induced and maintains anergy in T cells by targeting several proteins along the TCR signaling pathway or other pathways important in T cell activation and proliferation. Although there are conflicting studies, GRAIL’s targets include TCR/CD3- zeta, CD40L, Otubain-1 and ERK (90, 92, 94, 98, 99).

Other factors that promote and/or induce T cell anergy are inhibitory receptors that are normally expressed upon T cell activation. These include CTLA-4, PD-1, Lag 3 and Tim-3 that can negatively control TCR signaling and may induce anergy. CTLA-4, a receptor expressed on activated T cells, is induced during T cell activation. CTLA-4 can negatively regulate T cell activation through mechanisms that are still poorly understood

(101). CD86 and CD80 bind CTLA-4 with a higher affinity than with CD28, and induce inhibitory signaling cascades that ultimately block the activation of NFAT, NF-kB and

AP-1 (125). Signaling through CTLA-4 is also implicated in selective blocking of ERK and JNK in the MAPK pathway (126).

Other factors that confound anergy

Other regulatory mechanisms that decrease effector T cell function in peripheral tissues and may be mistaken for anergy are apoptosis, T cell exhaustion and downmodulation of TCR/CD3 after priming (127). Apoptosis is an organized process that controls the orderly death of damaged cells and exhausted cells. Apoptosis signals cells to self- destruct to allow cell renewal or control aberrant cell growth. Changes in the plasma membrane are one the earliest or first characteristics of the apoptotic process detected in live cells. Early apoptosis can be detected by presence of phosphatidylserine (PS), which is normally located on the cytoplasmic face of the plasma membrane. During apoptosis

PS translocates to the outer leaflet of the plasma membrane and can be detected by flow cytometry and cell imaging through binding to the fluorochrome-labeled Annexin V when calcium is present (128). Steroids such as dexamethasone induce apoptosis of T cells by upregulating pro-apoptotic proteins (129). Some pathogens including Mtb have been reported to induce apoptosis in macrophages (130). T cell exhaustion is associated with increased and sustained expression of inhibitory receptors, notably PD1 in chronic conditions, although it usually occurs later than anergy induction (127). In addition to expression of inhibitory receptors, down-modulation of TCR occurs after priming in the peak phase of T cell response to antigen. This process limits T cell activation and allows the contraction phase, without which autoimmunity would occur. Downmodulation of

TCR may result in hyporesponsiveness upon restimulation especially when restimulation is done after a short time of resting following priming (89). Work in chapter 3 also focus on these anergy confounders in the LAM-induced anergy model.

Pathogens and anergy induction

Recent studies show that pathogens such as HIV-1 and Schistosoma mansoni may take advantage of anergy induction to escape the host’s immune response (131-133).

Schistosoma mansoni worms have been shown to induce anergy in T cells (133), while anergy induction favors HIV-1 replication in human T cells (131). It is not known whether Mtb can induce anergy in T cells. After demonstrating that Mtb inhibits proximal

TCR signaling, we hypothesized that LAM-induced inhibition of T cell activation may be associated with T cell anergy induction. Chapter 3 addresses the role of LAM in anergy induction

Reversal of T cell anergy

In vitro anergy induction can be reversed by addition of exogenous IL-2 at restimulation (86). However, there are few concrete reports on in vivo reversal of anergy induction such as the use of IL-2 to treat some disease conditions. The mechanisms underlying IL-2-dependent reversal of T cell anergy are not clear, however, recent reports show that signaling through the IL-2 receptor activates mTOR pathway, which plays an important role in the integration of signals that not only determine the fate of T cells, but also reverse anergy.

In summary, we already determined that LAM inhibits polyclonal murine CD4+ T cell activation by downregulating ZAP-70 phosphorylation. In the following chapters 2 and 3, we further explore the mechanism and impact of inhibition of proximal TCR signaling in both murine and human CD4+ T cells using both polyclonal and antigen-specific system, and specifically determine if this inhibition is associated with anergy induction.

Chapter 2: Mycobacterium tuberculosis ManLAM inhibits T-cell-receptor signaling by interference with Zap70, Lck and LAT phosphorylation

Abstract

Immune evasion is required for Mycobacterium tuberculosis to survive in the face

of robust CD4+ T cell responses. We have shown previously that M. tuberculosis cell wall glycolipids, including mannose-capped lipoarabinomannan (ManLAM), directly

+ inhibit polyclonal murine CD4 T cell activation by blocking ZAP-70 phosphorylation.

We extended these studies to antigen-specific murine CD4+ T cells and primary human T cells and found that ManLAM inhibited them as well. Lck and LAT phosphorylation also were inhibited by ManLAM without affecting their localization to lipid rafts.

Inhibition of proximal TCR signaling was temperature sensitive, suggesting that

ManLAM insertion into T cell membranes was required. Thus, M. tuberculosis

ManLAM inhibits antigen-specific CD4+ T cell activation by interfering with very early events in TCR signaling through ManLAM’s insertion in T cell membranes.

Introduction

Mycobacterium tuberculosis infects and persists in a substantial portion of the world’s population making it one of the world’s most important pathogens (1). M. tuberculosis’ ability to survive in the host despite eliciting strong innate and adaptive immune responses is dependent on mechanisms of immune evasion (2, 3). These evasion mechanisms include resistance to macrophage killing, inhibition of phagosome maturation and indirectly suppressing CD4+ T cell recognition of M. tuberculosis infected cells by interfering with MHC-II antigen processing.

Recent reports have shown that M. tuberculosis also can directly inhibit T-cell function (56, 134). We recently demonstrated that glycolipids, specifically mannose- capped lipoarabinomannan inhibit T-cell receptor signaling through suppression of ZAP-

70 phosphorylation (135). These results are consistent with what has previously been reported (56, 136). However the mechanism of inhibition is unknown. Although

ManLAM binds host receptors including the mannose receptor, dendritic-cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), and CD14, these receptors are not expressed on T cells (137). ManLAM can interact with host cells, including T cells, independent of receptor binding by directly inserting into cell membranes (57, 138). Through their glycosylphosphatidylinositol (GPI)-anchor, M. tuberculosis glycolipids can insert themselves within GPI rich domains of cellular membranes such as lipid rafts, rich in cholesterol and sphingolipids that act as a platform for cell signaling (139, 140).

ManLAM insertion into GPI rich domains can modulate T cell and macrophage function (141). One study of LAM’s effect on Th1 cytokine mRNA expression found

LAM present in lipid rafts of Th1 cells resulting in increased activation of Lck and

Cbp/PAG, a negative regulator of Lck (56). Others have shown that LAM insertion into lipid rafts contributes to blocking phagosome maturation in macrophages with a similar effect recently reported with lipophosphoglycan from Leishmania donovani (138, 142).

In this study we extended our observation of direct inhibition of T cell activation by M. tuberculosis glycolipids in two directions. First we determined if ManLAM inhibition of murine primary CD4+ T cells could be extended to antigen-specific CD4+ T cell activation by antigen presenting cells and whether human CD4+ T cells were similarly inhibited. Second, we determined the mechanism of ManLAM-mediated inhibition of

TCR signaling in terms of its effect on Lck and LAT phosphorylation and lipid raft integrity.

Materials and Methods

Mice

8-10-week-old female C57Bl/6 mice were purchased from Charles River Laboratories

(Wilmington, MA). DO11.10 TCR transgenic mice were that express TCRs specific for

d the OVA323-339 presented in the context of I-A (15). Mice were housed under specific- pathogen-free conditions. Studies were approved by the Institutional Animal Care and

Use Committee at Case Western Reserve University.

Cells and medium

Unless otherwise specified, all experiments were performed at 37°C in 5% CO2 atmosphere and serum-free HL-1 media (BioWhittaker, East Rutherford, NJ) supplemented with 1 µM 2-ME, 10 mM HEPES buffer, nonessential amino acids, 2 mM

L-glutamine, 100 µg of streptomycin, and 100 U of penicillin (complete HL-1;

BioWhittaker). Spleen cells from 8-10-week old wild-type C57Bl/6 mice, OVA-specific

DO11.10 or M. tuberculosis 85B antigen-specific TCR transgenic P25 mice (ref) were isolated and red blood cells lysed in hypotonic lysis buffer (10 mM Tris-HCl and 0.83% ammonium chloride). Spleen cells were plated in 100 mm tissue culture plates and

+ allowed to adhere for 1 Mtb at 37°C. Untouched CD4 T cells were purified from nonadherent spleen cells using CD4+ T cells negative isolation kits (Miltenyi Biotec,

Germany) following manufacturer’s instructions. Purity of CD4+ T cells was confirmed by flow cytometry and ranged between 88-95% (135). T-hybridoma cells, DB-1 and

1T1A, were generated as previously described (144), and maintained in DMEM

(BioWhittaker, East Rutherford, NJ) supplemented as indicated for complete HL-1 with

27 the addition of 10% heat-inactivated fetal bovine serum (Hyclone, Logan, Utah). Prior to use in a stimulatory assay T-hybridoma cells were washed and re-suspended in complete

HL-1.

Human T lymphoblasts were a gracious gift from Dr. Alan Levine and prepared as previously described (143, 144). Briefly, PBMC were purified from the blood of healthy donors by Ficoll-Hypaque density separation (Sigma-Aldrich, St. Louis, MO). PBMC were stimulated with 0.5% PHA (Invitrogen Life Technologies, Carlsbad, CA) in the presence of 5 ng/ml IL-2 (R&D Systems, Minneapolis, MN) in RPMI 1640, 10% heat- inactivated FBS and 25 mM HEPES for 48 Mtb. Cells were treated with 5 ng/ml IL-2 for

1 wk and rested for 24 Mtb prior to use in T-cell activation assays.

Antibodies and reagents

The following mAbs and antibodies were purchased for murine CD4+ T-cell activation: hamster anti-mouse CD3 mAb (145-2C11), hamster anti-mouse CD28 mAb (L3T4) and mouse anti-hamster immunoglobulin G1 (IgG1) from BD Biosciences (San Jose, Ca).

For human T cell activation, murine anti-human CD3 (OKT3) and anti-human CD28 were purchased from BD Bioscience and cross-linking sheep anti-mouse F(ab́)2 from

Sigma-Aldrich. For immunoblotting rabbit mAbs or polyclonal antibodies against ZAP-

70, phosphorylated ZAP-70 (Tyr319), Lck, phosphorylated Src-Tyr416 (recognizing phosphorylated Lck-Tyr394), phosphorylated Lck-Tyr505, LAT, phosphorylated LAT-

Tyr171 and phosphorylated LAT-Tyr191 were purchased from Cell Signaling

Technologies (Danvers, MA). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used for detection. The

28 pharmacological inhibitor 8-Bromoadenosine 3’,5’-cyclic monophosphorothioate, Rp- isomer was purchased from Sigma-Aldrich. Anti-LAM producing hybridoma cell line and mannose-capped LAM (ManLAM) from M. tuberculosis H37Rv were obtained through the Tuberculosis Vaccine Testing and Research Materials Contract (NIAID

HHSN266200400091C) at Colorado State University (CSU).

ManLAM purification

ManLAM was also purified from M. tuberculosis H37Ra using same method as used at

CSU. M. tuberculosis cells were delipidated by organic extraction with 10:10:3 chloroform, methanol water (CHCl3:CH3OH:H2O). Delipidated cells were re-suspended in lysis buffer containing 8% TX-114 and lysed in a French press. Lysates were extracted with 8% TX-114 as described previously (145). The detergent phase was precipitated with cold ethanol overnight and digested with pronase. Remaining protein- free extract was separated with size exclusion columns connected in tandem (S-100, S-

200, GE Healthcare) in an AKTA purifier system (Pharmacia Biotech, GE Healthcare).

Fractions were analyzed in 15% Tris-glycine polyacrylamide gels and glycolipid bands revealed with acid-silver stain. Three pools of fractions containing 34-kDa (pool 1,

ManLAM), 14-kDa (pool 2, LM) and 4-kDa (pool 3, PIM6) bands, respectively, were collected. Presence and purity of ManLAM in pool 1 were confirmed by Western blotting with mAb CS-35. H37Ra ManLAM purified at CWRU had same bio-activity for

TCR inhibition as H37Rv ManLAM obtained from CSU.

CD4+ T cell assays

6 For polyclonal activation resting murine CD4+ T cells or human lymphoblasts (1x10 cells/well) were activated in 96-well, flat-bottomed microtiter plates with 1 µg/ml of soluble CD28 mAb in wells coated with 1 µg/ml of CD3 mAb. Cells were stimulated in the presence or absence of ManLAM for 48 Mtb. For antigen-specific activation assays, purified OVA-specific DO11.10 were treated with or without ManLAM and cultured in the presence of peptide or protein pulsed IFN-γ pretreated bone-marrow derived macrophages (BMM) for 24 Mtb before collecting supernatants for IL-2 measurements as previously described. Supernatants were assayed for IL-2 production in Immulon 4HBX flat-bottomed microtiter plates (Thermo) coated with purified capture IL-2 mAb (1µg/ml) and detected with biotinylated IL-2 mAb (1 µg/ml), followed by alkaline phosphatase- conjugated streptavidin (Jackson ImmunoResearch) and phosphatase substrate (Sigma-

Aldrich). Plates were read with a Versa Max turntable microplate reader and data analyzed with Soft Max Pro LS analysis software.

Measurement of tyrosine phosphorylation

CD4+ T cells were rested overnight in complete DMEM supplemented with 1% FBS.

6 Cells (3x10 cells/ml) were re-suspended in complete HL-1 medium in 1.5ml eppendorf tubes (LBS, Rochester, NY) in 1 ml. Cells were incubated with ManLAM (10 µg/ml) for

1 Mtb at 37°C before activation. Cells were washed (10 min at 4,000 rpm) and re- suspended in 100 µl of HL-1. The TCR complex was activated by the addition of a cross-linking mouse anti-hamster IgG1 (10 µg/ml) for 2 min before the addition of anti-

CD3 (10 µg/ml) for 2 min. CD4+ T-cell activation was stopped by adding 100 µl of 2X

Laemmli sample buffer and boiling samples for 10 min. Unstimulated cells, incubated with mouse anti-hamster IgG1 alone, served as control for non-specific TCR-CD3 activation.

Isolation of lipid rafts by sucrose gradient centrifugation

Lipid rafts were purified from T-hybridoma cells as previously described (19). T- hybridoma cells (~2.5x107) were suspended in 2 ml of complete HL-1 medium in a 15 ml conical tube. ManLAM (10 µg/ml) or medium alone was added and cells were incubated for 1 Mtb at 37°C. Cells were washed (4,000 rpm for 10 min) and re-suspended in 100

µL of fresh HL-1. Cells were stimulated for 2 min with cross-linking hamster-anti-mouse

IgG1 followed by 5 min with anti-CD3 as stated above and immediately placed on ice.

Cells were spun down (4,000 rpm for 10 min) and supernatant re-suspended in 500 µL of

TKM buffer (50 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl2, and 1 mM EDTA) containing 1% Triton X-100 (Sigma-Aldrich) and a cocktail of protease inhibitors

(Roche). Cell suspension was homogenized with a Dounce homogenizer (~25-30 strokes) and lysed on ice for 30 min. Post-nuclear supernatant was prepared by spinning lysate down (4,000 rpm for 10 min) twice. 500 µl of 80% wt/vol sucrose (Sigma

Aldrich) in TKM buffer was added to lysate and transferred to SW41 tubes and overlaid with 6 ml of 38% wt/vol sucrose and topped with 3.5 ml of 5% sucrose in TKM buffer.

Sucrose gradients were ultra-centrifuged at 39,000 rpm for 18 Mtb in an SW41 rotor

(Becton Dickinson, Franklin Lakes, NJ) and 1 ml fractions collected from the top of the gradient to the bottom. For Western Blot analysis, 20 µl of each fraction was mixed with

31 an equal volume of 2X Laemmli buffer and boiled for 10 min.

Western Blotting

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% gel (Bio-Rad, Hercules, CA) under reducing conditions and electro-transferred to nitrocellulose membranes (Bio-Rad) in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol. After transfer, membranes were incubated at room temperature for 1

Mtb in SuperBlock (Thermo Scientific, Rockford, IL). Primary and secondary antibodies were diluted in 1% nonfat milk, 0.05% Tween-20 in PBS at the concentrations recommended by the manufacturer. For detection of ManLAM within lipid rafts, 1:2 dilution of anti-ManLAM hybridoma CS35 supernatant was incubated with nitrocellulose membranes at 4°C overnight. Following multiple washes with 0.05% Tween-20, membranes were incubated with conjugated secondary antibody for 1 Mtb at room temperature. Detection of HRP-conjugated Abs was performed using West Pico

Supersignal (Thermo Scientific). Chemiluminescence was detected with BioMax film

(Kodak).

Statistical Analysis

Statistical analysis was performed by using a one-tailed student t test. A p value of <

0.05 was considered significant.

Results

ManLAM inhibits antigen-specific murine and polyclonal human T cell activation

We previously demonstrated that M. tuberculosis glycolipids directly inhibit polyclonal activation of primary naïve and memory mouse CD4+ T cells in the absence of antigen presenting cells and that ManLAM as a major M. tuberculosis glycolipid was particularly potent (135). To determine if ManLAM inhibits antigen-specific activation, we performed experiments with DO11.10 CD4+ TCR transgenic T cells specific for

d OVA323-339 peptide in the context of I-A and IFN-γ activated BMM as APC. As shown in Fig. 1A, pretreatment of DO11.10 CD4+ T cells with 40 µg/ml ManLAM for 1 Mtb before adding antigen pulsed APC resulted in decreased IL-2 production. To control for

ManLAM carryover effects on APC, BMM were fixed with 1% paraformaldehyde before adding T cells and again inhibition of T cell activation was measured along a range of

OVA323-339 peptide concentrations (Fig. 2.1B). Purity of CD4+ T cells for these and subsequent experiments in this manuscript was 88-95% by flow cytometry. Pretreatment of CD4+ T cells was sufficient to inhibit their subsequent activation by antigen, since extensive washing of DO11.10 cells after a 1 Mtb incubation with a range of ManLAM concentrations did not affect ManLAM’s ability to inhibit antigen-specific T cells activation (Fig. 2.1C).

Figure 2.1. ManLAM inhibits antigen-specific CD4+ T cell activation. A. Bone marrow-derived macrophages (1 × 105 cells/well) were activated with IFN-γ overnight and then incubated with ovalbumin for 4 Mtb. Purified CD4+ T cells (5 × 104 cells/well) from DO11.10 TCR transgenic mice, pre-incubated with ManLAM (40 µg/ml) or medium alone for 1 Mtb, were added to antigen-pulsed APC for 24 Mtb. Purified CD4+ T cells (CD4+ T cells alone) were cultured with antigen to confirm functional depletion of APC from purified T cells. IL-2 production was measured by ELISA. B. IFN-γ treated macrophages were fixed with 1% paraformaldehyde and incubated with OVA323-339 peptide for 4 Mtb. CD4+ T cells pre-incubated with or without ManLAM were added to peptide-pulsed fixed APC for 24 Mtb, and supernatants harvested for IL-2 measurement. C. CD4+ T cells (2 × 106 cells) were incubated with ManLAM in increasing concentrations for 1 Mtb, and divided into 2 groups (1x106 cells each). One was washed extensively and the other remained in ManLAM. 5 ×104 CD4+ T cells/well from each 3 group were added to OVA323-339 peptide-pulsed, fixed APC for 72 Mtb, and [ H]- thymidine incorporation measured. Data presented are the mean (+/-S.D.) for triplicate wells. Results are representative of at least three experiments. Reproduced from R.N. Mahon et al (146); Copyright © 2012; Elsevier Inc. All rights reserved.

To extend these findings further, we determined whether human T cells were inhibited by ManLAM. Human T lymphoblasts, generated by activating PBMC with

PHA and IL-2 for 7 d, were re-stimulated with plate-bound anti-CD3 and soluble anti-

CD28 mAbs in the presence or absence of 10 µg/ml of ManLAM. In the presence of

ManLAM, IL-2 production by human lymphoblasts was inhibited by more than 50%, and this was associated with decreased ZAP-70 phosphorylation (Fig. 2.2). Overall, these results indicate that ManLAM not only inhibits the polyclonal activation of murine and human T cells but also directly interferes with antigen-specific activation of CD4+ T cells independent of its effects on APC.

Figure 2.2. ManLAM inhibits the activation of human T cells. A. In serum-free medium human T lymphoblasts (1x105 cells/well) were stimulated with increasing concentrations of plate-bound anti-CD3 (3-10 µg/ml) and soluble anti-CD28 (1 µg/ml) for 48 Mtb. Cells were co-cultured in the presence or absence of ManLAM (10 µg/ml). Supernatants were harvested and IL-2 measured by ELISA. Data points and values are means +/-S.D. of triplicate wells, and representative of three experiments. *P < 0.01. B. 3x106 human T-lymphoblasts were incubated with 10 µg/ml of ManLAM or medium alone at 37°C for 1 Mtb prior to activation. Cells were washed and incubated for 2 min with cross-linking sheep anti-mouse F(ab́)2 (10 µg/ml) followed by anti-human CD3 mAb (10 µg/ml) for 2 min. Western analysis was performed with antibodies to phosphorylated ZAP-70-Tyr319 (upper panel) and total ZAP-70 (lower panel). Results presented are representative of three independent experiments. Reproduced from R.N. Mahon et al (146); Copyright © 2012, Elsevier Inc. All rights reserved.

ManLAM inhibits phosphorylation of Lck Tyr-394 and LAT Tyr-191/-171 in addition to that of ZAP-70

Our initial study demonstrated that ManLAM inhibits phosphorylation of ZAP-

70, an early step in TCR signaling (135). To further characterize ManLAMs effect on

TCR signaling, we analyzed signaling upstream and downstream of Zap-70. Upon binding of the TCR with peptide loaded MHC, Lck, a Src family kinase, is phosphorylated at Tyr-394 allowing the kinase domain to phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) found on the TCR complex. ZAP-70 binds to these motifs and is subsequently phosphorylated by Lck (147, 148). As shown in Fig.

2.3A, phosphorylation of Lck at Tyr-394 was inhibited when murine CD4+ T cells were pretreated with ManLAM (10 µg/ml) before anti-CD3 activation. Phosphorylation of

ITAMs was also inhibited (data not shown)

Activated ZAP-70 phosphorylates adaptor molecule LAT at Tyr-171 and Tyr-191.

Activated LAT binds several molecules, including PLC-γ1, Grb2 and SLP-76 that activate downstream secondary signaling pathways. ManLAM decreased phosphorylation of LAT at both Tyr-191 and Tyr-171 (Fig. 2.3B-C). These findings indicate that

ManLAM blocks the entire proximal TCR signaling pathway.

Figure 2.3. ManLAM inhibits Lck and LAT phosphorylation. CD4+ T cells (3x106) were pre-incubated with 10 µg/ml of ManLAM or medium alone for 1 Mtb at 37°C. Cells were washed and cultured for 2 min with mouse anti-hamster IgG1 (10 µg/ml) followed by hamster anti-mouse CD3 mAb (10 µg/ml) for 2 min. Western analysis was performed with antibody to Lck-Tyr394 A., phosphorylated LAT-191 and LAT-171 B-C. Total Lck and LAT were detected to control for protein loading. Results presented are representative of at least three independent experiments. Reproduced from R.N. Mahon et al (146); Copyright © 2012; Elsevier Inc. All rights reserved.

ManLAM does not alter Lck Tyr-505 phosphorylation

M. tuberculosis cell wall glycolipids can modulate Cbp/PAG, a transmembrane protein involved in regulating Lck (4). Cbp/PAG regulates membrane localization of

Csk, a protein kinase that phosphorylates Lck at Tyr-505 inducing Lck’s kinase domain into an inactive conformation. Csk binding to Cbp/PAG is regulated through phosphorylation of Cbp/PAG and involves cAMP/PKA (149-151). To determine if this pathway had a role, we utilized two approaches. First, we cultured T cells with

ManLAM (10 µg/ml) in the presence or absence of PKA inhibitor, 8-bromoadenosine 3’,

5’-cyclic monophosphorothioate, Rp-isomer (50 µM) for 48 Mtb. PKA inhibitor did not reverse ManLAM’s inhibition of IL-2 production (Fig. 2.4A). At higher concentrations

(500 µM), there was a slight increase in IL-2 production due to non-specific activation effects. As a positive control for PKA inhibition, CD4+ T cells were stimulated in the presence of prostaglandin E2, a known cAMP/PKA inhibitor, resulting in reversal (up to

50%) of 10-8 M prostaglandin E2-induced T cell inhibition (data not shown).

Next we analyzed phosphorylation of Lck Tyr-505 in T cells stimulated with cross-linked anti-CD3 after pre-incubation with ManLAM. There was no difference in

Tyr-505 phosphorylation between cells pre-treated with or without ManLAM (Fig. 2.4B).

Thus, ManLAM does not appear to inhibit TCR signaling through the Cbp/PAG pathway.

Figure 2.4. ManLAM does not activate the cAMP/PKA pathway or inhibit Lck- Tyr505 phosphorylation. A. In complete HL-1 medium, CD4+ T cells (1x105 cells/well) were activated with plate bound anti-CD3 (1 µg/ml) and soluble anti-CD28 (1 µg/ml) for 48 Mtb in the presence of either 50 µM PKA inhibitor 8-bromoadenosine 3’, 5’-cyclic monophosphorothioate, Rp-isomer or medium alone. Cells were co-cultured with ManLAM (10 µg/ml) or medium alone. Following incubation, supernatants were harvested for IL-2 measurement. Data points are expressed as means of triplicate samples +/-S.D. Results are representative of four independent experiments. B. CD4+ T cells (3x106 cells) were pre-incubated with ManLAM (10 µg/ml) or medium alone for 1 Mtb at 37°C, and stimulated as in Fig. 2.3. Western analysis was performed with antibody to phosphorylated Lck-Tyr505 (upper panel) or total Lck (lower panel). Results are representative of at least three independent experiments. Reproduced from R.N. Mahon et al (146); Copyright © 2012; Elsevier Inc. All rights reserved.

ManLAM insertion into CD4+ T cell membranes is necessary for inhibition of proximal

TCR signaling

Through their glycosylphosphatidylinositol (GPI)-anchor, ManLAM can insert into host cell membranes, including GPI-rich lipid rafts (56, 57). Lipid rafts are organized microdomains of sphingolipids and cholesterol, and their integrity and reorganization is required for T cell activation (139, 140). Inhibition of proximal TCR signaling by ManLAM required pre-incubation and inhibition of antigen-specific activation was not affected by washing ManLAM treated CD4+ T cells (Fig. 1C), suggesting that membrane insertion was required. ManLAM’s insertion into host membranes is perturbed when cells are cooled to 4°C, due to loss of membrane fluidity

(57). Murine CD4+ T cells were pretreated with ManLAM at both 4°C and 37°C. There was no decrease in ZAP-70 phosphorylation of CD4+ T cells pre-incubated with

ManLAM at 4°C compared to cells pre-incubated at 37°C (Fig. 2.5).

To determine the role of ManLAM insertion into lipid rafts, we used a MHC-II restricted T-hybridoma system due to the large numbers of cells required to purify lipid rafts. We first verified that ManLAM inhibited T hybridoma activation (Fig. 2.6A-B).

To verify that ManLAM inserted into lipid rafts, rafts from T-hybridoma cells were isolated using sucrose gradient ultracentrifugation. Lysates from T-hybridoma cells pre- incubated with ManLAM (10 µg/ml) were ultra-centrifuged in a sucrose gradient and fractions (n=9, 1 ml) analyzed by Western blot. Lipid raft were in fractions 2 through 5, as determined by the presence of LAT and Lck. ManLAM was found in the lipid rafts of

ManLAM treated T hybridoma cells, confirming previous studies (56, 57, 138) (Fig.

2.6C).

Figure 2.5. Inhibition of TCR signaling by ManLAM is temperature sensitive. CD4+ T cells (3x106) were pre-incubated with 10 µg/ml of ManLAM for 1 Mtb at either 37°C A. or 4°C B. and stimulated as in Fig. 2.3. Western analysis was performed for phosphorylated ZAP-70-Tyr319 (upper panels) or total ZAP-70 (lower panels). Results are representative of three independent experiments. Reproduced from R.N. Mahon et al (146); Copyright © 2012; Elsevier Inc. All rights reserved.

ManLAM does not affect localization of LAT and Lck to lipid rafts in resting and activated CD4+ T cells.

Modulating lipid raft integrity affects T cell activation (143, 152). Next we determined if

Lck and LAT levels in rafts were changed by ManLAM. In non-activated T cells, exposure to ManLAM did not affect Lck and LAT localization in raft and non-raft fractions (data not shown). For T cell activation, T-hybridoma cells were pre-incubated with ManLAM (10 µg/ml) for 1 Mtb at 37°C, washed and stimulated for 5 min with cross-linked anti-CD3 mAb. Western blot analysis of sucrose gradient fractions of unstimulated, stimulated and stimulated in the presence of ManLAM T cells showed no decrease or major alteration in the distribution of Lck (Fig. 2.6D) or LAT (data not shown) among the three experimental groups. In fact, rather than decreases, minimal but consistent increased LAT or Lck levels were observed in rafts of ManLAM treated cells.

Thus, inhibition of TCR signaling by ManLAM was not due to major disruptions of Lck and LAT localization to lipid rafts.

Figure 2.6. ManLAM is found in T cell membranes and does not affect localization of Lck in lipid rafts of activated T cells. A. T-hybridoma cells (1x105 cells/well) were stimulated with plate bound anti-CD3 (1 µg/ml) for 6 Mtb in the presence or absence of ManLAM (10 µg/ml). Supernatants were for IL-2 measurement. Data points and values are means +/-S.D. of triplicate wells, and representative of three experiments. *P < 0.01. B. 3x106 T-hybridoma cells were incubated with 10 µg/ml of ManLAM or medium alone at 37°C for 1 Mtb prior to activation. Cells were stimulated as in Fig 2.3. Western analysis was performed with antibody to phosphorylated ZAP-70-Tyr319 (upper panel) and total ZAP-70 (lower panel). C. T-hybridoma cells (2.5x107 cells) were incubated in the presence or absence of ManLAM (10 µg/ml) for 1 Mtb at 37°C. Cells were subjected to discontinuous sucrose density gradient ultracentrifugation. 1 ml fractions (Fract.) were collected from the top to the bottom and ManLAM distribution within fractions analyzed by Western with anti- ManLAM mAb CS-35. Results are representative of two independent experiments. D. T-hybridoma cells (2.5x107 cells) were incubated in the presence or absence of ManLAM (10 µg/ml) for 1 Mtb at 37°C. Cells were stimulated as in Fig. 2.3. Cells were lysed and subjected to discontinuous sucrose density gradient ultracentrifugation. 1 ml fractions were collected from top to the bottom from unstimulated (upper panel), stimulated (middle panel), and stimulated T cells in the presence of ManLAM (lower panel). Western analysis was performed for the distribution of Lck. Results are representative blots of four independent experiments. Reproduced from R.N. Mahon et al (146); Copyright © 2012; Elsevier Inc. All rights reserved.

Discussion

We previously reported that M. tuberculosis glycolipids, including ManLAM, directly inhibited polyclonal CD4+ T cell activation by blocking ZAP-70 phosphorylation. In this study, we focused on ManLAM, the major cell wall glycolipid of M. tuberculosis, and extended these observations to antigen-specific CD4+ T cell responses, to human T cells and further analysis of ManLAM’s effect on proximal TCR signaling. Phosphorylation of signaling proteins were inhibited both upstream (Lck) and downstream (LAT) of ZAP-70, indicating that ManLAM inhibited TCR signaling at the site of triggering. ManLAM did not affect phosphorylation at Lck-Tyr505 and was independent of PKA indicating that negative regulator Csk was not involved. As reported by others, ManLAM inserted into T cell membranes including lipid rafts. In addition, temperature sensitivity and washing experiments suggest that membrane insertion by ManLAM is required for inhibition of proximal TCR signaling. ManLAM insertion did not affect the presence Lck and LAT in lipid rafts in T cells.

Others have postulated that ManLAM’s insertion in membranes modulates host cell behavior (56, 138). However, no study has definitively shown that membrane insertion itself is responsible for ManLAM’s observed effects. Our results and others support two divergent hypotheses. The first is that insertion of ManLAM into T cell membranes is required and sufficient. In this model, membrane insertion activates an unknown signaling event(s) or inhibits the ability of membranes to reorganize themselves upon stimulation. This hypothesis is supported by Shabaana et al. of increased kinase activity within lipid rafts of Th1 cells (56). We did not observe increased

45 phosphorylation of Lck at either its positive or negative regulatory site. Additionally our data indicates that Csk, a negative regulator of Lck, is not activated by ManLAM. An alternative hypothesis is that membrane insertion is necessary but the effect is not dictated by lipid raft disruption. ManLAM insertion via its GPI motif acts as a tether allowing other components of ManLAM (such as its mannose cap) to bind or interfere with T cell surface molecule(s), possibly the TCR complex itself, and thus affect their function. This model has been proposed for ManLAM’s ability to inhibit phagosome maturation after insertion into macrophage lipid rafts and is supported by our previous finding that PIM, while having the phosphoinositol backbone of ManLAM, is much less efficient on a per molecule basis in inhibiting T cell activation (135, 138).

Lipid rafts are defined by their insolubility in detergent and their ability to float to low density in sucrose density gradient centrifugation (153, 154). Upon TCR engagement, these rafts coalesce at the site of activation, bringing with them signaling molecules such as Lck and LAT required for transducing the TCR signal. This model has been criticized because of the method used to isolate lipid rafts (155, 156). Sucrose density gradients give a static picture of rafts and only determine whether or not certain molecules are present. They do not address raft structure nor establish if proper microcluster formation is occurring properly at the T cell-APC synapse. Confocal microscopy and other more advanced imaging techniques have broadened our understanding of the dynamics and kinetics of T cell stimulation induced microcluster formation. Lillemeier et al. used a combination of photoactivated localization microscopy, fluorescence cross-correlation spectroscopy and transmission electron microscopy to show that TCR and LAT are found on separate protein islands that

46 coalesce upon activation (157). Total internal reflection fluorescence microscopy has been used to detect TCR microcluster formations that include ZAP-70, Lck, and other signaling components that form within few seconds of TCR engagement (158, 159).

Further studies of ManLAMs activity within rafts may identify a T cell regulatory mechanism that ManLAM hijacks to benefit M. tuberculosis survival.

In addition to blocking microcluster formation, ManLAM could induce phosphatase activity. Numerous phosphatases negatively regulate T cell activation by dephosphorylating Lck, ZAP-70 and ERK (160, 161). Studies of microbial inhibition of

T cells have identified several instances where pathogens either through the induction of phosphatase activity or through their own phosphatases inhibit proximal TCR signaling

(162-164). Knutson et al. has shown that LAM by increasing phosphatase activity suppressed MAPK signaling in macrophages (165). Phosphatase Src homology 2 containing tyrosine phosphatase (SHP-1) had a major role. SHP-1 regulates proximal

TCR signaling and its activity itself is regulated at several points that ManLAM could take advantage of (166). ManLAM did not inhibit IL-2 production by anti-CD3/CD28 activated Jurkat T cells (data not shown) known to be missing inositol phosphatases

SHIP-1 and PTEN (165, 167). These phosphatases regulate phosphorylation downstream of proximal TCR signaling. PTEN is involved in regulation of phosphoinositide 3-kinase

(PI3K) pathway, utilized by some co-stimulatory receptors.

Modulation of host immunity by glycolipids is uncommon except among intracellular pathogens such as Leishmania (lipophosphoglycan), Trypanosoma

(glycoinositol phospholipids, GIPL) and mycobacteria. These pathogens block phagosome maturation, suppress cytokine production and modulate surface expression of

MHC molecules (168, 169). While these glyolipids have unique head groups, they share a glycosylphosphatidylinositol (GPI) anchor which is important for their effects on host immunity (170-172). Tachado et al. purified GPIs from Plasmodium, Trypanosoma and

Leishmania and found that they induced protein kinase C activity (173). This study was performed in macrophages and to our knowledge no one has looked at their effects on T cells. In fact, Trypanosoma GIPL is the only other microbial glycolipid known to directly modulate T cell activation (174, 175). T-cell studies by others have suggested that ManLAM can induce PKC signaling to modulate host immunity, however, in our earlier study, PKC activation by phorbol myristate acetate (PMA) was not inhibited by

ManLAM (135, 174). Besides ManLAM, the only other microbial glycolipid known to directly modulate T cells is Trypanosoma GIPL. Since microbial glycolipids have similar effects on other cell types, microbial lipophosphoglycan and glycolipids may have similar inhibitory effects on T cell activation as seen for ManLAM. To our knowledge we are the first to determine that microbial glycolipids such as ManLAM can directly inhibit proximal TCR signaling. These findings not only provide insight into how M. tuberculosis evades host immunity but also how TCR activation may be regulated by microbes.

Chapter 3: Mannose-Capped Lipoarabinomannan from Mycobacterium tuberculosis

Induces CD4+ T cell Anergy via GRAIL

Abstract

Mycobacterium tuberculosis cell wall glycolipid, Lipoarabinomannan, can inhibit CD4+

T cell activation by down-regulating phosphorylation of key proximal TCR signaling molecules Lck, CD3ζ, ZAP70 and LAT. Inhibition of proximal TCR signaling can result in T cell anergy, in which T cells are inactivated following an antigen encounter, yet remain viable and hyporesponsive. We tested whether LAM-induced inhibition of CD4+

T cell activation resulted in CD4+ T cell anergy. The presence of LAM during primary stimulation of P25TCR-Tg murine CD4+ T cells with M. tuberculosis Ag85B peptide resulted in decreased proliferation and IL-2 production. P25TCR-Tg CD4+ T cells primed in the presence of LAM also exhibited decreased response upon re-stimulation with

Ag85B. The T cell anergic state persisted after the removal of LAM. Hypo- responsiveness to re-stimulation was not due to apoptosis, generation of FoxP3-positive regulatory T cells or inhibitory cytokines. Acquisition of the anergic phenotype correlated with up-regulation of GRAIL (gene related to anergy in lymphocytes) protein in CD4+ T cells. Inhibition of human CD4+ T cell activation by LAM also was associated with increased GRAIL expression. Small interfering RNA-mediated knockdown of GRAIL before LAM pre-treatment abrogated LAM induced hypo-responsiveness. In addition, exogenous IL-2 reversed defective proliferation by down-regulating GRAIL expression.

These results demonstrate that LAM up-regulates GRAIL to induce anergy in Ag- reactive CD4+ T cells. Induction of CD4+ T cell anergy by LAM may represent one mechanism by which M. tuberculosis evades T cell recognition.

Introduction

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen and leading cause of morbidity and mortality worldwide. Most individuals require adaptive T cell immunity to control Mtb but fail to eradicate the bacilli. T cells and infected antigen presenting cells (APC) are central for control of Mtb but also targets of its immune evasion strategies. Mtb infection results in the activation of multiple T cell subsets that recognize a very diverse repertoire of antigens. Paradoxically, despite this extensive T cell repertoire, small numbers of Mtb bacilli survive and persist in granulomas by evading immune recognition and elimination.

Major histocompatibility complex class II (MHC-II) molecule-restricted CD4+ T cells have a central role in the T cell response to Mtb. Recent studies have demonstrated that CD4+ T cells from persons who have controlled Mtb infection recognize a very diverse range of antigens (176-179). Antigenic variation among Mtb strains for CD4+ T cells is minimal and an unlikely mechanism of immune evasion (180). In light of these broad responses, it is likely that Mtb’s T cell immune evasion strategies involve direct effects on APC and/or CD4+ T cell function. Earlier studies determined that Mtb can inhibit MHC-II antigen processing in macrophages in a TLR-2 dependent manner and thus indirectly affect memory and effector CD4+ T cell function (10, 35, 36, 181-183).

Exosomes and microbial microvesicles provide a mechanism for Mtb molecules to be directly delivered to CD4+ T cells in the immediate microenvironment of Mtb infection. Mannose-Capped Lipoarabinomannan (LAM) is one of the most abundant glycolipids in the Mtb cell wall and readily found in Mtb microvesicles (37). Our earlier

51 studies showed that LAM can inhibit CD4+ T cell activation by down-regulating phosphorylation of the key proximal TCR signaling molecules Lck, CD3ζ, ZAP-70 and

LAT in a TLR-2 independent manner (135, 146). LAM can interact with host cells by directly inserting into cell membranes, in addition to binding to host receptors (MR, DC-

SIGN, Dectin-2, CD14) expressed on APC (56, 57, 137, 138).

Assays used to measure effects of LAM on CD4+ T cell activation were short- term and did not address long-term effects of LAM on T cell function. Was LAM inhibition a transient phenotype, were Tregs activated, was there evidence for apoptosis or anergy? Anergy is characterized by persistent defective proliferation and IL-2 production by previously activated T cells upon re-stimulation (89, 184). Different biochemical pathways initiate and maintain the anergic state, including blockade of the

Ras-MAPK pathway, and defects in ZAP70 and LAT phosphorylation (89, 184, 185).

Gene related to anergy in lymphocytes (GRAIL) negatively regulates IL-2 transcription

(90, 105, 186, 187) and up-regulation of GRAIL is associated with induction and maintenance of anergy (90, 94). Anergy also occurs when T cells are stimulated either in the presence of TGFbeta and IL-10, or suppression by regulatory T cells (Treg) (90, 94,

188). Anergy induction may be a mechanism of immune evasion in chronic infections by

SIV, HIV-1 and Schistosoma mansoni mostly due to manipulation of co-stimulatory molecules or up-regulated inhibitory cytokine production by APC (131, 133, 189-191).

Using an antigen specific system we determined the longer-term functional implication of

LAM inhibition of CD4+ T cell activation. P25 TCR Tg CD4+ T cells activated in the presence of LAM were anergized. Once anergy was established, LAM was no longer required. After 5 days of rest, LAM was no longer detectable in T cells, yet CD4+ T cells

52 previously treated with LAM proliferated poorly. Proliferation of anergic T cells was rescued by IL-2. The induction of anergy correlated with up-regulation of GRAIL in

CD4+ T cells. LAM treatment of human CD4+ T cells also induced GRAIL protein.

Inhibition of GRAIL mRNA with siRNA before LAM pre-treatment reduced T cell inhibition in naïve and Th1 polarized effector CD4+ T cells. Moreover, exogenous IL-2 reversed defective proliferation in LAM-anergized CD4+ T cells by down-regulating

GRAIL expression. We conclude that LAM up-regulates GRAIL expression to induce anergy in Mtb-reactive CD4+ T cells. Anergy induction by LAM is another mechanism by which Mtb can evade CD4+ T cell recognition.

Materials and Methods

Antigens and Antibodies

Mtb Ag85B encompasses the major epitope (aa 240-254) recognized by P25 TCR-Tg T cells (peptide 25). Peptide 25 (NH2-FQDAYNAAGGHNAVF-COOH) was purchased from Invitrogen. LAM, anti-LAM Ab (Cs-35, 1:250 titer) and biotinylated Cs-35 (anti-

LAM) from M. tuberculosis H37Rv were obtained from the Tuberculosis Vaccine

Testing and Research Materials contract (NIAID HHSN266200400091C) at Colorado

State University (CSU). The following mAbs and isotype controls were purchased for analysis of receptor expression: anti-CD3-PE, anti-CD4-APC, anti-CD28-APC, anti-

Tim3-PE, anti-CTLA4-PE, anti-CD25-alexa Fluor-488, anti-FoxP3-PE, anti-Annexin V-

Alexa Fluor 450, anti-cholera toxin subunit B-Alexa Fluor 647, anti-CD3-Alexa Fluor

647, anti-mouse IgG-Alexa Fluor 488, and LIVE/DEAD violet and yellow cell stain (all from ebioscience); anti-PD1-PE (BD Pharmingen); anti-Lag3-APC (Biolegend); anti-

TCR-Vβ-PE and anti-CD40L-PE (Miltenyi Biotech).

For T-cell activation, hamster anti-mouse CD3ε (145-2C11), anti-mouse CD28 (clone

37.51), mouse anti-human CD3 (clone HIT3a), mouse anti-human CD28 and mouse anti- hamster secondary immunoglobulin G1 (IgG1) were purchased from BD Biosciences.

For Western blotting and intracellular staining for GRAIL, rabbit anti-GRAIL antibodies were purchased from Thermoscientific and Abcam and goat anti-rabbit IgG-FITC from

Southern Biotech. Horseradish peroxidase (HRP)-conjugated secondary anti-rabbit mAb

(Jackson ImmunoResearch) was used for detection. For mouse and human IL-2 ELISA, primary and biotin-conjugated secondary antibodies were purchased from ebioscience.

Recombinant mouse IL-7 (407-ML-005) was purchased from R&D systems. Ionomycin

(I3909) and dexamethasone were purchased from Sigma-Aldrich.

Mice

Eight to ten-week-old female C57BL/6J were purchased from Charles River Laboratories

(Wilmington, MA). Mycobacterial Ag85B-specific TCR transgenic (P25 TCR-Tg) mice were provided by Kiyoshi Takatsu (University of Tokyo, Japan) (192). P25TCR-Tg T cells recognize peptide (NH2-FQDAYNAAGGHNAVF-COOH) derived from Mtb

Ag85B in the context of MHC II I-Ab (192). Mice were housed under specific pathogen- free conditions. All experiments were performed in compliance with the U.S. Department of Health and Human Services Guide for the care and use of Laboratory Animals and were approved by the Institutional Animal care and Use Committee at Case Western

Reserve University (protocol number: 2012-0020).

Isolation of mouse CD4+T cells

Mouse CD4+ T cells were isolated from spleens of 8- to 10-week old P25 TCR Tg mice or from the spleens of wild-type C57BL/6J mice. Tissues were dissociated, and RBC lysed in hypotonic lysis buffer (10 mM Tris-HCl and 0.83% ammonium chloride).

Splenocytes were plated in 100-mm tissue culture plates and allowed to adhere for 1 Mtb at 37o C. Untouched CD4+ T cells were purified from non-adherent splenocytes using a

CD4+ T-cell-negative isolation kit (Miltenyi Biotec) by following manufacturer’s instructions (purity > 95%). For purification of CD4+CD25-T cells, negatively selected

CD4+ T cells were positively selected for CD4 and then sorted by CD25 surface levels by flow sorting. For some experiments, highly purified naïve (CD25- CD44- CD62L+) CD4+

T cells were isolated from spleens using a combination of immune-MACS followed by

FACS as described with some modifications (34). The average purity was 98 to 99%.

CD4+ T cells were rested overnight in complete DMEM (BioWhittaker, East Rutherford,

NJ) supplemented with 1 µM 2-merchatoethanol, 10 mM HEPES buffer, nonessential amino acids, 2 mM L-glutamine, penicillin/streptomycin, 10% fetal bovine serum prior to use in assays. The primary stimulation and re-stimulation of CD4+ T cells were performed in serum-free HL-1 medium (BioWhittaker, East Rutherford, NJ) supplemented with 1 µM 2-merchatoethanol, 10 mM HEPES buffer, nonessential amino acids, 2 mM L-glutamine, penicillin/streptomycin). For re-stimulation experiments, primed CD4+ T cells were rested for 5 days in complete DMEM as above, containing 20 ng/ml IL-7 to maintain viability.

Cultures of bone marrow-derived macrophages

Bone marrow-derived macrophages (BMM) were generated by culturing bone marrows from C57BL/6J mice in complete DMEM containing 20% L929 culture supernatant for

7-10 days. At day 8, BMM were matured by treating with IFNγ (4 ng/ml) for 48 Mtb before use in assays. 2 x106 BMM per well in 6-well plates or 1 x105 BMM per well in

96-well plates were washed three times, lightly fixed with 1% paraformaldehyde in medium for 15 min at 37°C. Cells were washed extensively, and pulsed with Ag85B peptide for 4 Mtb at 37°C before incubation with untreated, LAM-treated or ionomycin- treated P25 TCR Tg CD4+ T cells.

CD4+ T cell stimulation (priming) and re-stimulation assays

To prime, 1x106 P25TCR Tg CD4+ T cells were left untreated (none) or pretreated with

LAM (1 µM) or lonomycin (Iono) [1 µM] (positive control for anergy induction), and incubated for 1 Mtb or 24 Mtb at 37°C. T cells were washed and initially stimulated by co-culturing with 2 x106 paraformaldehyde-fixed BMM pulsed with 1 µg/ml of Mtb Ag

85B peptide (APC + peptide) in 6-well plates for 48 Mtb. After 24 Mtb, supernatants were collected and IL-2 levels measured by ELISA as described previously (146). After

48 Mtb, primed CD4+ T cells were collected from culture by vigorous pipetting and extensively washed in DMEM (3 times). For polyclonal stimulation, CD4+ T cells pretreated with and without LAM were stimulated with plate-bound anti-CD3ε (1 µg/ml) and soluble anti-CD28 (1 µg/ml) for 24 Mtb to 48 Mtb. After 24 Mtb of stimulation, supernatants were collected, and IL-2 levels measured by ELISA. T cell proliferation was measured after 48 Mtb by [3H]-thymidine incorporation.

For re-stimulation, primed P25TCR Tg or polyclonal CD4+ T cells were washed and rested for 5 additional days in IL-7 to maintain viability. T cells were harvested, washed and live cells separated by density gradient centrifugation before re-stimulation. To re- stimulate, 5 x 104 P25TCR Tg CD4+ T cells per well were co-cultured with 1 x 105

Ag85B-pulsed fixed-BMM in triplicate wells in 96-well plates for 48 Mtb. Polyclonal

CD4+ T cells (5x104/well) were re-stimulated with plate-bound anti-CD3ε and soluble anti-CD28 as described above. After 24 Mtb of re-stimulation, supernatants were collected, and IL-2 levels measured by ELISA. T cell proliferation was measured after 48

Mtb by [3H]-thymidine incorporation.

57 siRNA experiments required proliferating cells and were performed with pre-activated naïve CD4+ T cells and in vitro generated Th1 effector cells. In brief, naïve (CD25-

CD44- CD62L+) CD4+ T cells were stimulated with plate bound anti-CD3e and soluble anti-CD28 for 3 days. Cells were washed and rested in IL-7 containing media for 48 hours before transfection with anti-GRAIL siRNA or non-target negative control. For

Th1 effector CD4+ T cell generation, naïve (CD25- CD44- CD62L+) CD4+ T cells were stimulated with plate bound anti-CD3e and soluble anti-CD28 for 3 days in the presence of IL-12 (10 ng/ml) and anti-IL-4 (5 µg/ml). Cells were washed and rested in IL-7 containing media for 48 hours before transfection with anti-GRAIL siRNA or non-target negative control.

Isolation and stimulation of human CD4+ T cells

Primary human CD4+ T cells from healthy donors were isolated as described previously

(34). Briefly, peripheral blood was obtained from healthy donors and the T cells isolated from the buffy coat following Hypaque-Ficoll (Amersham Biosciences) density gradient centrifugation, followed by isolation with miltenyi beads. Where required, flow sorting was used. T cells (> 95% purity) were cultured/rested in complete ex vivo media (Life

Technologies) until use. CD4+ T cells pretreated with or without LAM were stimulated with plate-bound anti-CD3ε (10 µg/ml) and soluble anti-CD28 (1 µg/ml). All human cell studies were approved by the Case Western Reserve University Institutional Review

Board and the National Institutes of Health (IRB number: 03-88-63). All adult subjects provided informed written consents, and a parent or guardian of any child participant provided informed consent on their behalf.

IL-2 secretion and T cell proliferation

Supernatants were assayed for IL-2 production in Immulon 4HBX flat-bottomed microtiter plates (Thermo) coated with purified capture IL-2 mAb (1 µg/ml) and detected with biotinylated IL-2 mAb (1 µg/ml), followed by alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch) and phosphatase substrate (Sigma-Aldrich).

Plates were read with a versa Max turntable microplate reader and data analyzed with soft

Max Pro LS analysis software. Cells were pulsed during the final 16 Mtb of culture with

1 µCi of [3H] thymidine (Amersham Pharmacia Biotech, Piscataway, NJ)/well. [3H] thymidine incorporation was measured by liquid scintillation counting, and the results expressed as mean counts per minute (cpm) of triplicate values.

Flow cytometry

For surface receptor expression, cells were stained with mAbs for the following receptors: CD3, CD4, CD28, CD25, TCR Vβ, CTLA4, PD1, Lag-3, Tim-3 and CD40L

(Biolegend). Live cell staining was performed with LIVE DEAD fixable violet or yellow dead cell stain (eBioscience). Cells were fixed and permeabilized with BD Cytofix

Cytoperm kit (BD PharMingen) according to the manufacturer’s instructions before flow cytometry. Intracellular cytokine staining was performed with anti-FoxP3 and anti-

CTLA4. For apoptosis assays, cells were stained with Annexin V eFluor 450 and LIVE

DEAD yellow fixable dead cell stain. For LAM staining assays, CD4+ T cells pretreated with LAM were harvested and stained with anti-LAM mAb (Cs35-biotin) or biotin mouse IgG3, k isotype control (BD Biosciences) for 30 min, followed by streptavidin- alexafluor-488 for 30 min on ice. For GRAIL expression, cells were stained for

59 intracellular GRAIL using rabbit anti-GRAIL primary antibody (Abcam) followed by goat anti-rabbit IgG-FITC secondary antibody (southern Biotech). After staining, cells were analyzed by flow cytometry on an LSR II (BD Biosciences) and data analyzed with

FlowJo software (TreeStar).

Confocal microscopy

Human CD4+ T cells were incubated with LAM (2 µM) for 30 min. at 37°C with gentle shaking. After incubation with LAM, lipid rafts in the T cell membrane were labeled by incubating with Alexa Fluor 647 conjugated-cholera toxin subunit B (CT-B, 1 µg/ml) for

20 min. on ice, washed extensively, labeled with anti-LAM mAb (clone Cs35) followed by Alexa Fluor 488 conjugated IgG and fixed with 1% paraformaldehyde. To label CD3-

TCR complex, after incubation with LAM, cells were labeled on ice with anti-LAM mAb

(clone Cs35) followed by Alexa Fluor 488 congugated-anti-mouse IgG. Then, Alexa

Fluor 647 congugated-anti-CD3 mAb was used to label the CD3-TCR complex. Cells were visualized in a Leica DM16000B confocal microscope (100x oil immersion lens) and images acquired.

Knockdown of GRAIL expression by small interfering RNA (siRNA)

Knockdown of GRAIL was performed as described previously (186). Briefly, preactivated naïve or in vitro differentiated Th1 CD4+ T cells were transfected with a solution of four GRAIL (i.e. ring finger protein-128, gene ID 66889) or one control siRNAs [Qiagen, Valencia, CA] to knockdown GRAIL expression by using the HiPerfect transfection reagent (Qiagen, Valencia, CA) following the manufacturer’s protocol. The target sequences for the mouse GRAIL siRNAs are:

1) 5ʹ-CTCGAAGATTACGAAATGCAA-3ʹ, 2) 5ʹ-CAGGATAGAAACTACCATCAA-

3ʹ, 3) 5ʹ-CTCTAATTACATGAAATTTAA-3ʹ, 4) 5ʹ-

CAGGGCCTCCTAGTTTACTATGAA-3ʹ.

For transfection of siRNA, a total of 3 x 106 CD4+ T cells plated at 3 x 105 cells per well in a 24-well plate were transfected with 75 nM or 100 nM each of these GRAIL siRNAs altogether using 6 µl HiPerfect dissolved in serum-free Opti-MEM medium (Invitrogen,

Carlsbad, CA). After 6 Mtb of transfection, an additional 400 µl DMEM medium with

10% FBS was added and cells allowed to incubate at 37°C for 24 Mtb. A non-target negative control (NC)-siRNA (5ʹ-AATTCTCCGAACGTGTCACGT-3ʹ, Qiagen,

Valencia, CA) was transfected into CD4+ T cells to serve as experimental control for non-specific effects. After 24 Mtb, levels of remaining protein were evaluated by

Western blot with β-actin as loading control. GRAIL knocked down or control CD4+ T cells were left untreated or treated with LAM for 1 Mtb at 37°C. Cells were washed and stimulated with plate-bound anti-CD3ε and soluble anti-CD28 for 24 Mtb to 48 Mtb.

After 24 Mtb and 48 Mtb of stimulation, supernatants were collected, and IL-2 and IFN-γ measured by ELISA. T cell proliferation was measured after 48 Mtb by [3H]-thymidine incorporation.

Western blotting

For analysis of GRAIL protein expression, T cell lysates obtained from primed, re- stimulated or knocked down CD4+ T cells were suspended in 2X sample buffer and heated to 95°C. Proteins (20 µg/well were separated by SDS-PAGE on 10% ready-to-use gels (Invitrogen) under reducing conditions and electro-transferred to nitrocellulose

61 membranes (Bio-Rad, Hercules, CA, USA). After transfer, membranes were incubated at room temperature for 1 Mtb in blocking buffer (1XPBS, 5% BSA and 0.1% Tween-20).

Primary (rabbit anti-GRAIL, Thermoscientific) and mouse anti-rabbit HRP conjugated secondary antibodies (Jackson ImmunoResearch) were diluted in same buffer containing

1XPBS, 5% BSA and 0.1% Tween-20 and incubated for 16 Mtb and 1 Mtb, respectively.

Western blots were analyzed with ImageJ software (NIH).

Statistical analyses

All data are presented as mean +/- SEM or +/- SD. Statistical analyses were performed as either student t test or two-way ANOVA (Graph-Pad Prism software, version 6.0 for

Mac). A p value of <0.05 was considered significant, and represented as ∗.

Results

Association of LAM with CD4+ T cell membrane correlates with inhibition of CD4+ T cell activation during priming

LAM can incorporate into the membranes of murine and human T cells within 30 min, with maximal incorporation occurring within several hours (56, 57). We reported previously that pretreatment of CD4+ T cells with LAM inhibits activation upon stimulation (146). However, the correlation between LAM association with the T cell membrane and inhibition of T cell function has not been analyzed. CD4+ T cells pretreated with LAM for 1 Mtb and washed were cultured for different intervals before activation with anti-CD3/anti-CD28 and LAM binding to the cell membrane determined in parallel by flow cytometry. Because more than 40% of mouse CD4+ T cells ex-vivo will die within 24 Mtb, unless stimulated or maintained viable by addition of cytokines,

IL-7 was used to maintain a CD4+ T cell viability of more than 80% (Fig. 3.1A). Eighty percent of LAM-pretreated CD4+ T cells also remained viable in IL-7 at 0, 24 and 48 Mtb time points and over 70% at 96 Mtb after LAM pretreatment (Fig. 3.1B). Treatment with

IL-7 did not render CD4+ T cells refractory to LAM-inhibition of T cell activation (Fig.

3.1C). LAM was detectable in the T cell membrane at 0 and 24 Mtb after pretreatment, but did not remain associated with the cell membrane for more than 24 Mtb, as demonstrated by the time-dependent loss of anti-LAM mAb staining (Fig. 3.2A).

Whereas, 50-70% inhibition of IL-2 secretion and proliferation was observed for CD4+ T cells stimulated at 0 and 24 Mtb after LAM pretreatment, T cells stimulated 48 and 96

Mtb after LAM pretreatment were not inhibited (Fig. 3.2B and 3.2C), demonstrating a correlation between the period when LAM was present in the membrane and the period

63 when inhibition of T cell responses was observed. The viability of untreated and LAM- treated T cells were similar (Fig. 3.1B). Untreated CD4+ T cells were analyzed in parallel, stained negative for LAM and activated normally (data not shown). These results indicate that LAM has to be associated with the CD4+ T cell membrane at the time of primary

TCR/CD3 stimulation for LAM to inhibit T cell activation.

Figure 3.1.Viability and function of CD4+ T cells with IL-7 treatment. (A) CD4+ T cells remain viable in IL-7 for more than 5 d. 1 x 106 CD4+ T cells were incubated in IL-7 (20 ng/ml) for indicated time points. Cells were washed, stained at each time point with LIVE/DEAD violet stain and analyzed by flow cytometry. (B) CD4+ T cells (1x106) were left untreated (none) or pre-treated with LAM (1 µM) for 1 Mtb at 37οC, washed and incubated with IL-7 (20 ng/ml). Cells were chased for 0, 24, 48 and 96 Mtb. At the end of each chase period, cells were removed and washed. Cells were stained with LIVE/DEAD yellow dye, and analyzed by flow cytometry. (C) Treatment with IL-7 does not make T cells refractory to LAM inhibition. 1 x 106 CD4+ T cells were incubated inIL-7 (20 ng/ml) for indicated time points. After each time point, cells were washed and either left untreated or pretreated with LAM for 1 Mtb. Cells were stimulated with anti-CD3/CD28. After 24 Mtb, IL-2 in the supernatant was measured by ELISA. Error bars indicate mean +/- SD of triplicate wells of one representative experiment (N=3). Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

Figure 3.2. Presence of LAM on CD4+ T cell membranes is required for inhibition of CD4+ T cell activation after primary stimulation. (A) CD4+ T cells (1x106) were pretreated with LAM (1 µM) or media (untreated) for 1 Mtb at 37οC, washed and incubated with IL-7 (20 ng/ml). Cells were chased for 0, 24, 48 and 96 Mtb. At the end of each chase period, cell aliquots were removed and washed with media, prior to LAM staining. LAM-treated T cells were stained with anti-LAM mAb or isotype control and analyzed by flow cytometry. (B, C) LAM-treated and untreated CD4+ T cells at the end of each chase period were activated with plate-bound anti-CD3 (1 µg/ml) and soluble anti-CD28 (1 µg/ml). IL-2 was measured in 24 Mtb culture supernatants by ELISA (B) and T cell proliferation was measured after 48 Mtb by [3H] thymidine incorporation (C). Percent inhibition reflects the ratio of IL-2 (B) or proliferation (C) between LAM-treated and untreated T cells x 100. Error bars indicate mean +/- SD of triplicate wells of one representative experiment (n=3). Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

LAM induces CD4+ T cell anergy

Because suppression of IL-2 expression and T cell proliferation are associated with induction of anergy, we next determined if LAM-induced inhibition during primary stimulation of CD4+ T cells (priming in our experimental system) resulted in anergy. We utilized an in vitro T cell-and APC-based system to induce functional anergy (93) (Fig.3.

3A). LAM has inhibitory effects on BMM that could indirectly inhibit T cell proliferation and cytokine production (138). In addition, activated viable Mtb-infected BMM can secrete cytokines that are inhibitory and T cell anergizing such as IL-10 and TGFβ. To rule out these inhibitory effects, BMM were fixed before use in priming and re- stimulation experiments (see methods). Fixed BMM were pulsed with Mtb Ag85B peptide (APC + peptide) and used to prime LAM-pretreated P25 TCR Tg CD4+ T cells.

Calcium ionophore ionomycin served as a positive control for anergy induction (131). As shown before, CD4+ T cells primed by Ag85B peptide-pulsed BMM in the presence of

LAM secreted lower amounts of IL-2 (Fig. 3.3C), and this correlated with detection of

LAM on the cell surface (Fig. 3.3B, left histogram). More importantly, T cells primed in the presence of LAM produced significantly lower amounts of IL-2 and proliferated less compared to control cells after antigenic re-stimulation 7 days later (Fig. 3.3D, upper and lower panels), even though LAM was not present on the cell membrane at this point (Fig.

3.3B, right histogram). The level of inhibition by LAM during priming upon re- stimulation was similar to that measured by the positive control, ionomycin.

Figure 3.3. LAM induces anergy in P25 CD4+ T cells. (A) Experimental design. (B) P25 TCR-Tg CD4+ T cells (1 x 106) pretreated with LAM for 1 Mtb were stained with anti-LAM mAb or with an isotype control mAb before priming (left histogram) or 5 d after priming and before re-stimulation (right histogram), and analyzed by flow cytometry. (C) 1 Mtb after pretreatment, untreated (none), LAM-treated or ionomycin- treated T cells were primed with Ag 85B peptide-pulsed APC for 48 Mtb. IL-2 was measured in 24-Mtb culture supernatants by ELISA. (D) Primed P25 TCR-Tg CD4+ T cells (in C) were cultured for 5 d in IL-7. Cells were washed and re-stimulated with Ag85B peptide-pulsed APC. IL-2 was measured in 24 Mtb-culture supernatants by ELISA (upper panel) and cell proliferation was determined after 48 Mtb by [3H] thymidine incorporation (lower panel). Data in C through D are means +/- SEM of three independent experiments conducted in triplicate. ***P<0.001 (paired t test). Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

Because suboptimal or high antigen concentrations are an alternative potential cause of hypo-responsiveness upon TCR stimulation (194), we stimulated CD4+ T cells over a range of Ag85B peptide concentrations in the presence of LAM, and observed

LAM-induced inhibition over a wide range of antigen concentrations, indicating that exposure to higher concentrations of antigen did not affect LAM-induced anergy (Fig.

3.4A). Maximum anergy induction was observed with concentrations of LAM as low as

0.62 µM based on LAM response experiments (Fig. 3.4B). These data suggest that defective proliferation in cells stimulated in the presence of LAM is a result of functional anergy. The results also suggest that the presence of LAM in the CD4+ T cell membrane is required during priming to induce functional T cell anergy, but once anergy is induced, the presence of LAM is no longer required to maintain anergy.

Figure 3.4. LAM-induced P25 CD4+ T cell anergy occurs over a range of Ag85B peptide and LAM concentrations. (A) LAM induces anergy in P25 CD4+ T cells primed with a range of Ag85B concentrations. 1 x 106 P25TCR-Tg CD4+ T cells were left untreated (none) or pre-treated with LAM (1 µM) for 1 Mtb. Cells were washed and primed with Ag 85B peptide-pulsed or mock-pulsed APC (resting). After 48 Mtb cells were washed and rested in media containing IL-7 for 5 days. CD4+ T cells were then washed and re-stimulated for 48 Mtb. Cell proliferation was measured by [3H] thymidine incorporation. (B) 1 x 106 P25 CD4+ T cells were left untreated or pretreated with increasing LAM concentrations for 1 Mtb. Cells were washed and primed with Ag85B peptide-pulsed APC for 48 Mtb. IL-2 was measured in 24-Mtb culture supernatants by ELISA. Primed cells were washed and rested in media containing IL-7 for 5 days. CD4+ T cells were washed and re-stimulated with Ag85B peptide-pulsed APC. After 24 Mtb, IL-2 was measured by ELISA in the supernatant. Error bars indicate mean +/- SD of triplicate wells of one representative experiment. Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

Tregs or apoptosis is not responsible for LAM-induced CD4+ T cell anergy

Other mechanisms responsible for defective T cell proliferation are inactivation of antigen-reactive CD4+ T cells by Tregs or apoptosis (188, 195-197). We used flow cytometry to measure the number of FoxP3+ CD4+ T cells 48 hours after primary stimulation and 5 days of rest. The number of FoxP3+ CD4+ T cells detected in both

LAM-treated and untreated CD4+ T cells ranged from 3-6% (Fig. 3.5A). This falls within the 5-15% level of natural Tregs found in spleens of healthy mice, and is not sufficient to suppress conventional CD4+ T cells (188). In flow purified CD3+CD4+CD25- T cells, anergy was still induced by LAM and ionomycin, even though nTregs had been depleted

(Fig. 3.5B). There also was no increase in IL-10 production by LAM treated CD4+ T cells

(data not shown).

To determine whether LAM induced apoptosis and whether apoptosis accounted for hypo-responsiveness upon re-stimulation, Annexin V was measured by flow cytometry 48 Mtb after re-stimulation. Eight to 12% of CD4+ T cells were Annexin V- positive (Fig. 3.5C), with similar levels in LAM-treated and untreated T cells. Moreover,

LAM-induced anergy was not related to cell death (Fig. 3.6), indicating that decreased

IL2 production and proliferation upon re-stimulation of LAM-treated CD4+ T cells was not due to loss of T cell viability. Altogether these results exclude involvement of newly generated FoxP3+ cells, Tregs, secretion of inhibitory anergy-inducing cytokines, and apoptosis as causes of LAM-induced T cell anergy.

Figure 3.5. LAM does not induce FoxP3-positive regulatory T cells and/or increase activation-induced cell death (apoptosis). (A) 1x106 P25 TCR-Tg CD4+ T cells left untreated (none) or treated with LAM (as described in Fig. 3.3) were primed with APC- Ag85B peptide for 48 Mtb. Cells were washed and cultured in IL-7 for 5 d. Cells were washed, and Foxp3-positive T cells were determined by intracellular staining and analyzed by flow cytometry. (B) 1 x 106 T reg depleted P25 TCR-Tg CD4+ T cells were left untreated (none) or pre-treated with LAM (1 µM) for 1 Mtb. Cells were washed and primed with Ag 85B peptide-pulsed (APC + Pep) or mock-pulsed APC (APC). After 48 Mtb cells were washed and rested in media containing IL-7 for 5 days. CD4+ T cells were then washed and re-stimulated for 24 Mtb. IL-2 was measured in 24-Mtb culture supernatants by ELISA. (C) P25 TCR-Tg CD4+ T cells suppressed through treatment with LAM or ionomycin (as described in Fig.2) were re-stimulated with APC-Ag85B peptide. After 24 Mtb, T cells were stained with Annexin V eFluor 450 and apoptosis was measured by flow cytometry analysis of annexin V-positive T cells. At re- stimulation, T cells treated with 1 µM of dexamethasone (Dexa) were used as a positive control for apoptosis. Error bars indicate means +/- SD of triplicate wells of one representative experiment (n=3). ***P<0.001 (paired t test). Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

Figure 3.6. LAM-induced CD4+ T cell anergy is not due to cell death. P25 TCR-Tg CD4+ T cells (1x106) were left untreated (none) or treated with LAM (1 µM) or ionomycin (1 µM) for 1 Mtb. Cells were washed and primed with Ag 85B peptide-pulsed APC. After 48 Mtb, cells were washed and rested in media containing IL-7 for 5 days. CD4+ T cells were washed and re-stimulated with Ag85B peptide-pulsed APC. Viability of CD4+ T cells was determined by flow cytometric analysis of CD4+ T cells stained with violet LIVE/DEAD dye. Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

LAM does not affect TCR/CD3 and co-signaling receptor expression

Other pathways that have been associated with the initiation and/or promotion of

T cell anergy are inhibitory receptors PD-1, CTLA-4, Lag-3 and Tim-3, that are induced after 48 Mtb of T cell priming (89, 198-201, 202). Previous reports have shown that intracellular pathogens can manipulate co-signaling molecules to evade the immune response (190). To determine if there was a role for these receptors in LAM-induced anergy, primary P25TCR-Tg T cells were stimulated with Ag85-pulsed BMM for 48 hours. This was followed by measurement of proliferation and surface expression of the aforementioned receptors. Although LAM-treated CD4+ T cells exhibited the expected decrease in proliferation, there was no significant increase in the expression of PD-1,

CTLA-4, Lag-3 or Tim-3 in LAM-treated compared to non-treated T cells (Fig. 3.7A, upper histograms). CD28 is the co-stimulatory molecule essential for productive T cell activation, while CD40L also regulates T cell function and has been associated with up- regulation of the gene related to anergy in lymphocytes (GRAIL) (203). No differences in CD28 or CD40L expression in LAM treated vs. non-treated T cells were observed

(Fig. 3.7A, lower histograms).

An inhibitory environment may induce down-regulation of TCR-CD3 expression after priming, which could result in hypo-responsiveness at re-stimulation (92). At the time of Ag85B re-challenge, LAM-treated and non-treated CD4+ T cells had equivalent

TCR and CD3 levels (Fig. 3.7A, lower histograms), indicating that decreased IL-2 production and proliferation upon re-stimulation in LAM-treated T cells was not due to endocytosis or internalization of the TCR-CD3 complex. The levels of IL-2Rα expression in LAM-treated and untreated T cells at re-stimulation were similar (data not shown).

Although we observed a small increase in PD-1 expression in LAM-treated T cells, the difference as compared to untreated T cells was not significant (Fig. 3.7B), suggesting that the slight increase in PD-1 expression cannot account for LAM-induced anergy.

Figure 3.7. LAM does not affect receptor expression on LAM-anergized P25 TCR- Tg CD4+ T cells. (A) 1 x 106 CD4+ T cells were pre-treated with either LAM or left untreated (none) and co-cultured with Ag85B-pulsed APCs. After 48 Mtb, cells were collected, washed and labeled with anti-PD1, anti-Lag3 or anti-Tim 3, anti-CD28, anti- CD40L mAbs. Intracellular staining was performed for CTLA4. Gating was performed on live CD4+ T cells. Alternatively, after 48 Mtb cells were collected, washed and cultured in IL-7 for 5 d. Cells were then stained for surface expression of TCR and CD3. Gating was performed on live CD4+ T cells. Representative histograms of at least three independent experiments are shown. (B) The mean fluorescence intensity (MFI) of PD-1 staining in LAM-treated (LAM) and untreated (none) T cells was quantified after 48 Mtb of primary stimulation. Data are means +/- SEM of three independent experiments. Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

LAM-induced anergy correlates with up-regulation of GRAIL protein expression

The initiation and maintenance of CD4+ T cell anergy has been associated with increased expression of anergy-related genes, including GRAIL (90, 94, 105, 191, 203).

GRAIL is a protein involved in induction and maintenance of CD4+ T cell anergy by negatively regulating IL-2 transcription (90, 94, 186, 188). GRAIL is inducible upon stimulation of peripheral CD4+ T cells, and increased expression may occur as early as 3 to 6 Mtb (94). Therefore, we examined whether LAM-treated CD4+ T cells display elevated expression levels of GRAIL 6, 12 and 24 Mtb after priming and 24 Mtb after re- stimulation. Significant up-regulation of GRAIL protein in LAM-treated T cells was observed both after priming (Fig. 3.8A) and upon re-stimulation (Fig. 3.8B), which correlated inversely with the ability of the T cells to produce IL-2 (data not shown).

These data suggest that the initiation and maintenance of anergy in LAM-treated T cells is associated with increased expression of GRAIL upon T cell activation.

Figure 3.8. LAM induces increased GRAIL protein expression both during priming and upon re-stimulation. (A) Untreated and LAM-pre-treated P25 TCR-Tg CD4+ T cells were stimulated with Ag85B peptide-pulsed APC for the indicated times. GRAIL protein expression was measured in cell lysates from re-purified CD4+ T cells by Western blot. Blots were analyzed with ImageJ software (NIH) and the ratios of band intensities of GRAIL/beta-actin expressed as relative densities (bar graph). (B) P25 TCR-Tg CD4+ T cells were left untreated (none) or pretreated with LAM or ionomycin and primed with Ag85B peptide-pulsed APC. Cells were washed and cultured in IL-7 for 5 d. Cells were re-stimulated with Ag85B peptide-pulsed APC for 24 Mtb. T cell lysates were obtained from re-stimulated CD4+ T cells, and Western blot performed for GRAIL protein. Data presented are the means +/- SEM of three independent experiments conducted in triplicate. ***P<0.001 (paired t test). Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

Knockdown of GRAIL expression reduces LAM’s ability to inhibit CD4+ T cell activation.

GRAIL deficient T cells hyper-proliferate upon stimulation with anti-CD3 and anti-CD28 and are defective in anergy induction (90, 186, 191). Optimal up-regulation of

GRAIL occurred within 6 Mtb to 24 Mtb after LAM pretreatment and primary T cell stimulation (Fig. 3.9A). To test if GRAIL up-regulation was responsible for LAM- induced anergy, we knocked down GRAIL expression in pre-activated naïve CD4+ T cells before LAM pretreatment and anti-CD3/CD28 re-stimulation. As shown in Fig.

3.9A and 9B, the knockdown efficiency of GRAIL siRNA was approximately 75 to 92%.

As a result of GRAIL knockdown, LAM’s ability to induce anergy was significantly reduced as shown by the reversal of the IL2 (Fig. 3.9C) and proliferation (Fig. 3.9D) defects in cells transfected with GRAIL siRNA compared to cells transfected with non- targeting siRNA (NC). GRAIL knockdown also resulted in an increase in IL-2 and cell proliferation in untreated cells, although the effect was not as significant as that observed in LAM-treated cells.

GRAIL is not only expressed in naïve T cells, but also in effector T cell subsets and controls their activation. Kriegel et al reported that Grail knockout Th1 effector

CD4+ T cells overproduce IFN-γ. We reported previously that LAM inhibits activation of effector T cells (135). To determine whether LAM can induce anergy in effector T cells,

GRAIL expression was knocked down by siRNA in in vitro differentiated Th1 effector

CD4+ T cells. The knockdown efficiency of GRAIL siRNA in Th1 effector CD4+ T cells was 75 to 86% (Fig. 3.9E and Fig. 3.9F). After GRAIL knockdown Th1 cells and control cells were pre-treated with LAM before anti-CD3/CD28 re-stimulation. There was reversal of IL-2 and IFN-γ inhibition by LAM in GRAIL deficient-T cells compared to

79 control Th1 cells (Fig. 3.9G and Fig. 3.9H). As observed with naïve T cells, there was a modest effect of GRAIL knockdown on spontaneous IL-2 and IFN-γ production in untreated T cells compared to the large effect on LAM-treated cells. These results indicate that GRAIL is not only a gatekeeper of T cell activation, but GRAIL expression is essential for LAM-induced inhibition of primary activation of naïve and effector CD4+

T cells and thus anergy.

Figure 3.9. Knockdown of GRAIL expression by siRNA prevents inhibition of CD4+ T cell activation by LAM. (A, B) A total of 3 x 106 pre-activated naïve CD4+ T cells plated at 3 x 105 cells per well in a 24-well plate were transfected with 75 nM or 100 nM of siRNAs targeting GRAIL (GRAIL siRNA) or non-targeting control siRNA (NC) using 6 µl HiPerfect dissolved in serum-free Opti-MEM medium. After 6 Mtb of transfection, an additional 400 µl DMEM medium with 10% FBS was added and cells allowed to incubate at 37°C for 24 Mtb. After 24 Mtb, GRAIL was measured by Western blot with β-actin as loading control. (C, D) GRAIL knocked-down or control naïve CD4+ T cells were left untreated (None) or treated with LAM for 1 Mtb at 37°C. Cells were washed and re-stimulated with plate-bound anti-CD3ε and soluble anti-CD28 for 48 Mtb. IL-2 was measured in 24 Mtb culture supernatants by ELISA (C) and T cell proliferation after 48 Mtb by [3H] thymidine incorporation (D). For A-D, representative blots and densitometry data are shown. (E, F) A total of 3 x 106 Th1 effector CD4+ T cells plated at 3 x 105 cells per well in a 24-well plate were transfected and cultured as in (A, B) above. After 24 Mtb, GRAIL was measured by Western blot. (G, Mtb) GRAIL knocked down or control Th1 effector CD4+ T cells were left untreated (None) or treated with LAM for 1 Mtb at 37°C. Cells were washed and re-stimulated as above for 48 Mtb. IL-2 (G) and IFN-γ (Mtb) were measured in 24 Mtb and 72 Mtb culture supernatants respectively by ELISA. IL-2 and proliferation data shown are means +/- SEM of three independent experiments. *P< 0.05, **P< 0.01 (paired t test). Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

Exogenous IL-2 reverses LAM-induced anergy and down-regulates GRAIL in antigen- specific CD4+ T cells

Anergy in CD4+ T cells can be reversed by the addition of high concentrations of exogenous IL-2 at the time of re-stimulation (86, 89). P25 TCR-Tg CD4+ T cells pretreated with LAM (1 µM) or ionomycin (1 µM) were primed and rested for 5 days in media containing IL-7. Cells were re-stimulated with Ag85B peptide-pulsed paraformaldehyde-fixed APC with or without exogenous IL-2. Exogenous IL-2 at time of re-stimulation partially restored proliferation in LAM-anergized CD4+ T cells (Fig.

3.10A), and this was comparable to reversal of ionomycin-induced anergy.

IL-2 can down-regulate GRAIL expression (186). We determined the effect of exogenous IL2 on GRAIL expression in LAM-anergized T cells. P25 TCR-Tg CD4+ T cells pretreated with LAM were primed and rested as described above. Cells were re- stimulated with Ag85B peptide-pulsed paraformaldehyde-fixed APC with or without exogenous IL-2. Addition of IL2 resulted in decreased GRAIL expression by Western blot (Fig. 3.10B), which correlated with reversal of defective proliferation. Thus exogenous IL2 reversed LAM-induced anergy by down-regulating GRAIL expression.

Figure 3.10. Exogenous IL-2 down-regulates GRAIL expression and restores T cell proliferation in LAM-anergized CD4+ T cells. (A) P25 TCR-Tg CD4+ T cells (1 x 106) were left untreated (none) or treated with LAM (1 µM) or ionomycin (1 µM) for 1 Mtb. Cells were washed and primed with Ag85B peptide-pulsed APC. After 48 Mtb, cells were washed and rested in media containing IL-7 for 5 days. CD4+ T cells were washed and re-stimulated with Ag85B peptide-pulsed APC with or without addition of exogenous rIL-2 (20 ng/ml). Cell proliferation was measured after 48 Mtb by [3H] thymidine incorporation. Data are means +/- SEM of three independent experiments conducted in triplicates. *P< 0.05, **P< 0.01 (paired t test). (B) P25 TCR-Tg CD4+ T cells were pretreated with LAM and primed with Ag85B peptide-pulsed APC. Cells were washed and cultured in IL-7 for 5 d. Cells were re-stimulated with Ag85B peptide-pulsed APC with or without addition of exogenous rIL-2 (20 ng/ml) for 48 Mtb. T cell lysates were obtained and GRAIL protein measured by Western blot. A representative blot and densitometry for 3 experiments is shown. Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

LAM pretreatment up-regulates GRAIL expression in activated human CD4+ T cells

Anergy-inducing proteins have been reported in both mouse and human anergized CD4+

T cells (94, 131). Since LAM also inhibits human CD4+ T cell activation (146), we first determined if LAM binds to resting human CD4+ T cell membranes. We used confocal microscopy to analyze LAM pretreated T cells labeled with anti-LAM mAb and with either lipid raft marker, Alexa Fluor 647-conjugated cholera toxin subunit B or CD3-TCR complex marker, Alexa Fluor 647 conjugated anti-CD3 mAb. This revealed that LAM was not only bound to the T cell membrane, but also associated with lipid rafts and the

CD3-TCR complex (Fig. 3.11A). We next determined if LAM pretreatment of human

CD4+ T cells induces GRAIL expression upon stimulation. One hour after LAM pretreatment, CD4+ T cells from four different donors were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of a wide range of LAM concentrations for 24 Mtb, followed by measurement of IL-2 levels by ELISA and flow cytometry for intracellular GRAIL protein expression. As shown (Fig. 3.11B), all individuals were inhibited albeit with variable sensitivity to LAM. GRAIL expression also was clearly upregulated in LAM-treated T cells compared to untreated T cells from 3 donors (Fig.

3.11C and 3.11D) and minimally in 1 donor, indicating that LAM may induce anergy in human CD4+ T cells. These results suggest in this small pool of donors that there may be variable sensitivity of human CD4+ T cells to LAM’s inhibitory effect and ability to upregulate GRAIL.

Figure 3.11. LAM associates with lipid rafts and CD3 on human CD4+ T cells, and up-regulates GRAIL upon activation with anti-CD3/CD28. (A) Human CD4+ T cells were incubated with LAM for 1 Mtb at 37°C. Top panels, after incubation with LAM, cells were incubated with Alexa Fluor 647 conjugated-cholera toxin subunit B (CT-B, 1 µg/ml) for 20 min on ice, washed extensively, labeled with anti-LAM mAb (clone Cs35) followed by Alexa Fluor 488 conjugated anti-mouse IgG and fixed with 1% paraformaldehyde. Lower panels, after incubation with LAM, cells were labeled on ice with anti-LAM mAb (clone Cs35) followed by Alexa Fluor 488 conjugated-anti-mouse IgG. Then, Alexa Fluor 647 congugated-anti-CD3 mAb was used to label the CD3-TCR complex. Cells were visualized and images merged in a Leica DM16000B confocal microscope (100X oil immersion lens). (B) Human CD4+ T cells isolated from four different Mtb uninfected adult donors were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of indicated LAM concentrations for 24 Mtb, followed by measurement of IL-2 levels by ELISA. (C) Human CD4+ T cells (1 x 106) were left untreated (none) or pretreated with LAM (1 µM), followed by stimulation as above. Cells were stained for surface CD4 and intracellular GRAIL using APC-CD4 mAb and rabbit anti-GRAIL primary antibody, respectively. FITC-labeled anti-rabbit secondary antibody was used, followed by FACS analysis. (D) The mean fluorescence intensity (MFI) of GRAIL staining in LAM-treated (LAM) and untreated (none) T cells was quantified after 48 Mtb of primary stimulation. Error bars indicate mean +/- SD of triplicate wells of one representative experiment. Reproduced from Sande OJ et al (193); Copyright © 2016. The American Association of Immunologists, Inc. All rights reserved.

Discussion

LAM can incorporate itself into the membrane of CD4+ T cells within 30 min of exposure to soluble LAM (56, 57). We have reported before that Mtb LAM directly inhibits early TCR signaling in CD4+ T cells by blocking ZAP-70, CD3ζ, Lck and LAT phosphorylation and that this inhibition is rapid (146). In this study, we established that

LAM-induced inhibition of CD4+ T cell activation is not an isolated event from which T- cells easily recover, but rather a powerful anergizing stimulus with long-term functional consequences. As shown with other T-cell anergy inducing molecules, anergy induction required the presence of LAM in the T cell membrane at the time of primary stimulation, but LAM was not required during the re-stimulation step that revealed the T-cell anergic state. While several T-cell anergy models have been described before, this study is the first demonstration of anergy induction by a microbial glycolipid in the context of primary T-cell activation by a strong agonist antigen.

Anergy induction is thought to occur in two steps: First, an anergizing molecule elicits suboptimal or partial activation of the T cell. Second, the sub-optimally activated T cell undergoes a state of hypo-responsiveness and becomes refractory to re-stimulation

(86, 87). Our results demonstrate that LAM first inhibits primary activation, which is followed by hypo-responsiveness upon optimal re-stimulation with an immunogenic antigen. Anergy induction and maintenance correlated with increased expression of the

E3 ubiquitin ligase, GRAIL in both mouse and human CD4+ T cells. GRAIL is an anergy-related gene whose increased expression has been shown to down-regulate IL-2 transcription (90, 94, 186). These findings indicate that following blockade of proximal

TCR signaling and activation, as reported previously, LAM inhibition of T cells

86 facilitates induction of anergy-related genes that render T cells hypo-responsive to optimal re-stimulation, resulting in long-term CD4+ T cell dysfunction. Whether such a biological phenomenon occurs in vivo and could be responsible for the delayed response to mycobacterial antigens characteristic of Mtb infection needs to be determined.

A previous study demonstrated that LAM can be detected by immuno- histochemical staining on macrophages and lymphocytes isolated from the lungs and spleens of Mtb infected mice (204). Moreover, exogenous administration of LAM by intravenous injection of mice revealed that LAM was distributed to splenic immune cells

(205). In further support of LAM association with immune cells, our results demonstrated that LAM pretreatment of human CD4+ T cells not only results in LAM binding to T cell membranes, but also associates with lipid rafts and the CD3-TCR complex. This strengthens the model of LAM insertion into the T-cell membrane via its lipid moieties, although further studies are necessary to exclude a specific receptor for LAM on T cells.

A recent report suggests that the lack of mannose cap in M. tuberculosis LAM does not affect its virulence in vivo or its interaction with macrophages in vitro (206). Although we did not examine the molecular structure of LAM to determine which parts of the molecule are necessary for its interaction and/or inhibitory effects on T cells, we assessed the effects of related mycobacterial lipolgycans including PILAM from M. smegmatis and lipomannan (LM) which lack a mannose cap, but have a similar lipid structure as

LAM from M. tuberculosis. Whereas PILAM exhibited slight inhibition at high concentrations, LM was stimulatory for CD4+ T cells (unpublished data), suggesting that the lipid moieties are necessary, but not sufficient for LAM’s interaction with and inhibitory effects on T cells.

Most studies of T cell anergy established that anergy results from TCR stimulation in an inhibitory environment including increased co-inhibition or decreased co-stimulation, or TCR engagement with a weak agonist peptide such as altered- or self- peptides (89). Others have shown that T cell anergy results from persistent agonist antigen stimulation (194). We did not observe differences in expression of co-stimulatory receptor, CD28 or co-inhibitory receptors, PD1, CTLA4, Lag-3 and Tim-3 between

LAM-treated and untreated T cells. Therefore, modulation of the aforementioned co- signaling receptors were not responsible for T-cell anergy induction in our model.

Instead, our results reveal a novel mechanism of T-cell anergy induction in which the association of LAM with the T cell membrane at the time of primary TCR engagement by strong agonistic antigen triggers a hypo-responsive state. At the moment, it is not clear if LAM-induced anergy results from direct interference with TCR signaling, TCR membrane mobility, T cell-APC conjugate formation or a combination of effects. Further studies will establish if the presence of LAM in T cell membranes physically interferes with the formation of an effective immunological synapse upon stimulation, resulting in incomplete activation.

Additional studies are needed to understand how LAM inhibition of T-cell activation leads to anergy via GRAIL. A separate study in our laboratory using proteomics to determine the effects of LAM on CD4+ T cell activation revealed down- regulation of Otub-1 in LAM-treated T cells (Karim AF, unpublished). Otub-1, regulated by the Akt-mTOR pathway, is a known negative regulator of GRAIL function (94, 105).

This suggests that LAM may disrupt the Akt-mTOR pathway resulting in Otub-1 down- regulation, which in turn may induce GRAIL. CD3 and CD40L are targets of GRAIL

88 regulation. Up-regulation of GRAIL in T cells led to degradation of CD3 (92), whereas ectopic expression of GRAIL in naïve T cells from CD40-/- mice resulted in down- regulation of CD40L (203). However, we did not find differences in CD3- or CD40L- expression between LAM-treated and non-treated T cells. Our data do not support a model of LAM anergy induction by GRAIL-mediated- CD3- or CD40L- down- regulation, which may be due to differences in experimental set-up and/or the ligand used to induce anergy. GRAIL can be upregulated in FoxP3-positive regulatory cells, and is sufficient to convert T cells to a regulatory phenotype. Tregs also can mediate suppression by inducing anergy in conventional CD4+ T cells through secretion of anergizing cytokines such as IL-10 (105, 207). We found no evidence for up-regulation of FoxP3 or induction of Tregs by LAM during priming and re-stimulation of purified

CD4+ T cells. Removal of nTregs had no impact on LAM’s ability to induce CD4+ T cell anergy. Thus, Tregs were not responsible for the T-cell anergy induction observed. We did demonstrate that exogenous IL2 down-regulates GRAIL expression in LAM anergized CD4+ T cells and restores the proliferative ability of these anergized T cells.

This is consistent with a recent report by Aziz et al. demonstrating inhibition of GRAIL by IL-2 in anergized T cells (186).

We have shown previously that LAM inhibits activation of naïve and effector

CD4+ T cells, in addition to pre-activated T cells (135, 146). Our current studies demonstrate the development of anergy when LAM is present during priming of naïve T cells, which may occur in secondary lymphoid tissues where the bacteria may be few. In addition we also show that effector T cells are similarly affected, suggesting that such an event may happen at the site of infection in the lungs where the bacterial loads are higher.

Altogether, these findings suggest that LAM can interfere with CD4+ T cell function at different differentiation and activation states during Mtb infection and disease.

Previous and current results demonstrate T cell inhibition over a wide range of

LAM concentrations down to 100 nM. The concentration of LAM in the immediate microenvironment of extracellular or intracellular Mtb is difficult to determine. However recent studies by others and our group indicate that small vesicles produced by Mtb alone

(microvesicles) or released by infected cells (exosomes) contain large amounts of LAM in addition to other mycobacterial molecules (37, 49, 208). These microvesicles may represent efficient means to deliver LAM to T cells in the immediate environment of Mtb infected cells.

In addition to its importance in cancer, autoimmunity and organ transplantation, T cell anergy is a mechanism of immune evasion in chronic infections due to HIV-1 and

Schistosoma mansoni (131, 133). In these infections T cell anergy was caused by protein antigens. Modulation of host immunity by glycolipids is rare except among intracellular pathogens such as trypanosomes and leishmanial species. Besides LAM, the only other microbial glycolipid known to directly inhibit T cell activation is trypanosomal glycoinositolphospholipids (GIPL) (209). Whether these pathogen glycolipids induce anergy is unknown. To our knowledge we are the first to show that a microbial glycolipid directly induces CD4+ T cell anergy by interfering with proximal TCR signaling.

In summary, we established a novel link between LAM association with the T cell membrane, LAM-induced T cell anergy and the expression of GRAIL in CD4+ T cells. It would be important to extend these studies to answer the following questions: 1) What

90 are GRAIL’s targets in LAM-anergized T cells? 2) Can LAM be detected on T cells in vivo, and on T cells from active or latent TB patients, and are these T cells anergic? 3)

How efficient are LAM-laden Mtb microvesicles from Mtb-infected macrophages in transferring LAM to T cells? 4) Could differential sensitivity to LAM inhibition of T cells explain the heterogeneous clinical manifestations of Mtb infection or the differential susceptibility to TB reactivation among humans? A recent report suggests that anergy induction in CD4+ T cells favors HIV-1 replication (131). It will be important to explore the biology and implications of LAM-anergized CD4+ T cells in Mtb infection or disease, and TB/HIV co-infection. Finally, these data provide novel insight into how CD4+ T cells despite sensitization to a broad repertoire of Mtb antigens may not respond optimally to their cognate antigens. T cell immune evasion strategies likely contribute to the host’s inability to eliminate Mtb and allow it to survive in the heart of a robust cellular immune system of macrophages and T cells such as the granuloma.

Chapter 4: Discussion and future directions

LAM trafficks outside M. tuberculosis-infected macrophages and inhibits T cell activation We investigated the role of M. tuberculosis LAM in evasion of CD4+ T cell responses by M. tuberculosis. Based on our data, we propose that M. tuberculosis LAM can directly modulate CD4+ T cell function above and beyond the known inhibitory effects that LAM has on antigen processing and presentation by M. tuberculosis-infected

APCs.

How then might M. tuberculosis LAM directly access CD4+ T cells? Several observations may help provide support. The intracellular localization of M. tuberculosis had originally been thought to preclude direct interaction between mycobacterial glycolipids including LAM and uninfected immune cells, such as CD4+ T cells. In M. tuberculosis-infected macrophages, mycobacterial LAM-containing vesicles can traffic outside phagosomes as bacterial vesicles into the extracellular microenvironment of the infection site and are then available for fusion with plasma membranes of nearby cells, including CD4+ T cells (37). In a mouse model of TB, a previous study by Mustafa et al demonstrated that LAM can be detected by immuno-histochemical staining on macrophages and lymphocytes isolated from the lungs and spleens of M. tuberculosis- infected mice (204). Moreover, exogenous administration of LAM by intravenous injection of mice revealed that LAM was distributed to splenic immune cells (205), suggesting that purified LAM may associate with immune cells including lymphocytes.

In further support of LAM association with immune cells, our in vitro data demonstrate that LAM pretreatment of CD4+ T cells results in LAM association with the T cell membranes.

How much LAM would physiologically be required for inhibition? Previous and current results demonstrate T cell inhibition over a wide range of LAM concentrations down to 100 nM. The concentration of LAM in the immediate microenvironment of extracellular or intracellular Mtb is difficult to determine. However recent studies by others and our group indicate that small vesicles produced by bacterial Mtb alone or released by infected cells as exosomes and /or microvesicles contain large amounts of

LAM in addition to other mycobacterial molecules (37, 49, 208). These bacterially derived microvesicles may represent efficient means to concentrate and deliver LAM to T cells in the immediate environment of Mtb infected cells. We propose that since local concentrations of LAM and other M. tuberculosis molecules will be highest around infected cells, direct inhibition of CD4+ T cell activation most likely will occur in the immediate vicinity of these M. tuberculosis-infected APCs. However, there is need to determine the efficiency of LAM-laden bacterial vesicles from M. tuberculosis-infected macrophages in transferring LAM to CD4+ T cells.

LAM and inhibition of TCR signaling

We discovered and characterized another likely mechanism that may limit the efficacy of the adaptive immune response to M. tuberculosis. We discovered that M. tuberculosis mannose-capped lipoarabinomannan (LAM) inhibits antigen-specific CD4+

T cell activation by interfering with very early events in TCR signaling through LAM’s insertion in T cell membranes. Our earlier study demonstrated that M. tuberculosis glycolipids, including LAM, directly inhibited polyclonal CD4+ T cell activation by blocking ZAP-70 phosphorylation, independent of TLR2. In chapter 2, we focused on

LAM, the major cell wall glycolipid of M. tuberculosis, and extended the observations to

94 antigen-specific CD4+ T cell responses, to human CD4+ T cells and further analysis of

LAM’s effect on proximal TCR signaling. We found that phosphorylation of signaling proteins was inhibited both upstream (Lck) and downstream (LAT) of ZAP-70, indicating that LAM inhibited TCR signaling at the site of triggering. LAM did not affect phosphorylation at Lck-Tyr505 and was independent of PKA indicating that the negative regulator of Lck-Tyr505, Csk was not involved. In addition, as reported by others (56,

57), LAM associated with the T cell membranes including lipid rafts (Figure 2.6).

Washing after 1 hour LAM pretreatment before stimulation with anti-CD3/CD28 did not affect inhibition of CD4+ T cell activation. Moreover, presence of LAM with CD4+ T cell membrane correlated with inhibition of CD4+ T cell activation, as demonstrated by the time-dependent loss of anti-LAM mAb staining and T-cell inhibition (Figure 3.1)

Together, the binding, washing and temperature sensitivity experiments suggest that LAM requires a specific membrane environment (fluid membrane) for association with the T cell membrane, and they also suggest that presence of LAM in the membrane is required for inhibition of proximal TCR signaling. LAM insertion did not affect the presence Lck and LAT in lipid rafts in T cells.

How does LAM mediate its inhibitory activity? Other studies have postulated that

LAM’s association with T cell membrane modulates host cell function (56, 138).

However, no study has definitively demonstrated that LAM association with the T cell membrane is responsible for LAM’s observed effects. Our results and others support three divergent hypotheses. The first is that association of LAM with the CD4+ T cell membranes is necessary and sufficient to induce inhibition of T cell activation. In this model, LAM association with the membrane activates an unknown signaling event(s) or 95 interferes with the ability of T cell membranes to assemble or reorganize themselves upon stimulation. In support of this hypothesis Shabaana et al observed that pretreatment with LAM led to increased kinase activity within lipid rafts of human Th1 cells (56). We did not observe increased phosphorylation of Lck at either its positive or negative regulatory sites. Additionally our data indicates that Csk, a negative regulator of Lck

(210), is not activated by ManLAM. Other studies have shown that ZAP70, the downstream molecule of Lck was hypophosphorylated upon disruption of lipid raft and membrane assembly, suggesting interference with upstream events in the membrane

(145).

The second possibility may be related to the mannose cap or other unique structural features that LAM possesses. The alternative hypothesis would be that LAM’s association with the T cell membrane is necessary but the effect is not dictated by interference with lipid raft function. LAM’s insertion via its GPI motif acts as a tether allowing other components of LAM (such as its mannose cap) to bind or interfere with T cell surface molecule(s), possibly the TCR complex itself, and thus affect its function.

This model has been proposed because of LAM’s ability to inhibit phagosome maturation after insertion into macrophage lipid rafts and is supported by our previous finding that

PIM, while having the phosphoinositol backbone of LAM, is much less efficient on a per molecule basis in inhibiting T cell activation (135, 138). LAM’s inhibitory effects on

CD4+ T cells are TLR2-independent (135), while LM is a TLR2 agonist (34, 211), suggesting that LAM may actually associate with different T cell molecules than other related Mtb glycolipids. LAM not only co-localized with lipid rafts, but also the CD3

96 complex on the human CD4+ T cell membrane (Figure 3.8). This observation raises the possibility of direct interference by LAM with T cell surface molecule function.

However, further studies are required to exclude the possibility that there are receptors for LAM on the T cell surface.

The third hypothesis is that LAM is internalized by T cells, resulting in direct interference with molecules in the TCR pathway. The time-dependent loss of LAM in the

LAM binding experiments (Figure 3.2) suggest that LAM may be internalized by T cells, allowing LAM to directly interfere with TCR signaling molecules or other molecules in other pathways that cooperate with the TCR signaling pathway. We have not ruled out the latter possibility. The experiment required would be to pretreat T cells with labeled

LAM and follow LAM into various subcellular fractions by western or intracellular cytokine staining.

Sucrose density gradients give a static picture of rafts and only determine whether or not certain molecules are present. They do not address raft structure nor establish if proper microcluster formation is occurring at the T cell-APC synapse. Confocal microscopy and other more advanced imaging techniques have broadened our understanding of the dynamics and kinetics of T cell stimulation induced microcluster formation. Lillemeier et al. used a combination of photoactivated localization

97 microscopy, fluorescence cross-correlation spectroscopy and transmission electron microscopy to show that TCR and LAT are found on separate protein islands that coalesce upon activation (157). Total internal reflection fluorescence microscopy has been used to detect TCR microcluster formations that include ZAP-70, Lck, and other signaling components that form within seconds of TCR engagement (158, 159). These advanced technologies may help elucidate the exact molecular mechanisms by which

LAM interferes with proximal TCR signaling.

In addition to blocking microcluster formation, ManLAM could induce phosphatase activity or ubiquitinylation. Numerous phosphatases negatively regulate T cell activation by dephosphorylating Lck, ZAP-70 and ERK (160, 161, 166). Studies of microbial inhibition of T cells have identified several instances where pathogens either through the induction of phosphatase activity or through their own phosphatases inhibit proximal TCR signaling (162-164). Knutson et al. has shown that LAM by increasing phosphatase activity suppressed MAPK signaling in macrophages (165). Phosphatase

Src homology 2 containing tyrosine phosphatase (SHP-1) had a major role. SHP-1 regulates proximal TCR signaling and its activity itself is regulated at several points that

ManLAM could take advantage of (166). ManLAM did not inhibit IL-2 production by anti-CD3/CD28 activated Jurkat T cells (data not shown) known to be missing inositol phosphatases SHIP-1 and PTEN (165, 167). These phosphatases regulate phosphorylation downstream of proximal TCR signaling. PTEN is involved in regulation of the phosphoinositide 3-kinase (PI3K) pathway, utilized by some co-stimulatory receptors. Ubiquitin ligases have emerged as one of the most important molecules playing a negative T cell regulatory role in immune response and inflammation in a wide

98 range of conditions including immune response to pathogens (91). Therefore more studies are needed to rule out this mechanism.

In general, the studies described in chapter 2 and 3 attempted to examine the lipid raft hypothesis in the effort to understand the concepts in LAM-induced inhibition of T cell activation. Although we demonstrated LAM association with the T cell membrane

(Figure 3.2A and Figure 3.11A) these data does not conclusively prove lipid raft disruption. According to a previous report, the elemental lipid raft is very small (<70 nanometers in diameter) (140), and yet as shown in Figure 3.11A lipid raft markers clearly appear to be more than this size, suggesting that association of LAM with the T cell membrane facilitates lipid rafts to coalesce instead of disruption. Therefore, in order to specifically elucidate this hypothesis, one needs, in detail, to dwell on the following important concepts in LAM-induced inhibition T cell activation: First, does LAM physically interfere with lipid raft assembly and/or block microcluster formation during the initiation of TCR signaling? Second, how does LAM do it? Is this via association with T cell membrane/lipid rafts or endocytosis? Third, the part(s) of the LAM molecule is/are required or sufficient to mediate this interaction.

While direct inhibition of CD4+ T cell activation via interference with TCR signaling is a novel mechanism for M. tuberculosis, it has been reported to occur with other pathogens including viruses, bacteria and protozoan parasites. The herpes family of viruses also can inhibit proximal TCR signaling: Herpesvirus saimiri inhibits ZAP-70 phosphorylation by sequestering Lck (212), while herpes simplex virus down-regulates

LAT phosphorylation (163). Whole Neisseria gonorrhoeae and other outer membrane vesicles have been shown to interact with CEACAM1, a co-inhibitory receptor that

99 stimulates SHP-1 and -2 phosphatase activity that negatively regulates the TCR signaling pathway (162, 213). Bacterial toxins including those from Bacillus anthracis and

Helicobacter pylori can inhibit TCR signaling. Bacillus anthracis lethal and edema toxins inhibit downstream T cell signaling via the MAP kinase and calcium signaling pathways, respectively (214-216). Mtb. pylori produces exotoxin VacA, which can block calcium influx and induces the activation of the stress kinase p38 (217). Salmonella enterica can also express proteins that can downregulate TCR expression (218). Yersinia pestis, the causative agent of plague, produces a protein called YopH, which acts as a tyrosine phosphatase and can dephosphorylate multiple components of the proximal TCR signaling pathway, including Lck, LAT and SLP-76 (219).

Modulation of host immunity by glycolipids is uncommon except among intracellular pathogens such as Leishmania (lipophosphoglycan), Trypanosoma

(glycoinositol phospholipids, GIPL) and mycobacteria. These pathogens block phagosome maturation, suppress cytokine production and modulate surface expression of

MHC molecules (168, 169). While these glyolipids have unique head groups, they share a glycosylphosphatidylinositol (GPI) anchor which is important for their effects on host immunity (170, 171). Tachado et al. purified GPIs from Plasmodium, Trypanosoma and

Leishmania and found that they induced protein kinase C activity (175). This study was performed in macrophages and to our knowledge no one has evaluated their effects on T cells. In fact, Trypanosoma GIPL is the only other microbial glycolipid known to directly modulate T cell activation (209). T cell studies by others have suggested that

ManLAM can induce PKC signaling to modulate host immunity, however, in our earlier study, PKC activation by phorbol myristate acetate (PMA) was not inhibited by

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ManLAM. Besides ManLAM, the only other microbial glycolipid known to directly modulate T cells is Trypanosoma GIPL (209). Since microbial glycolipids have similar effects on other cell types, microbial lipophosphoglycan and glycolipids may have similar inhibitory effects on T cell activation as seen for ManLAM. To our knowledge we are the first to determine that a microbial glycolipid such as ManLAM can directly inhibit proximal TCR signaling. These findings not only provide insight into how M. tuberculosis evades adaptive immunity but also how TCR activation and productive immune response may be regulated by pathogenic microorganisms.

A Model of Anergy Induction by LAM

Based on the data presented in chapter 2 and chapter 3, we propose that the presence of LAM in the CD4+ T cell membrane during primary stimulation plays a central role, not only in inhibition of T cell activation, but also in induction of anergy in antigen-reactive CD4+ T cells. Our proposed model of anergy induction by LAM is depicted in Figure 4.1. In the absence of LAM, the engagement of the TCR by a strong agonist antigen and full costimulation results in strong TCR signaling, IL-2 gene expression and IL-2 production (Figure 4.1A). In contrast, the presence of LAM in the T cell membrane at the time of primary stimulation results in weak TCR signaling, increased GRAIL and partial activation (Figure 4.1B) which is refractory to restimulation, even though LAM is no longer present in the T cell membrane at the time of restimulation. The addition of exogenous IL-2 at restimulation down-regulates GRAIL and reverses LAM-induced anergy (not shown here).

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NFAT + AP1 = IL-2 NFAT alone = GRAIL

Figure 4.1. A model of anergy induction by LAM. For details, see text.

All the known in vitro models of T cell anergy induction including pretreatment of T cells with the calcium ionophore ionomycin, anergizing cytokines (IL-10, TGF-β) and suppression via regulatory T cells mimic the typical anergy model, ie TCR engagement without full costimulation. The ionomycin-induced anergy model is the most studied and established of all the in vitro anergy models (86, 89, 90), while the mechanisms for other models including anergizing cytokines IL-10, TGF-β are not clear.

Pretreatment with ionomycin results in increased Ca2+ flux into the cytoplasm, higher activation of the Ca2+/calcineurin arm of the TCR signaling pathway relative to

PKC/IKK/Ras/MAP kinase pathway, resulting in increased production of anergy- inducing proteins including GRAIL. The outcome of LAM-induced anergy is consistent and comparable to the aforementioned in vitro anergy models, however, the mechanism seems to mimic anergy induction by weak TCR ligands or persistent antigen stimulation

102 in vivo in which blockage of ZAP-70 or LAT and sometimes increased expression of anergy-inducing proteins have been observed (185). Our supporting experimental data are as follows:

1. LAM pretreated CD4+ T cells exhibit partial activation and down-

regulation of proximal TCR signaling pathway, consistent with previous

findings of anergy induction associated with blockade of the proximal

TCR signal transduction pathway (89, 93).

2. CD4+ T cells anergized by LAM show defective IL-2 production and T

cell proliferation at restimulation, and as shown before with other T-

cell anergy inducing models, anergy induction required pretreatment

with LAM before primary stimulation, but presence of LAM in the T

cell membrane was not required during the re-stimulation step that

revealed the T-cell anergic state (90).

3. The anergy-associated protein GRAIL was upregulated in CD4+ T cells

rendered anergic by LAM during both priming and restimulation.

4. Anergy was reversible by exogenous IL-2 at restimulation (discussed

below in another section).

Therefore, the outcome (expression of anergy inducing genes and reversal of anergy) in LAM-induced anergy, although the mechanism may be different,is comparable to the ionomycin-induced anergy model. This is the most established model used to study anergy induction in vitro, confirms that LAM is a potent anergy inducer. It would be interesting to further explore the difference in mechanisms between LAM-induced anergy

103 and other anergy models especially those induced by weak TCR ligands and persistent antigen stimulation.

Anergy induction is rapid and occurs during priming and is thought to follow two steps: First, an anergizing molecule elicits suboptimal or partial activation of the T cell.

Second, the sub-optimally activated T cell undergoes a state of hypo-responsiveness and becomes refractory to re-stimulation (86, 87). Our results demonstrate that LAM first inhibits primary activation, which is followed by hypo-responsiveness upon optimal re- stimulation with an immunogenic antigen. Anergy induction and maintenance correlated with increased expression of the E3 ubiquitin ligase, GRAIL in both mouse (Figure 3.8) and human CD4+ T cells (Figure 3.11). Moreover, GRAIL-deficient CD4+ T cells were resistant to anergy induction by LAM (Figure 3.9) and addition of exogenous IL-2 down- regulates GRAIL and reversed LAM-induced anergy (Figure 3.10). GRAIL is an anergy- related gene whose increased expression has been shown to down-regulate IL-2 transcription (90, 94, 186). These findings indicate that following blockade of proximal

TCR signaling and activation, as reported previously, LAM inhibition of T cells facilitates induction of anergy-related genes that render T cells hypo-responsive to optimal re-stimulation, resulting in long-term CD4+ T cell dysfunction. While several T- cell anergy models have been described before, this study is the first demonstration of anergy induction by a microbial glycolipid in the context of primary T-cell activation by a strong agonist antigen. However, as seen with ionomycin and other models, it would be interesting to determine the effect of LAM on Ca2+ signaling and other pathways that cooperate with the TCR signaling pathway for productive T cell activation. It would also be important to determine whether other anergy-associated genes/proteins such as Cbl-b,

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Itch are induced in this model. It will be important to determine whether this biological phenomenon occurs in vivo and could be responsible for the delayed response to mycobacterial antigens characteristic of Mtb infection.

Other factors or stimuli that lead to and can confound anergy

LAM-induced anergy was not due to other external stimuli such as T regulatory cells or anergizing cytokines (Figure 3.5A and 3.5B). GRAIL can be upregulated in

FoxP3-positive regulatory cells, and is sufficient to convert T cells to a regulatory phenotype. Tregs also can mediate suppression by inducing anergy in conventional CD4+

T cells through secretion of anergizing cytokines such as IL-10 (105, 207). We found no evidence for up-regulation of FoxP3 or induction of Tregs by LAM during priming and re-stimulation of purified CD4+ T cells. Removal of nTregs had no impact on LAM’s ability to induce CD4+ T cell anergy. Thus, Tregs were not responsible for the T-cell anergy induction observed.

Most studies of T cell anergy established that anergy results from TCR stimulation in an inhibitory environment including increased co-inhibition or decreased co-stimulation, or TCR engagement with a weak agonist peptide such as altered- or self- peptides (89). Others have shown that T cell anergy results from high dose of antigen and persistent agonist antigen stimulation (194). We did not observe differences in expression of co-stimulatory receptor, CD28 or co-inhibitory receptors, PD1, CTLA4, Lag-3 and

Tim-3 between LAM-treated and untreated T cells. Therefore, modulation of the aforementioned co-signaling receptors were not responsible for T-cell anergy induction in our model. Moreover, our data shows that LAM-induced anergy can occur over a wide

105 range of Ag85B concentrations (Figure 3.4A), indicating that high antigen concentration did not contribute to anergy induction. Instead, our results reveal a novel mechanism of

T-cell anergy induction in which the association of LAM with the T cell membrane at the time of primary TCR engagement by Mtb Ag85B triggers a hypo-responsive state. At the moment, it is not clear if LAM-induced anergy results from direct interference with TCR signaling, TCR membrane mobility, T cell-APC conjugate formation or a combination of effects. Further studies will establish if the presence of LAM in T cell membranes physically interferes with the formation of an effective immunological synapse upon stimulation, resulting in incomplete activation.

Activation-induced cell death (apoptosis) and necrosis are other factors that can frequently be associated with anergy (127). Mice injected with high doses of superantigens (proteins that interact simultaneously with MHC class II and the Vβ region of the TCR) or soluble antigen delete large numbers of reactive T cells, but the surviving

T cells remain hypo-responsive to subsequent stimulation (220). Moreover, some pathogens, including Mtb can induce apoptosis of macrophages (221). We maintained the viability of murine CD4+ T cells for over 5 days during rest via culture in IL-7 (Figure

3.1A and 3.1B), thus necrosis was greatly minimized. IL-7 is a homeostatic cytokine that has been proven to efficiently maintain long term survival of naïve, memory and effector

T cells without antigen stimulation (127). After removal of IL-7 from the culture, significant apoptosis was only observed in T cells that were pretreated with dexamethasone, compared to non-treated T cells prior to restimulation (Figure 3.5C), indicating LAM is not associated with deletion of T cells via apoptosis. Dexamethasone can induce apoptosis by promoting the functions of some caspases (222). In further

106 support of our findings, Kriegel et al showed that Grail knockout CD4+ T cells exhibit enhanced survival after two days of stimulation (90).

T cell exhaustion is another biological phenomenon during chronic infections and cancer that can mimic T cell anergy (127). The inhibitory receptors PD1, Lag-3, and

Tim-3 are important negative regulatory pathways that control autoreactivity and immunopathology (127). The expression of these inhibitory receptors correlates with activation of functional T cells, however, higher and sustained expression is a hallmark of

T cell exhaustion. The inhibitory signaling pathway mediated by PD1 in response to binding to PDL1 and/or PDL2 is the most reported example (223). We did not observe a significant difference in expression of PD1 and other inhibitory receptors in LAM-treated and non-treated T cells in our experiments (Figure 3.7A and 3.7B). Altogether, these results indicate that LAM-induced anergy was not confounded by T regulatory cells, inhibitory receptors, apoptosis or T cell exhaustion.

Role and induction of GRAIL by LAM

The biochemical pathways that lead to anergy induction are still being elucidated, however, there are common and unifying observations in most anergy models:

1. Under anergizing conditions, TCR stimulation leads to increased

expression and production of anergy-inducing proteins notably E3

ubiquitin ligases including GRAIL, which block proximal TCR signaling

or their downstream signaling pathways (eg Ras MAPK) and also block

cytokine transcription (90, 186).

2. The anergy-inducing protein, specifically GRAIL prevents transcription of

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IL-2 and other cytokines and may block proliferative pathways when the

cell is restimulated.

3. The induction of GRAIL is normally antagonized or reversed by

stimulation of the TCR/CD3 with a strong agonist antigen with full

costimulation, and with proliferation induced by signaling through IL-2Rα

(186).

Our data support interference with proximal TCR signaling pathway and upregulation of GRAIL as the biochemical pathways that may be underlying LAM- induced anergy. We demonstrate that GRAIL induces and maintains the anergic state after the removal of LAM (Figure 3.3 and Figure 3.8). However, how GRAIL mediates and maintains anergy in CD4+ T cells will require further study to help obtain more mechanistic insight as to how anergy is maintained after removal of LAM. One of the implicated mechanisms GRAIL uses to induce and maintain anergy is repression of IL-2 and other cytokine transcription. GRAIL upregulation results in decrease in IL-2 mRNA.

We did not measure IL-2 mRNA levels in our studies, however, one would propose that decreased IL-2 production at priming (Figure 3.3A) indirectly supports this mechanism.

GRAIL targets key proteins in the proximal TCR signaling pathway (TCR, CD3-zeta,

ZAP-70), Akt-mTOR pathway (Otubain-1), MAP kinase pathway (total and phosphorylated Erk), costimulation (CD40L), actin cytoskeleton (RhoA GTPase), tetraspanins (CD81, CD151) (90, 94, 105, 224) for proteosomal degredation. Nurieva et al showed that GRAIL induces and maintains anergy by degrading TCRβ, CD3-zeta (92), thus decreasing TCR specific signals. Our data reveal downregulation of phosphorylated

ZAP-70 while total ZAP-70 was unchanged at primary stimulation. A previous report

108 showed that levels of both total and phosphorylated ZAP-70 were unchanged in T cells from both Grail-/- and wild type mice (90, 92), This is likely due to the difference in assays or ligands used. The assay we used here to determine both phosphorylated and total ZAP-70 levels was short-lived (4 min). The decrease in phosphorylated ZAP-70 in our experimental set up is an early event likely due to interference with membrane assembly by LAM during TCR stimulation. One would therefore be reluctant to suggest that high levels of GRAIL would be achieved within this short time to effect degradation of total ZAP-70. Lineberry et al demonstrated that GRAIL ubiquitinates and leads to degradation of the co-stimulatory molecule CD40L (224). We tried to address this mechanism in figure. 3.4, and did not find differences in surface expression of TCR,

CD40L in LAM-treated compared to non-treated T cells, suggesting that these molecules are not GRAIL targets in our system. Although, we did not specifically look for levels of

CD3-zeta, which Nurieva determined, we did not find differences in surface expression of another important component of CD3 complex, CD3e in LAM-treated compared to non-treated T cells. Our data therefore, do not support a model of LAM anergy induction by GRAIL-mediated- TCR/CD3- or CD40L- down-regulation. We, and the aforementioned authors used different experimental set-up and/or the ligand to induce

GRAIL and anergy, which may explain our different findings. Otubain-1, a de- ubiquitinating enzyme, facilitates degradation of GRAIL. We have preliminary data from a proteomic analysis of LAM’s effect on CD4+ T cells that Otubain-1 is downregulated in LAM-treated CD4+ T cells (Karim AF and Sande OJ, unpublished), providing one of the likely mechanisms for how GRAIL is up-regulated. Otubain-1, regulated by the Akt-mTOR pathway, is a known negative regulator of GRAIL function

109

(94, 105). This suggests that LAM may disrupt the Akt-mTOR pathway during primary stimulation, resulting in Otub-1 down-regulation, which in turn may lead to sustained high GRAIL levels after anergy induction (94).

The MAP kinase signaling pathway in T cells is one of the pathways that cooperate with proximal TCR signaling and is crucial for T cell proliferation, differentiation and cytokine secretion, and its kinase, ERK, is another GRAIL target. In an effort to understand the effects of GRAIL deficiency in CD4+ T cells, for example,

Kriegel et al (90) showed that GRAIL deficiency leads to increased total and phosphorylated levels of ERK1/2. Naïve CD4+ T cells from Grail-/- mice had augmented levels of ERK1/2 compared to T cells from wild type mice, suggesting that GRAIL promotes degradation of ERK. Others have shown that GRAIL modulates cytoskeletal function and reduces T/APC conjugate efficiency. Our data shows GRAIL upregulation in LAM-treated T cells after both antigen-specific and polyclonal stimulation, consistent with observations by others, suggesting that this mechanism may not be a prominent feature in GRAIL-mediated anergy induction and maintenance. Although we did not specifically examine the state of ERK and other molecules in LAM-anergized cells in our studies, our data add more support to observations by others that demonstrate that anergy induction via any model involves elevation of anergy mediators including GRAIL that induce and maintain anergy by interfering with the proximal TCR specific signals, its downstream signaling pathways and other pathways, as well as cytokine transcription, all of which must cooperate during T cell stimulation. We therefore need to study in detail the various signaling pathways, and transcription factors involved in primary T cell activation to better understand the mechanisms leading to anergy via GRAIL in LAM-

110 induced anergy. It would be interesting to examine the Ca2+/calcineurin/NFAT arm of

TCR signaling, for example.

Reversal of LAM-induced anergy by IL-2

T cell anergy can be reversed by stimulating T cells in the presence of high levels of IL-2.

We did demonstrate that exogenous IL2 down-regulates GRAIL expression in LAM anergized CD4+ T cells and restores the proliferative ability of these anergized T cells

(Figure 3.10A and 3.10B). This is consistent with a recent report by Aziz et al. demonstrating inhibition of GRAIL by IL-2 in anergized T cells (186). This reversal is not just an outgrowth of small numbers of T cells that may have failed to be anergized, as we would not expect to see such difference in GRAIL levels in LAM-anergized T cells with or without IL-2 at restimulation.

The mechanisms underlying IL-2-dependent regulation of T cell anergy are still not fully elucidated. The IL-2 reversal in in vitro T cell anergy models was first reported in the early 1990s as a process that occurs at both the level of cytokine production and transcriptional activation of the IL-2 gene (89). Signaling through the IL-2 receptor has been shown to activate mTOR, which plays an important role in the integration of signals that determine the fate of T cells. Macian et al, in a previous study showed that IL-2 receptor signaling mediated through mTOR inhibits the expression of anergy-inducing genes independently of any effect on cell cycle progression (225). In addition they showed that this effect is likely due to changes in the levels of AP-1 activation induced by IL-2 receptor signaling in T cells, thus supporting control at the level of transcription of IL-2 gene (Figure 4.1). Moreover, study showed that IL-2 signaling downregulated

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GRAIL and reversed anergy by via mTOR and Otubain-1 activation (94). Otubain-1 is known to promote rapid degradation of GRAIL during T cell activation. In our unpublished data (Ahmad F. Karim et al) we found that mTOR phosphorylation is downregulated and Otubain-1 levels are decreased when CD4+ T cells are stimulated in the presence of LAM during priming, suggesting that the presence of LAM in the membrane is not only interfering with TCR signaling, but may also affect mTOR signaling pathways. We did not determine the effect of exogenous IL-2 on mTOR phosphorylation at re-stimulation. However, we propose that since LAM is no longer on the membrane at the time of re-stimulation, IL-2 would activate mTOR, resulting in inhibition of GRAIL.

Our data not only confirms other reports that show and try to explain how IL-2 may prevent or reverse the establishment of anergy in T cells, but also reveals for the first time that this occurs in the context of anergy induced by a microbial glycolipid. This, therefore, helps to understand how the cytokine environment can be a determinant that shapes the outcome of T cell responses – anergy versus activation-when antigen is encountered.

Although the phenomenon of IL-2 reversal is not yet clear in in vivo studies, the ability to reverse LAM-induced anergy raises the question of the in vivo relevance of this state in the realm of M. tuberculosis infection. Under which circumstances would high levels of IL-2 occur, and what would be its effect on progression of infection and disease? One would be tempted to propose that IL-2 reversal can occur at the site of infection (granuloma), generating hyperactivated T cells, resulting in hyperinflammation and tissue destruction. This could partly explain the reactivation of TB with severe symptoms of immune reconstitution inflammatory syndrome (IRIS) seen in patients with

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HIV/TB co-infection in whom anti-retroviral therapy is initiated. Strong vaccines might also be able to promote hyperinflammation by increasing IL-2 production by T cells.

Relevance of IL-2 is reflected in clinical studies of IL-2 use. High doses of IL-2 have been used as a form of immunotherapy to treat renal cell carcinoma after surgical intervention (226). High doses of IL-2 reverse anergy and increase the population and function of effector T cells (CD4+ and CD8+ T cells) and natural killer cells, in addition to its effects on B cells, dendritic cells and macrophages in the cancer microenvironment, resulting in improved killing of cancer cells. Low-dose IL-2 can increase Treg populations resulting in suppression of conventional effector T cell function. And to this end, there are active phase ½ trials aimed at repriming analogous Tregs to treat autoimmunity and to prevent graft versus host disease in solid organ transplant (226).

Low-dose recombinant human IL-2 in combination with anti-tuberculous drugs has been tried before to treat multi-drug resistant, with mixed results (227). In a mouse model of

TB, a combination of IL-2, granulocyte-monocyte colony stimulating factor (GM-CSF) and anti-tuberculous agents isoniazid and rifampicin to treat multi-drug resistant TB resulted in lower bacterial load and lesions in treated compared to control mice (228).

However, follow up studies to confirm these findings have not been forthcoming. All in all, our findings reveal another factor that can be further elucidated to establish its relevance in pathogenesis, immune homeostasis and treatment of not only TB, but other conditions as well.

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GRAIL and anergy induction by pathogens

While anergy induction via GRAIL is novel in M. tuberculosis, GRAIL is just one of the many proteins in the elaborate ubiquitination system in both mice and humans, that a wide range of pathogens exploit to evade host defenses (229). Other proteins that are implicated are the E3 ubiquitin ligases Cbl-b and Itch. In a recent report, upregulation of

Cbl-b and Itch was associated with anergy induction in human CD4+ T cells and this promoted increased HIV-1 replication (31). In addition to its importance in cancer, autoimmunity and maintenance of glucose metabolism in the liver, GRAIL is implicated in immune evasion in chronic infection due to the parasitic worm, Schistosoma mansoni

(131, 133). In these infections T cell anergy was facilitated by protein antigens from the pathogen. Modulation of host immunity by glycolipids is rare except among intracellular pathogens such as trypanosomes and leishmanial species. Besides LAM, the only other microbial glycolipid known to directly inhibit T cell activation is trypanosomal glycoinositolphospholipids (GIPL) (209). Whether these pathogen glycolipids induce anergy is unknown. To our knowledge we are the first to show that a microbial glycolipid directly induces CD4+ T cell anergy via GRAIL. Together, our data also underscores the role of ubiquitin the pathway in the regulation of immune responses and inflammation in chronic infections.

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Conclusion and future directions

It would be important to extend these studies to answer the following questions:

1. What is the role of LAM’s structure in inhibition of CD4+ T cells? While we have demonstrated that LAM exerts its suppressive effects by associating with lipid rafts of CD4+ T cell membranes, the role of LAM structure, specifically the mannose cap and mechanisms linking lipid raft association with TCR signaling defect and anergy are still not clear. Based on data in chapter 2 and 3, we would hypothesize that association of

LAM with the T cell membrane via its lipid tails is necessary and sufficient to induce inhibition of T cell activation. LAM association with the T cell membrane interferes with the ability of T cell membranes to assemble themselves upon TCR engagement of peptide-MHC. Alternatively, LAM association with the T cell membrane is necessary but not sufficient. LAM via lipid moieties acts as tether allowing other components such as the mannose cap to bind or interfere with T cell surface molecules, and thus affect their function. T cells lack mannose receptors. There are three types of LAM described based on the presence and structure of the motifs capping the non-reducing termini of the arabinan domain: mannose-capped LAM (ManLAM), phospho-myo-inositol (PI)-capped

LAM (PILAM) and non-capped LAM (AraLAM). In addition variations in structure of

ManLAM itself from Mtb clinical isolates are known. These are M. tuberculosis strains

HN885 and HN1554 that contain truncated ManLAM, and have a low association with mannose receptor on macrophages, and Erdman, the laboratory strain used to study the pathogenesis of TB, and H37Rv that bind mannose receptors on macrophages. Therefore we can utilize these LAM structural variations to gain more insight in the molecular mechanism associated with the mannose cap and other LAM structures in LAM-induced

115 suppression of T cell function, for example we can examine LAM association with T cell membrane, TCR mobility/microcluster formation and CD4+ T cell function in terms of inhibition of T cell activation and anergy induction. We would expect that ManLAM will either, 1) associate with the T cell membrane more stably than the other types and variants of ManLAM, 2) show similar levels of association. In the first case, if stronger inhibition of T cells is observed with ManLAM than other LAM structures, the data would suggest that LAM association via its lipid moieties are necessary, but not sufficient for LAM’s observed inhibitory effects, pointing to the possibility of other requirements such as the mannose cap on LAM. However, if the effects are similar, then LAM association via its lipid moieties, and not other requirements, are necessary and sufficient.

Unlike LAM, LM is a TLR2 ligand. Moreover activated T cells can express TLR2 that serves as a co-stimulatory receptor. It would be important to use activated T cells to further compare the effects of ManLAM and all the aforementioned LAM structures. We predict that a stimulatory effect will be observed with LM compared to ManLAM, for example. In addition LAM colocalization with CD3 does not exclude the possibility of a receptor for LAM on T cells. There is therefore, need to do more studies towards determining if there is a receptor for LAM on T cells.

2. What is the link between LAM-induced inhibition of CD4+ T cell activation,

TCR/CD3 signaling and anergy? Additional studies are needed to identify GRAIL’s targets in LAM-anergized T cells and understand how LAM inhibition of T-cell activation leads to anergy via GRAIL. One possible method would be to utilize T cells from Grail-/- mice to determine the effects on key proteins in the TCR signaling pathway and associated downstream pathways including Ca2+ signaling and CD28 signaling. Our

116 unpublished data revealed that LAM downregulates phosphorylation of Akt and mTOR, which together with CD28 signaling facilitate expression of transcription factor, activator protein 1 (AP-1). AP-1 binds to NFAT to promote IL-2 and other cytokine gene expression. Absence of AP-1 allows NFAT to promote alternative gene expression including anergy-inducing genes (Figure 4.1). Based on the expression of GRAIL in our data in chapter 3, we would expect a significant decrease in expression of AP-1.

3. What is the physiological relevance of LAM-induced T cell suppression? The physiological relevamce of LAM-induced anergy needs to be demonstrated both in vitro and in vivo. Our experiments and data are based on the concept that LAM-laden bacterial vesicles from M. tuberculosis-infected macrophages transfer LAM to the T cell membrane. But how efficient are LAM-laden Mtb microvesicles from Mtb-infected macrophages in transferring LAM to T cells? Are the vesicles able to inhibit T cell activation and induce anergy? Jaffre Athman in the Harding Lab. already started addressing this issue. One possible method to determine whether macrophages transfer

LAM to T cell membrane would be culture of wild type Mtb-infected macrophages with wild type CD4+ T cells or RFP P25 TCR transgenic CD4+ T cells. Then flow sort the

CD4+ T cells, and perform Western blotting on the lysates or stain the CD4+ T cells with directly conjugated anti-LAM antibody (Cs35-Alexafuor-488) or biotinylated anti-LAM antibody (Cs35-biotin) and do flow cytometry, immunofluorescence or confocal microscopy.

There is a need to determine the in vivo effects of LAM and LAM-laden membrane vesicles. Membrane vesicles and supernatants recovered from Mtb-infected bone marrow macrophages and alveolar macrophages contain both Mtb cell wall

117 molecules (LAM, PIM, LprG, LpqH), and these vesicles showed immunomodulatory effects on macrophages both in vitro and in vivo (50). The in vivo relevance of these glycolipid and lipoprotein-containing bacterial vesicles on CD4+ T cell response against

M. tuberculosis is largely unknown. Based on the fact that the in vivo microenvironment may have other confounders including other pro-inflammatory cytokines such as TNFα or anti-inflammatory (T cell inhibitory) cytokines such as IL-10, it is would be important to further explore the effects of LAM-laden vesicles in vivo. Are these vesicels still inhibitory or stimulatory? Can LAM be detected on T cells in vivo, and on T cells from active or latent TB patients, and are these T cells anergic? Previous studies using mouse models of tuberculosis showed that LAM is distributed on macrophages in the lung.

Studies to determine the in vivo association of LAM and T cells and its effects have not been done. We could use M. tuberculosis-infected mice and TB patients to determine the in vivo role of LAM. For example, we could use T cells from the lungs of Mtb-infected mice, or T cells from Mtb-infected lymph nodes from TB patients. We could perform

Western blot from the lysates of the T cells or analyze by flow cytometry, confocal microscopy after staining with anti-LAM antibody, and function assays in parallel.

Because of the complexities of the in vivo environment, we predict that the level of detection of LAM on T cell membrane will be low, requiring use of high detection signal such as biotinylated anti-LAM antibody. Another in vivo alternative would be to infect

Grail-/- mice with Mtb and determine the outcome. One would predict that Grail-/- mice would be resistant to Mtb infection in the lungs, with lower bacillary load (colony- forming units) than wild type animals. However, there is also the possibility of severe disease and reduced survival in Mtb-infected Grail-/- mice due to hyperinflammation.

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It is also important to extend LAM and GRAIL studies in humans to include effects on T cell inhibitory receptors (PD1, Lag 3, Tim 3, etc), costimulatory receptors

(CD40L, CD28) and other anergy-inducing proteins such as Itch, Cbl-b and others. We need to determine whether LAM inhibits other T cell subsets such as CD8+ T cells, γδ T cells and other innate T cells that carry CD3/TCR complex in both mice and humans. We propose that induction of anergy in both CD4+ T cells and CD8+ T cells could have a significant effect on not only Mtb infection outcome, but also other coinfections. A recent report suggests that anergy induction in human CD4+ T cells favors HIV-1 replication. It will be important to explore the biology and implications of LAM-anergized CD4+ T cells in Mtb infection or disease, and TB/HIV co-infection.

M. tuberculosis is characterized by multiple clinical states. Could differential sensitivity to LAM inhibition of T cells explain the heterogeneous clinical manifestations of Mtb infection or the differential susceptibility to TB reactivation among humans?

Our findings may have a wide range of implications. We have shown that LAM inhibits activation of naïve and effector CD4+ T cells, in addition to pre-activated T cells.

Our current studies demonstrate the development of anergy when LAM is present during priming of naïve T cells, which may occur in secondary lymphoid tissues where the bacteria may be few. In addition we also show that effector T cells are similarly affected, suggesting that such an event may happen at the site of infection in the lungs where the bacterial loads are higher. Altogether, these findings suggest that LAM can interfere with

CD4+ T cell function at different differentiation and activation states during Mtb infection and disease.

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Since the bacilli are rarely eradicated, direct and indirect regulation of CD4+ T cell responses by LAM and other M. tuberculosis molecules may play predominant roles at different stages in M. tuberculosis infection and disease (ie, latent Mtb infection, primary progressive disease or reactivation). Indirect regulatory mechanisms may be predominant early in infection when activated APCs constitute the main target of Mtb control by interfering with activation of naïve CD4+ T cells in the regional lymph nodes.

When Mtb infection becomes latent in granulomas or progresses to primary disease or reactivation, it may imply that M. tuberculosis molecules (eg, inhibitors such as LAM) that traffick outside infected macrophages to the extracellular environment may play a role in the continuous inhibition or activation of memory and effector T cells, thereby maintaining latency and possibly leading to reactivation.

We propose further that our data may have implications in latent Mtb detection, anti-tuberculosis drug development and TB vaccine design. There are currently attempts to tame GRAIL expression to design therapies for autoimmune disorders including allergies. Upregulated GRAIL and LAM detection in T cells may not only be used as markers of infection, but also may have a role in preventing progressive disease or reactivation. TB vaccine designs that exclude LAM or include adjuvants that help override LAM inhibitory effects may be more effective than BCG, the only current vaccine. As IFN-γ production by Th1 cells is considered to be host-protective, our data would support the hypothesis that release of LAM during M. tuberculosis infection promotes an environment that is more favorable for infection and suggests further that highly efficacious antibiotics targeting LAM, such as benzothiazinones, may significantly

120 enhance Th1 cell function in the lung resulting in significant killing and reduction in bacterial burden (230)

In all, we established a novel link between LAM association with the T cell membrane, LAM-induced T cell anergy and the expression of GRAIL in CD4+ T cells.

We established that LAM-induced inhibition of CD4+ T cell activation is not an isolated event from which T-cells easily recover, but rather a powerful anergizing stimulus with long-term functional consequences. These data provide new insight into how CD4+ T cells despite sensitization to a broad repertoire of M. tuberculosis antigens may not respond optimally to their cognate antigens. T cell immune evasion strategies likely contribute to the host’s inability to eliminate M. tuberculosis and allow it to survive in the heart of a robust cellular immune system of macrophages and T cells such as the granuloma.

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Appendix 1: Correlation of dissertation figures with experiments

Figure Experiment number (s) 1.1 N/A 1.2 N/A 1.3 N/A 2.1 OJS011, OJS016, OJS019 2.2 OJS0121, OJS0123 2.3 OJS0126, OJS0127, OJS0128 2.4 Robert N. Mahon 2.5 Robert N. Mahon 2.6 Robert N. Mahon 3.1 OJS143, OJS165, OJS234 3.2 OJS432, OJS157, OJS205, OJS321 3.3 OJS431, OJS145, OJS435 3.4 OJS1117, OJS452 3.5 OJS223, OJS315 3.6 OJS0165, OJS228, OJS119, OJS212, OJS200, OJS351, OJS412, OJS206 3.7 OJS159, OJS167 3.8 OJS168, OJS481, OJS112, OJS184 3.9 OJS400, OJS348, OJS208, 3.10 OJS209, OJS1007 3.11 OJS376 4.1 N/A

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Appendix 2: List of publications and abstracts

Published papers

• Obondo J. Sande, Ahmad F. Karim, Qing Li, Xuedong Ding, Clifford V. Harding, Roxana E. Rojas, and W. Henry Boom. Mannose-Capped Lipoarabinomannan from Mycobacterium tuberculosis Induces CD4+ T cell Anergy via GRAIL. J Immunol. 2016 Jan 15; 196 (2):691-702. doi: 10.4049/jimmunol.1500710.

• Robert N. Mahon*, Obondo J. Sande*, Roxana E. Rojas, Alan D. Levine, Clifford V. Harding and W. Henry Boom. Mycobacterium tuberculosis ManLAM inhibits T-cell-receptor signaling by interference with Zap70, Lck and LAT phosphorylation. Cell Immunol. 2012 Jan-Feb; 275(1-2):98-105. doi: 10.1016/j.cellimm.2012.02.009. * Shared authorship

• Ahmad F. Karim, Obondo J. Sande, Sara E.Tomeckho, Xuedong Ding, Rob M. Ewing, Clifford V. Harding, Mark R. Chance, Roxana E. Rojas and W. Henry Boom. Proteomics analysis reveals inhibition of Akt-mTOR signaling in CD4+ T cells by Mycobacterium tuberculosis Mannose-capped Lipoarabinomannan. (Manuscript Communicated).

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Abstracts, posters, and presentations

• Obondo J. Sande, Ahmad F. Karim, Qing Li, Xuedong Ding, Clifford V. Harding, Roxana E. Rojas and W. Henry Boom. Mannose-Capped Lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis Induces CD4+ T cell Anergy. Keystone Symposium on Host Response in Tuberculosis, 2015 January 23-26, Santa Fe, New Mexico, USA. (Poster)

• Ahmad F. Karim, Obondo J. Sande, Sara E. Tomeckho, Xeudong Ding, Rob M. Ewing, Mark Chance, Roxana E. Rojas and W. Henry Boom. Label-free mass spectrometric analysis of M. tuberculosis ManLAM’s effect on the proteome of activated CD4+ T cells. Keystone symposia on Host Response in Tuberculosis. 2015 January 23-26, Santa Fe, New Mexico, USA. (Poster)

• Lipoarabinomannan from Mycobacterium tuberculosis Induces CD4+ T cell anergy. Center for Aids Research Retreat. Case Western Reserve University, Cleveland, OH. 2015 January 20. Invited Speaker (Oral presentation).

• Obondo J. Sande, Ahmad F. Karim, Sara Tomechko, Robert Ewing, Qing Li, Robert N. Mahon, Jaffre Athman, Pam Wearsch, Clifford V. Harding, Mark Chance, Roxana E. Rojas and W. Henry Boom. Inhibition of T cell function by Mycobacterium tuberculosis ManLAM: insight from T cell proteomics. 2013. Poster, Keystone Symposium on Host Response in Tuberculosis, Whistler, BC, Canada. (Poster)

• Obondo J. Sande, Robert N. Mahon, Roxana E. Rojas, Clifford V. Harding and W. Henry Boom. Mycobacterium tuberculosis ManLAM directly inhibits CD4+ T cell activation by interference with Lck and ZAP-70 phosphorylation. 40th Annual Autumn Immunology Conference, 2011 November 18-21, Chicago, USA. (Oral presentation and Poster).

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Works cited

1. Dye, C. 2006. Global epidemiology of tuberculosis. The Lancet 367:938-940. 2. Ottenhoff, T. H., and S. H. Kaufmann. 2012. Vaccines against tuberculosis: where are we and where do we need to go? PLoS Pathogens 8:e1002607. 3. Philips, J. A., and J. D. Ernst. 2012. Tuberculosis pathogenesis and immunity. Annual review of pathology 7:353-384. 4. Zumla, A., A. George, V. Sharma, N. Herbert, and I. Baroness Masham of. 2013. WHO's 2013 global report on tuberculosis: successes, threats, and opportunities. The Lancet 382:1765-1767. 5. Blomgran, R., L. Desvignes, V. Briken, and J. D. Ernst. 2012. Mycobacterium tuberculosis inhibits neutrophil apoptosis, leading to delayed activation of naive CD4 T cells. Cell host & microbe 11:81-90. 6. Grace, P. S., and J. D. Ernst. 2016. Suboptimal Antigen Presentation Contributes to Virulence of Mycobacterium tuberculosis In Vivo. Journal of immunology 196:357-364. 7. Arinaminpathy, N., T. Cordier-Lassalle, K. Lunte, and C. Dye. 2015. The Global Drug Facility as an intervention in the market for tuberculosis drugs. Bulletin of the World Health Organization 93:237-248A. 8. Dooley, K. E., E. A. Obuku, N. Durakovic, V. Belitsky, C. Mitnick, and E. L. Nuermberger. 2013. World Health Organization group 5 drugs for the treatment of drug-resistant tuberculosis: unclear efficacy or untapped potential? The Journal of infectious diseases 207:1352-1358. 9. Sebastien Gagneux., Clara Davis Long, Peter M. Small, Tran Van, Gary K. Schoolnik, and B. J. M. Bohannan. The Competitive Cost of Antibiotic Resistance in Mycobacterium tuberculosis. Science 312. 10. Harding, C. V., and W. H. Boom. 2010. Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors. Nat. Rev. Microbiol. 8:296-307. 11. Warren, A. E., K. Wyss, G. Shakarishvili, R. Atun, and D. de Savigny. 2013. Global health initiative investments and health systems strengthening: a content analysis of global fund investments. Globalization and health 9:30. 12. Hayward, D., P. D. van Helden, and I. J. Wiid. 2009. Glutamine synthetase sequence evolution in the mycobacteria and their use as molecular markers for Actinobacteria speciation. BMC evolutionary biology 9:48. 13. Ventura, M., C. Canchaya, A. Tauch, G. Chandra, G. F. Fitzgerald, K. F. Chater, and D. van Sinderen. 2007. Genomics of Actinobacteria: tracing the evolutionary history of an ancient phylum. Microbiology and molecular biology reviews : MMBR 71:495-548. 14. Menendez, M. C., M. J. Garcia, M. C. Navarro, J. A. Gonzalez-y-Merchand, S. Rivera-Gutierrez, L. Garcia-Sanchez, and R. A. Cox. 2002. Characterization of an rRNA Operon (rrnB) of Mycobacterium fortuitum and Other Mycobacterial Species: Implications for the Classification of Mycobacteria. Journal of Bacteriology 184:1078-1088.

125

15. Brown-Elliott, B. A., K. A. Nash, and R. J. Wallace, Jr. 2012. Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clinical microbiology reviews 25:545-582. 16. Jane Atesoh Awuha., Markus Hauga, Jennifer Mildenbergera, Anne Marstada, Chau Phuc Ngoc Do, Claire Loueta, Jørgen Stenvika, Magnus Steigedala, Jan Kristian Damåsa, Øyvind Halaasb, and Trude Helen Floa. 2014. Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages. PNAS. 17. Tortoli, E. 2014. Microbiological features and clinical relevance of new species of the genus Mycobacterium. Clinical microbiology reviews 27:727-752. 18. Swaim, L. E., L. E. Connolly, H. E. Volkman, O. Humbert, D. E. Born, and L. Ramakrishnan. 2006. Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity. Infection and immunity 74:6108-6117. 19. Cambau, E., and M. Drancourt. 2014. Steps towards the discovery of Mycobacterium tuberculosis by Robert Koch, 1882. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 20:196-201. 20. Torrelles, J. B., and L. S. Schlesinger. 2010. Diversity in Mycobacterium tuberculosis mannosylated cell wall determinants impacts adaptation to the host. Tuberculosis 90:84-93. 21. Harding, C. V., and W. H. Boom. 2010. Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors. Nat Rev Microbiol 8:296-307. 22. Russell, D. G., P. J. Cardona, M. J. Kim, S. Allain, and F. Altare. 2009. Foamy macrophages and the progression of the human tuberculosis granuloma. Nat Immunol 10:943-948. 23. Torrelles, J. B., and L. S. Schlesinger. 2010. Diversity in Mycobacterium tuberculosis mannosylated cell wall determinants impacts adaptation to the host. Tuberculosis (Edinb) 90:84-93. 24. Flynn, J. L., and J. Chan. 2001. Immunology of tuberculosis. Annu Rev Immunol 19:93-129. 25. Frieden, T. R., T. R. Sterling, S. S. Munsiff, C. J. Watt, and C. Dye. 2003. Tuberculosis. Lancet 362:887-899. 26. Kaufmann, S. H. 2006. Tuberculosis: back on the immunologists' agenda. Immunity 24:351-357. 27. Russell, D. G. 2006. Who puts the tubercle in tuberculosis? Nature Reviews Microbiology 5:39-47. 28. Canaday, D. H., R. J. Wilkinson, Q. Li, C. V. Harding, R. F. Silver, and W. H. Boom. 2001. CD4(+) and CD8(+) T cells kill intracellular Mycobacterium tuberculosis by a perforin and Fas/Fas ligand-independent mechanism. J Immunol 167:2734-2742. 29. Kwan, C. K., and J. D. Ernst. 2011. HIV and tuberculosis: a deadly human syndemic. Clinical microbiology reviews 24:351-376. 30. Frieden, T. R., T. R. Sterling, S. S. Munsiff, C. J. Watt, and C. Dye. 2003. Tuberculosis. The Lancet 362:887-899.

126

31. Knechel, N. A. 2009. Tuberculosis: pathophysiology, clinical features, and diagnosis. Critical care nurse 29:34-43; quiz 44. 32. Burgers, W. A., C. Riou, M. Mlotshwa, P. Maenetje, D. de Assis Rosa, J. Brenchley, K. Mlisana, D. C. Douek, R. Koup, M. Roederer, G. de Bruyn, S. A. Karim, C. Williamson, and C. M. Gray. 2009. Association of HIV-specific and total CD8+ T memory phenotypes in subtype C HIV-1 infection with viral set point. Journal of immunology 182:4751-4761. 33. Lancioni, C., M. Nyendak, S. Kiguli, S. Zalwango, T. Mori, H. Mayanja-Kizza, S. Balyejusa, M. Null, J. Baseke, D. Mulindwa, L. Byrd, G. Swarbrick, C. Scott, D. F. Johnson, L. Malone, P. Mudido-Musoke, W. H. Boom, D. M. Lewinsohn, and D. A. Lewinsohn. 2012. CD8+ T cells provide an immunologic signature of tuberculosis in young children. American journal of respiratory and critical care medicine 185:206-212. 34. Lancioni, C. L., Q. Li, J. J. Thomas, X. Ding, B. Thiel, M. G. Drage, N. D. Pecora, A. G. Ziady, S. Shank, C. V. Harding, W. H. Boom, and R. E. Rojas. 2011. Mycobacterium tuberculosis lipoproteins directly regulate human memory CD4(+) T cell activation via Toll-like receptors 1 and 2. Infect. Immun. 79:663- 673. 35. Gehring, A. J., K. M. Dobos, J. T. Belisle, C. V. Harding, and W. H. Boom. 2004. Mycobacterium tuberculosis LprG (Rv1411c): A Novel TLR-2 Ligand That Inhibits Human Macrophage Class II MHC Antigen Processing. J. Immunol. 173:2660-2668. 36. Noss, E. H., R. K. Pai, T. J. Sellati, J. D. Radolf, J. Belisle, D. T. Golenbock, W. H. Boom, and C. V. Harding. 2001. Toll-Like Receptor 2-Dependent Inhibition of Macrophage Class II MHC Expression and Antigen Processing by 19-kDa Lipoprotein of Mycobacterium tuberculosis. J. Immunol. 167:910-918. 37. Athman, J. J., Y. Wang, D. J. McDonald, W. H. Boom, C. V. Harding, and P. A. Wearsch. 2015. Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages. J. immunol. 195:1044-1053. 38. Prados-Rosales, R., A. Baena, L. R. Martinez, J. Luque-Garcia, R. Kalscheuer, U. Veeraraghavan, C. Camara, J. D. Nosanchuk, G. S. Besra, B. Chen, J. Jimenez, A. Glatman-Freedman, W. R. Jacobs, Jr., S. A. Porcelli, and A. Casadevall. 2011. Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice. The Journal of clinical investigation 121:1471-1483. 39. Cao, B., and S. J. Williams. 2010. Chemical approaches for the study of the mycobacterial glycolipids phosphatidylinositol mannosides, lipomannan and lipoarabinomannan. Natural Product Reports 27:919. 40. Rajni, N. Rao, and L. S. Meena. 2011. Biosynthesis and Virulent Behavior of Lipids Produced by Mycobacterium tuberculosis: LAM and Cord Factor: An Overview. Biotechnology Research International 2011:1-7. 41. Briken, V., S. A. Porcelli, G. S. Besra, and L. Kremer. 2004. Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response. Molecular Microbiology 53:391-403.

127

42. Rojas, R. E., J. J. Thomas, A. J. Gehring, P. J. Hill, J. T. Belisle, C. V. Harding, and W. H. Boom. 2006. Phosphatidylinositol mannoside from Mycobacterium tuberculosis binds alpha5beta1 integrin (VLA-5) on CD4+ T cells and induces adhesion to fibronectin. J Immunol 177:2959-2968. 43. Kaur, D., M. E. Guerin, H. Škovierová, P. J. Brennan, and M. Jackson. 2009. Chapter 2 Biogenesis of the Cell Wall and Other Glycoconjugates of Mycobacterium tuberculosis. 69:23-78. 44. Kallenius, G., M. Correia-Neves, H. Buteme, B. Hamasur, and S. B. Svenson. 2016. Lipoarabinomannan, and its related glycolipids, induce divergent and opposing immune responses to Mycobacterium tuberculosis depending on structural diversity and experimental variations. Tuberculosis (Edinb) 96:120-130. 45. Deatherage, B. L., J. C. Lara, T. Bergsbaken, S. L. Rassoulian Barrett, S. Lara, and B. T. Cookson. 2009. Biogenesis of bacterial membrane vesicles. Mol Microbiol 72:1395-1407. 46. Kuehn, M. J., and N. C. Kesty. 2005. Bacterial outer membrane vesicles and the host-pathogen interaction. Genes Dev 19:2645-2655. 47. Beveridge, T. J. 1999. Structures of gram-negative cell walls and their derived membrane vesicles. J Bacteriol 181:4725-4733. 48. Marsollier, L., P. Brodin, M. Jackson, J. Kordulakova, P. Tafelmeyer, E. Carbonnelle, J. Aubry, G. Milon, P. Legras, J. P. Andre, C. Leroy, J. Cottin, M. L. Guillou, G. Reysset, and S. T. Cole. 2007. Impact of Mycobacterium ulcerans biofilm on transmissibility to ecological niches and Buruli ulcer pathogenesis. PLoS Pathog 3:e62. 49. Bhatnagar, S., K. Shinagawa, F. J. Castellino, and J. S. Schorey. 2007. Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo. Blood 110:3234-3244. 50. Prados-Rosales, R., A. Baena, L. R. Martinez, J. Luque-Garcia, R. Kalscheuer, U. Veeraraghavan, C. Camara, J. D. Nosanchuk, G. S. Besra, B. Chen, J. Jimenez, A. Glatman-Freedman, W. R. Jacobs, S. A. Porcelli, and A. Casadevall. 2011. Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice. Journal of Clinical Investigation 121:1471- 1483. 51. Strohmeier, G. R., and M. J. Fenton. 1999. Roles of lipoarabinomannan in the pathogenesis of tuberculosis. Microbes Infect 1:709-717. 52. Schlesinger, L. S., S. R. Hull, and T. M. Kaufman. 1994. Binding of the terminal mannosyl units of lipoarabinomannan from a virulent strain of Mycobacterium tuberculosis to human macrophages. J Immunol 152:4070-4079. 53. Torrelles, J. B., R. Knaup, A. Kolareth, T. Slepushkina, T. M. Kaufman, P. Kang, P. J. Hill, P. J. Brennan, D. Chatterjee, J. T. Belisle, J. M. Musser, and L. S. Schlesinger. 2008. Identification of Mycobacterium tuberculosis clinical isolates with altered phagocytosis by human macrophages due to a truncated lipoarabinomannan. J Biol Chem 283:31417-31428. 54. Vercellone, A., J. Nigou, and G. Puzo. 1998. Relationships between the structure and the roles of lipoarabinomannans and related glycoconjugates in tuberculosis pathogenesis. Front Biosci 3:e149-163.

128

55. Birch, H. L., L. J. Alderwick, B. J. Appelmelk, J. Maaskant, A. Bhatt, A. Singh, J. Nigou, L. Eggeling, J. Geurtsen, and G. S. Besra. 2010. A truncated lipoglycan from mycobacteria with altered immunological properties. Proceedings of the National Academy of Sciences 107:2634-2639. 56. Shabaana, A. K., K. Kulangara, I. Semac, Y. Parel, S. Ilangumaran, K. Dharmalingam, C. Chizzolini, and D. C. Hoessli. 2005. Mycobacterial lipoarabinomannans modulate cytokine production in human T helper cells by interfering with raft/microdomain signalling. Cell. Mol. Life. Sci. 62:179-187. 57. Subburaj, I., S. Ami, M. Poincelet, T. Jean-Marc, P. J. Brennan, Nasir-ud-Din, and D. C. Hoessli. 1995. Integration of Mycobacterial Lipoarabinomannans into Glycosylphosphatidylinositol-Rich Domains of Lymphomonocytic Cell Plasma Membranes. J. Immunol. 155: 1334-1342. 58. Kathryn Haskins, John Kappler, and P. Marrack. 1984. The Major Histocompatibility Complex-Restricted Antigen Receptor on T cells. Ann. ReI'. Immunol 2:51-66. 59. Smith-Garvin, J. E., G. A. Koretzky, and M. S. Jordan. 2009. T cell activation. Annu Rev Immunol 27:591-619. 60. Houtman, J. C., R. A. Houghtling, M. Barda-Saad, Y. Toda, and L. E. Samelson. 2005. Early phosphorylation kinetics of proteins involved in proximal TCR- mediated signaling pathways. J Immunol 175:2449-2458. 61. Mustelin, T., and K. Tasken. 2003. Positive and negative regulation of T-cell activation through kinases and phosphatases. Biochem J 371:15-27. 62. Razzaq, T. M., P. Ozegbe, E. C. Jury, P. Sembi, N. M. Blackwell, and P. S. Kabouridis. 2004. Regulation of T-cell receptor signalling by membrane microdomains. Immunology 113:413-426. 63. Iwashima, M. 2003. Kinetic perspectives of T cell antigen receptor signaling. A two-tier model for T cell full activation. Immunol Rev 191:196-210. 64. Qian, D., and A. Weiss. 1997. T cell antigen receptor signal transduction. Curr Opin Cell Biol 9:205-212. 65. van Leeuwen, J. E., and L. E. Samelson. 1999. T cell antigen-receptor signal transduction. Curr Opin Immunol 11:242-248. 66. Wange, R. L., and L. E. Samelson. 1996. Complex complexes: signaling at the TCR. Immunity 5:197-205. 67. Chan, A. C., M. Dalton, R. Johnson, G. H. Kong, T. Wang, R. Thoma, and T. Kurosaki. 1995. Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function. EMBO J 14:2499-2508. 68. Di Bartolo, V., D. Mege, V. Germain, M. Pelosi, E. Dufour, F. Michel, G. Magistrelli, A. Isacchi, and O. Acuto. 1999. Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling. J Biol Chem 274:6285-6294. 69. Wange, R. L., R. Guitian, N. Isakov, J. D. Watts, R. Aebersold, and L. E. Samelson. 1995. Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70. J Biol Chem 270:18730-18733. 70. Lewis, R. S. 2001. Calcium signaling mechanisms in T lymphocytes. Annu Rev Immunol 19:497-521.

129

71. Rothenberg, E. V., and S. B. Ward. 1996. A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation. Proc Natl Acad Sci U S A 93:9358-9365. 72. Acuto, O., S. Mise-Omata, G. Mangino, and F. Michel. 2003. Molecular modifiers of T cell antigen receptor triggering threshold: the mechanism of CD28 costimulatory receptor. Immunol Rev 192:21-31. 73. McAdam, A. J., A. N. Schweitzer, and A. H. Sharpe. 1998. The role of B7 costimulation in activation and differentiation of CD4+ and CD8+ T cells. Immunol Rev 165:231-247. 74. Sharpe, A. H., and G. J. Freeman. 2002. The B7-CD28 superfamily. Nat Rev Immunol 2:116-126. 75. Brown, D. A., and E. London. 1998. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 14:111-136. 76. Simons, K., and D. Toomre. 2000. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31-39. 77. Janes, P. W., S. C. Ley, and A. I. Magee. 1999. Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J Cell Biol 147:447-461. 78. Williams, B. L., K. L. Schreiber, W. Zhang, R. L. Wange, L. E. Samelson, P. J. Leibson, and R. T. Abraham. 1998. Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70- deficient Jurkat T-cell line. Mol Cell Biol 18:1388-1399. 79. Alonso, A., N. Bottini, S. Bruckner, S. Rahmouni, S. Williams, S. P. Schoenberger, and T. Mustelin. 2004. Lck dephosphorylation at Tyr-394 and inhibition of T cell antigen receptor signaling by Yersinia phosphatase YopH. J Biol Chem 279:4922-4928. 80. Boncristiano, M., S. R. Paccani, S. Barone, C. Ulivieri, L. Patrussi, D. Ilver, A. Amedei, M. M. D'Elios, J. L. Telford, and C. T. Baldari. 2003. The Helicobacter pylori vacuolating toxin inhibits T cell activation by two independent mechanisms. J Exp Med 198:1887-1897. 81. Mahon, R. N., R. E. Rojas, S. A. Fulton, J. L. Franko, C. V. Harding, and W. H. Boom. 2009. Mycobacterium tuberculosis Cell Wall Glycolipids Directly Inhibit CD4+ T-Cell Activation by Interfering with Proximal T-Cell-Receptor Signaling. Infection and Immunity 77:4574-4583. 82. Nakata, K., Y. Suzuki, T. Inoue, C. Ra, H. Yakura, and K. Mizuno. 2011. Deficiency of SHP1 leads to sustained and increased ERK activation in mast cells, thereby inhibiting IL-3-dependent proliferation and cell death. Mol Immunol 48:472-480. 83. Stefanova, I., B. Hemmer, M. Vergelli, R. Martin, W. E. Biddison, and R. N. Germain. 2003. TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways. Nat Immunol 4:248-254. 84. Germain R. 2003. Ligand-Dependent Regulation of T Cell Development and Activation. Immunologic Research:277-286. 85. Stefanova, I., B. Hemmer, M. Vergelli, R. Martin, W. E. Biddison, and R. N. Germain. 2003. TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways. Nat Immunol 4:248-254.

130

86. Macian, F., F. Garcıa-Cozar, I. Sin-Hyeog, H. F. Horton, M. C. Byrne, and A. Rao. 2002. Transcriptional Mechanisms Underlying Lymphocyte Tolerance. Cell 109:719-731. 87. Wells, A. D., M. C. Walsh, D. Sankaran, and L. A. Turka. 2000. T Cell Effector Function and Anergy Avoidance Are Quantitatively Linked to Cell Division1. J. Immunol. 165:2432–2443. 88. Jiang, H., and L. Chess. 2009. Chapter 2 How the Immune System Achieves Self– Nonself Discrimination During Adaptive Immunity. 102:95-133. 89. Schwartz, R. H. 2003. T cell anergy. Annu. Rev. Immunol. 21:305-334. 90. Kriegel, M. A., C. Rathinam, and R. A. Flavell. 2009. E3 ubiquitin ligase GRAIL controls primary T cell activation and oral tolerance. Proc. Natl. Acad. Sci. USA 106:16770-16775. 91. Miller, S. D., D. M. Turley, and J. R. Podojil. 2007. Antigen-specific tolerance strategies for the prevention and treatment of autoimmune disease. Nat Rev Immunol 7:665-677. 92. Nurieva, R. I., S. Zheng, W. Jin, Y. Chung, Y. Zhang, G. J. Martinez, J. M. Reynolds, S. L. Wang, X. Lin, S. C. Sun, G. Lozano, and C. Dong. 2010. The E3 ubiquitin ligase GRAIL regulates T cell tolerance and regulatory T cell function by mediating T cell receptor-CD3 degradation. Immunity 32:670-680. 93. Hundt, M., H. Tabata, M. S. Jeon, K. Hayashi, Y. Tanaka, R. Krishna, L. De Giorgio, Y. C. Liu, M. Fukata, and A. Altman. 2006. Impaired activation and localization of LAT in anergic T cells as a consequence of a selective palmitoylation defect. Immunity 24:513-522. 94. Lin, J. T., N. B. Lineberry, M. G. Kattah, L. L. Su, P. J. Utz, C. G. Fathman, and L. Wu. 2009. Naive CD4 t cell proliferation is controlled by mammalian target of rapamycin regulation of GRAIL expression. J. Immunol. 182:5919-5928. 95. Baksh, S., and S. J. Burakoff. 2000. The role of calcineurin in lymphocyte activation. Semin Immunol 12:405-415. 96. Bergman, M., T. Mustelin, C. Oetken, J. Partanen, N. A. Flint, K. E. Amrein, M. Autero, P. Burn, and K. Alitalo. 1992. The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity. EMBO J 11:2919-2924. 97. Chen, D., and E. V. Rothenberg. 1994. Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles. J Exp Med 179:931-942. 98. Gaffen, S. L., and K. D. Liu. 2004. Overview of interleukin-2 function, production and clinical applications. Cytokine 28:109-123. 99. Hollander, G. A. 1999. On the stochastic regulation of interleukin-2 transcription. Semin Immunol 11:357-367. 100. Mary, D., C. Aussel, B. Ferrua, and M. Fehlmann. 1987. Regulation of interleukin 2 synthesis by cAMP in human T cells. J Immunol 139:1179-1184. 101. Saito, T., and S. Yamasaki. 2003. Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors. Immunol Rev 192:143-160. 102. Yamasaki, S., K. Nishida, M. Hibi, M. Sakuma, R. Shiina, A. Takeuchi, H. Ohnishi, T. Hirano, and T. Saito. 2001. Docking protein Gab2 is phosphorylated

131

by ZAP-70 and negatively regulates T cell receptor signaling by recruitment of inhibitory molecules. J Biol Chem 276:45175-45183. 103. Zhu, M., O. Granillo, R. Wen, K. Yang, X. Dai, D. Wang, and W. Zhang. 2005. Negative regulation of lymphocyte activation by the adaptor protein LAX. J Immunol 174:5612-5619. 104. Niroshana Anandasabapathy., Gregory S, Debra Bloom, Claire Holness, Violette Paragas, Christine Seroogy, Heidi Skrenta, Marie Hollenhorst, and C. Garrison Fathman, * and Luis Soares1,2. 2003. GRAIL: An E3 Ubiquitin Ligase that Inhibits Cytokine Gene Transcription Is Expressed in Anergic CD4 T Cells. Immunity 18:535–547. 105. Whiting, C. C., L. L. Su, J. T. Lin, and C. G. Fathman. 2011. GRAIL: a unique mediator of CD4 T-lymphocyte unresponsiveness. The FEBS J. 278:47-58. 106. Nakamichi, S., Y. Senga, H. Inoue, A. Emi, Y. Matsuki, E. Watanabe, R. Hiramatsu, W. Ogawa, and M. Kasuga. 2009. Role of the E3 ubiquitin ligase gene related to anergy in lymphocytes in glucose and lipid metabolism in the liver. Journal of molecular endocrinology 42:161-169. 107. Jin, X., H. Cheng, J. Chen, and D. Zhu. 2011. RNF13: an emerging RING finger ubiquitin ligase important in cell proliferation. The FEBS journal 278:78-84. 108. Mukai, A., H. Iijima, S. Hiyama, H. Fujii, S. Shinzaki, T. Inoue, E. Shiraishi, S. Kawai, M. Araki, Y. Hayashi, J. Kondo, T. Mizushima, T. Kanto, S. Egawa, T. Nishida, M. Tsujii, and T. Takehara. 2014. Regulation of anergy-related ubiquitin E3 ligase, GRAIL, in murine models of colitis and patients with Crohn's disease. Journal of gastroenterology 49:1524-1535. 109. Brockdorff, J., S. Williams, C. Couture, and T. Mustelin. 1999. Dephosphorylation of ZAP-70 and inhibition of T cell activation by activated SHP1. Eur J Immunol 29:2539-2550. 110. Croker, B. A., R. S. Lewis, J. J. Babon, J. D. Mintern, D. E. Jenne, D. Metcalf, J. G. Zhang, L. H. Cengia, J. A. O'Donnell, and A. W. Roberts. 2011. Neutrophils require SHP1 to regulate IL-1beta production and prevent inflammatory skin disease. J Immunol 186:1131-1139. 111. Fowler, C. C., L. I. Pao, J. N. Blattman, and P. D. Greenberg. 2010. SHP-1 in T cells limits the production of CD8 effector cells without impacting the formation of long-lived central memory cells. J Immunol 185:3256-3267. 112. Iype, T., M. Sankarshanan, I. S. Mauldin, D. W. Mullins, and U. Lorenz. 2010. The protein tyrosine phosphatase SHP-1 modulates the suppressive activity of regulatory T cells. J Immunol 185:6115-6127. 113. Kim, D. J., M. L. Tremblay, and J. Digiovanni. 2010. Protein tyrosine phosphatases, TC-PTP, SHP1, and SHP2, cooperate in rapid dephosphorylation of Stat3 in keratinocytes following UVB irradiation. PLoS One 5:e10290. 114. Koike, T., H. Yamagishi, Y. Hatanaka, A. Fukushima, J. W. Chang, Y. Xia, M. Fields, P. Chandler, and M. Iwashima. 2003. A novel ERK-dependent signaling process that regulates interleukin-2 expression in a late phase of T cell activation. J Biol Chem 278:15685-15692. 115. Lorenz, U. 2009. SHP-1 and SHP-2 in T cells: two phosphatases functioning at many levels. Immunol Rev 228:342-359.

132

116. Mustelin, T., J. Brockdorff, L. Rudbeck, A. Gjorloff-Wingren, S. Han, X. Wang, P. Tailor, and M. Saxena. 1999. The next wave: protein tyrosine phosphatases enter T cell antigen receptor signalling. Cell Signal 11:637-650. 117. Mustelin, T., G. S. Feng, N. Bottini, A. Alonso, N. Kholod, D. Birle, J. Merlo, and H. Huynh. 2002. Protein tyrosine phosphatases. Front Biosci 7:d85-142. 118. Paling, N. R., and M. J. Welham. 2002. Role of the protein tyrosine phosphatase SHP-1 (Src homology phosphatase-1) in the regulation of interleukin-3-induced survival, proliferation and signalling. Biochem J 368:885-894. 119. Poole, A. W., and M. L. Jones. 2005. A SHPing tale: perspectives on the regulation of SHP-1 and SHP-2 tyrosine phosphatases by the C-terminal tail. Cell Signal 17:1323-1332. 120. Ramachandran, I. R., W. Song, N. Lapteva, M. Seethammagari, K. M. Slawin, D. M. Spencer, and J. M. Levitt. 2011. The phosphatase SRC homology region 2 domain-containing phosphatase-1 is an intrinsic central regulator of dendritic cell function. J Immunol 186:3934-3945. 121. Ren, L., X. Chen, R. Luechapanichkul, N. G. Selner, T. M. Meyer, A. S. Wavreille, R. Chan, C. Iorio, X. Zhou, B. G. Neel, and D. Pei. 2011. Substrate specificity of protein tyrosine phosphatases 1B, RPTPalpha, SHP-1, and SHP-2. Biochemistry 50:2339-2356. 122. Zhang, J., A. K. Somani, and K. A. Siminovitch. 2000. Roles of the SHP-1 tyrosine phosphatase in the negative regulation of cell signalling. Semin Immunol 12:361-378. 123. Zhang, J., A. K. Somani, D. Yuen, Y. Yang, P. E. Love, and K. A. Siminovitch. 1999. Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell selection. J Immunol 163:3012-3021. 124. Sahoo, A., A. Alekseev, L. Obertas, and R. Nurieva. 2014. Grail controls Th2 cell development by targeting STAT6 for degradation. Nature communications 5:4732. 125. Fraser, J. H., M. Rincon, K. D. McCoy, and G. Le Gros. 1999. CTLA4 ligation attenuates AP-1, NFAT and NF-kappaB activity in activated T cells. Eur J Immunol 29:838-844. 126. Calvo, C. R., D. Amsen, and A. M. Kruisbeek. 1997. Cytotoxic T lymphocyte antigen 4 (CTLA-4) interferes with extracellular signal-regulated kinase (ERK) and Jun NH2-terminal kinase (JNK) activation, but does not affect phosphorylation of T cell receptor zeta and ZAP70. J Exp Med 186:1645-1653. 127. Wherry, E. J., and M. Kurachi. 2015. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 15:486-499. 128. Lee, S. H., X. W. Meng, K. S. Flatten, D. A. Loegering, and S. H. Kaufmann. 2013. Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm. Cell Death Differ 20:64- 76. 129. F Mor, and I. R. Cohen. 1996. IL-2 rescues antigen-specific T cells from radiation or dexamethasone-induced apoptosis. Correlation with induction of Bcl-2. J Immunol 156:515-522.

133

130. Behar, S. M., C. J. Martin, M. G. Booty, T. Nishimura, X. Zhao, H. X. Gan, M. Divangahi, and H. G. Remold. 2011. Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis. Mucosal Immunol 4:279-287. 131. Dominguez-Villar, M., A. S. Gautron, M. de Marcken, M. J. Keller, and D. A. Hafler. 2015. TLR7 induces anergy in human CD4(+) T cells. Nat. Immunol. 16:118-128. 132. Prendergast, C. T., D. E. Sanin, P. C. Cook, and A. P. Mountford. 2015. CD4+ T cell hyporesponsiveness after repeated exposure to Schistosoma mansoni larvae is dependent upon interleukin-10. Infection and Immunity 83:1418-1430. 133. Smith, P., C. M. Walsh, N. E. Mangan, R. E. Fallon, J. R. Sayers, A. N. J. McKenzie, and P. G. Fallon. 2004. Schistosoma mansoni Worms Induce Anergy of T Cells via Selective Up-Regulation of Programmed Death Ligand 1 on Macrophages. J. Immunol. 173:1240-1248. 134. Wang, X., P. F. Barnes, K. M. Dobos-Elder, J. C. Townsend, Y. T. Chung, H. Shams, S. E. Weis, and B. Samten. 2009. ESAT-6 inhibits production of IFN- gamma by Mycobacterium tuberculosis-responsive human T cells. Journal of immunology 182:3668-3677. 135. Mahon, R. N., R. E. Rojas, S. A. Fulton, J. L. Franko, C. V. Harding, and W. H. Boom. 2009. Mycobacterium tuberculosis cell wall glycolipids directly inhibit CD4+ T-cell activation by interfering with proximal T-cell-receptor signaling. Infect. Immun. 77:4574-4583. 136. C.Moreno, Angela Mehlert, and J.Lamb. 1988. The inhibitory effects of mycobacterial lipoarabinomannan andpolysaccharidesupon polyclonalandmonoclonal human T cellproliferation. Clin.exp.Immunol. 74:206- 210. 137. Briken, V., S. A. Porcelli, G. S. Besra, and L. Kremer. 2004. Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response. Mol. Microbiol. 53:391-403. 138. Welin, A., M. E. Winberg, H. Abdalla, E. Sarndahl, B. Rasmusson, O. Stendahl, and M. Lerm. 2008. Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block. Infect. Immun. 76:2882-2887. 139. Miguel A. Alonso, and J. Millán. 2001. The role of lipid rafts in signalling and membrane trafficking in T lymphocytes. Journal of Cell Science 114:3957-3965. 140. Pizzo, P., and A. Viola. 2003. Lymphocyte lipid rafts: structure and function. Current opinion in immunology 15:255-260. 141. Manes, S., G. del Real, and A. C. Martinez. 2003. Pathogens: raft hijackers. Nat Rev Immunol 3:557-568. 142. Winberg, M. E., A. Holm, E. Sarndahl, A. F. Vinet, A. Descoteaux, K. E. Magnusson, B. Rasmusson, and M. Lerm. 2009. Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts. Microbes Infect 11:215-222. 143. Das, L., and A. D. Levine. 2008. TGF- Inhibits IL-2 Production and Promotes Cell Cycle Arrest in TCR-Activated Effector/Memory T Cells in the Presence of Sustained TCR Signal Transduction. The Journal of Immunology 180:1490-1498.

134

144. Gehring, A. J., R. E. Rojas, D. H. Canaday, D. L. Lakey, C. V. Harding, and W. H. Boom. 2003. The Mycobacterium tuberculosis 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Regulated HLA-DR and Fc R1 on Human Macrophages through Toll-Like Receptor 2. Infection and Immunity 71:4487- 4497. 145. Schade, A. E., and A. D. Levine. 2002. Lipid Raft Heterogeneity in Human Peripheral Blood T Lymphoblasts: A Mechanism for Regulating the Initiation of TCR Signal Transduction. The Journal of Immunology 168:2233-2239. 146. Mahon, R. N., O. J. Sande, R. E. Rojas, A. D. Levine, C. V. Harding, and W. H. Boom. 2012. Mycobacterium tuberculosis ManLAM inhibits T-cell-receptor signaling by interference with ZAP-70, Lck and LAT phosphorylation. Cell. Immunol. 275:98-105. 147. Byron B. Au-Yeung, Sebastian Deindl, Lih-Yun Hsu, Emil H. Palacios, S. E. Levin, John Kuriyan, and Arthur Weiss. 2009. The structure, regulation, and function of ZAP-70. Immunological Reviews 228:41-57. 148. Subburaj Ilangumaran, Stephan Arni, Gerhild van Echten-Deckert, Bettina Borisch, and Daniel C. Hoessli. 1999. Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes. Molecular Biology of the Cell 10:891-905. 149. By Torkel Vang, Knut Martin Torgersen, Vibeke Sundvold, Manju Saxena, Finn Olav Levy, Bjørn S. Skålhegg, Vidar Hansson, Tomas Mustelin, and Kjetil Taskén. 2001. Activation of the COOH-terminal Src kinase (Csk) by cAMP- dependent Protein Kinase Inhibits Signaling through the T Cell Receptor. J. Exp. Med 193:497-507. 150. Samelson, L. E. 2002. Signal transduction mediated by the T cell antigen receptor: the role of adapter proteins. Annu Rev Immunol 20:371-394. 151. Tomas Mustelin, and Kjetil Taskey. 2003. Positive and negative regulation of T- cell activation through kinases and phosphatases. Biochem. J 131:15-27. 152. Vang, T., H. Abrahamsen, S. Myklebust, V. Horejsi, and K. Tasken. 2003. Combined spatial and enzymatic regulation of Csk by cAMP and protein kinase a inhibits T cell receptor signaling. J Biol Chem 278:17597-17600. 153. Daniel C. Hoessli, and E. Rungger-Brandle. 1983. Isolation of plasma membrane domains from murine T lymphocytes. Proc. NatL Acad. Sci. USA 80:439-443. 154. Ramnik Xavier, Todd Brennan, Qingqin Li, Christine McCormack, and Brian Seed. Membrane Compartmentation Is Required for Efficient T Cell Activation. Immunity 8:723-732. 155. Kai Simons , and Elina Ikonen. Functional rafts in cell membranes. Nature 387:569-572. 156. Kenworthy, A. K. 2008. Have we become overly reliant on lipid rafts? Talking Point on the involvement of lipid rafts in T-cell activation. EMBO Rep 9:531-535. 157. Shaw, A. S. 2006. Lipid rafts: now you see them, now you don't. Nat Immunol 7:1139-1142. 158. Campi, G., R. Varma, and M. L. Dustin. 2005. Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling. J Exp Med 202:1031-1036.

135

159. Lillemeier, B. F., M. A. Mortelmaier, M. B. Forstner, J. B. Huppa, J. T. Groves, and M. M. Davis. 2010. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat Immunol 11:90-96. 160. Takashi Saito, and Sho Yamasaki. 2003. Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors. Immunological Reviews 192:143-160. 161. Yokosuka, T., K. Sakata-Sogawa, W. Kobayashi, M. Hiroshima, A. Hashimoto- Tane, M. Tokunaga, M. L. Dustin, and T. Saito. 2005. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat Immunol 6:1253-1262. 162. Boulton, I. C., and S. D. Gray-Owen. 2002. Neisserial binding to CEACAM1 arrests the activation and proliferation of CD4+ T lymphocytes. Nat Immunol 3:229-236. 163. Sloan, D. D., J. Y. Han, T. K. Sandifer, M. Stewart, A. J. Hinz, M. Yoon, D. C. Johnson, P. G. Spear, and K. R. Jerome. 2006. Inhibition of TCR Signaling by Herpes Simplex Virus. The Journal of Immunology 176:1825-1833. 164. Tonks, N. K. 2006. Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol 7:833-846. 165. Keith L. Knutson, Zakaria Hmama, Patricia Herrera-Velit, Rosemary Rochford, and N. E. Reiner. 1998. Lipoarabinomannan of Mycobacterium tuberculosis Promotes Protein Tyrosine Dephosphorylation and Inhibition of Mitogenactivated Protein Kinase in Human Mononuclear Phagocytes. J Biol Chem 273:645-652. 166. Iwashima, M. 2003. Kinetic perspectives of T cell antigen receptor signaling: A two-tier model for T cell full activation. Immunological Reviews 191:196-210. 167. Robert T. Abraham, and A. Weiss. 2004. Jurkat T cells and development of the T- cell receptor signalling paradigm. Nat. Rev. Immunolol. 4:301-308. 168. A Descoteaux, S J Turco, D L Sacks, and G. Matlashewski. 1991. Leishmania donovani lipophosphoglycan selectively inhibits signal transduction in macrophages. J Immunol 146:2747-2753. 169. Brodskyn, C., J. Patricio, R. Oliveira, L. Lobo, A. Arnholdt, L. Mendonca- Previato, A. Barral, and M. Barral-Netto. 2002. Glycoinositolphospholipids from Trypanosoma cruzi Interfere with Macrophages and Dendritic Cell Responses. Infection and Immunity 70:3736-3743. 170. Arrighi, R. B., F. Debierre-Grockiego, R. T. Schwarz, and I. Faye. 2009. The immunogenic properties of protozoan glycosylphosphatidylinositols in the mosquito Anopheles gambiae. Dev Comp Immunol 33:216-223. 171. By Michel Desjardins, and A. Descoteaux. 1997. Inhibition of Phagolysosomal Biogenesis by the Leishmania Lipophosphoglycan. J. Exp. Med. 185:2061-2068. 172. Igor C. Almeida, and R. T. Gazzinelli. 2001. Proinflammatory activity of glycosylphosphatidylinositol anchors derived from Trypanosoma cruzi: structural and functional analyses. J. Leuk. Biol. 70:467-477. 173. Chawla, M., and R. A. Vishwakarma. 2003. Alkylacylglycerolipid domain of GPI molecules of Leishmania is responsible for inhibition of PKC-mediated c-fos expression. J Lipid Res 44:594-600. 174. Richard Bernier, Benoit Barbeau, Martin Olivier, and M. J. Tremblay. 1998. Mycobacterium tuberculosis mannose-capped lipoarabinomannan can induce NF-

136

κB-dependent activation of human immunodeficiency virus type 1 long terminal repeat in T cells. J. Gen. Virol. 79:1353-1361. 175. Souvenir D. Tachado, Peter Gerold, Ralph Schwartz, Suzanna Novakovic, Malcolm McConville, and L. Schofield. 1997. Signal transduction in macrophages by glycosylphosphatidylinositols of Plasmodium, Trypanosoma, and Leishmania: Activation of protein tyrosine kinases and protein kinase C by inositolglycan and diacylglycerol moieties. Proc. Natl. Acad. Sci. USA 94:4022- 4027. 176. Commandeur, S., K. E. van Meijgaarden, C. Prins, A. V. Pichugin, K. Dijkman, S. J. van den Eeden, A. H. Friggen, K. L. Franken, G. Dolganov, I. Kramnik, G. K. Schoolnik, F. Oftung, G. E. Korsvold, A. Geluk, and T. H. Ottenhoff. 2013. An unbiased genome-wide Mycobacterium tuberculosis gene expression approach to discover antigens targeted by human T cells expressed during pulmonary infection. J. immunol. 190:1659-1671. 177. Lindestam Arlehamn, C. S., A. Gerasimova, F. Mele, R. Henderson, J. Swann, J. A. Greenbaum, Y. Kim, J. Sidney, E. A. James, R. Taplitz, D. M. McKinney, W. W. Kwok, H. Grey, F. Sallusto, B. Peters, and A. Sette. 2013. Memory T cells in latent Mycobacterium tuberculosis infection are directed against three antigenic islands and largely contained in a CXCR3+CCR6+ Th1 subset. PLoS Pathogens 9:e1003130. 178. Lindestam Arlehamn, C. S., S. Paul, F. Mele, C. Huang, J. A. Greenbaum, R. Vita, J. Sidney, B. Peters, F. Sallusto, and A. Sette. 2015. Immunological consequences of intragenus conservation of Mycobacterium tuberculosis T-cell epitopes. Proc. Natl. Acad. Sci. USA 112:E147-155. 179. Sutherland, J. S., M. K. Lalor, G. F. Black, L. R. Ambrose, A. G. Loxton, N. N. Chegou, and D. Kassa. 2013. Analysis of Host Responses to Mycobacterium tuberculosis Antigens in a Multi-Site Study of Subjects with Different TB and HIV Infection States in Sub-Saharan Africa. PLOS ONE 8:e74080. 180. Comas, I., J. Chakravartti, P. M. Small, J. Galagan, S. Niemann, K. Kremer, J. D. Ernst, and S. Gagneux. 2010. Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved. Nat. Genet. 42:498-503. 181. Bhatt, K., and P. Salgame. 2007. Host innate immune response to Mycobacterium tuberculosis. J. clin. immunol. 27:347-362. 182. Jacobs, M., D. Togbe, C. Fremond, A. Samarina, N. Allie, T. Botha, D. Carlos, S. K. Parida, S. Grivennikov, S. Nedospasov, A. Monteiro, M. Le Bert, V. Quesniaux, and B. Ryffel. 2007. Tumor necrosis factor is critical to control tuberculosis infection. Microbes. infect. 9:623-628. 183. Pecora, N. D., A. J. Gehring, D. H. Canaday, W. H. Boom, and C. V. Harding. 2006. Mycobacterium tuberculosis LprA Is a Lipoprotein Agonist of TLR2 That Regulates Innate Immunity and APC Function. J. Immunol. 177:422-429. 184. Chappert, P., and R. H. Schwartz. 2010. Induction of T cell anergy: integration of environmental cues and infectious tolerance. Curr. Opin. Immunol. 22:552-559. 185. Chiodetti, L., S. Choi, D. L. Barber, and R. H. Schwartz. 2006. Adaptive Tolerance and Clonal Anergy Are Distinct Biochemical States. J. Immunol. 176:2279-2291.

137

186. Aziz, M., W. L. Yang, S. Matsuo, A. Sharma, M. Zhou, and P. Wang. 2014. Upregulation of GRAIL is associated with impaired CD4 T cell proliferation in sepsis. J. Immunol. 192:2305-2314. 187. Choi, S., and R. H. Schwartz. 2007. Molecular mechanisms for adaptive tolerance and other T cell anergy models. Semin. Immunol. 19:140-152. 188. Ermann, J., V. Szanya, G. S. Ford, V. Paragas, C. G. Fathman, and K. Lejon. 2001. CD4+CD25+ T Cells Facilitate the Induction of T Cell Anergy. J. Immunol. 167:4271-4275. 189. Bostik, P., A. E. Mayne, F. Villinger, K. P. Greenberg, J. D. Powell, and A. A. Ansari. 2001. Relative Resistance in the Development of T Cell Anergy in CD4+ T Cells from Simian Immunodeficiency Virus Disease-Resistant Sooty Mangabeys. J. Immunol. 166:506-516. 190. Nargis, K., U. Gowthaman, S. Pahari, and J. N. Agrewala. 2012. Manipulation of Costimulatory Molecules by Intracellular Pathogens: Veni, Vidi, Vici!! PLoS Pathogens 8:e1002676. 191. Taylor, J. J., C. M. Krawczyk, M. Mohrs, and E. J. Pearce. 2009. Th2 cell hyporesponsiveness during chronic murine schistosomiasis is cell intrinsic and linked to GRAIL expression. J. Clin. Invest. 119:1019-1028. 192. Tamura, T., H. Ariga, T. Kinashi, S. Uehara, T. Kikuchi, M. Nakada, T. Tokunaga, W. Xu, A. Kariyone, T. Saito, T. Kitamura, G. Maxwell, S. Takaki, and K. Takatsu. 2004. The role of antigenic peptide in CD4+ T helper phenotype development in a T cell receptor transgenic model. Int. Immunol. 16:1691-1699. 193. Sande, O. J., A. F. Karim, Q. Li, X. Ding, C. V. Harding, R. E. Rojas, and W. H. Boom. 2016. Mannose-Capped Lipoarabinomannan from Mycobacterium tuberculosis Induces CD4+ T Cell Anergy via GRAIL. Journal of immunology 196:691-702. 194. Wolchinsky, R., M. Hod-Marco, K. Oved, S. S. Shen-Orr, S. C. Bendall, G. P. Nolan, and Y. Reiter. 2014. Antigen-dependent integration of opposing proximal TCR-signaling cascades determines the functional fate of T lymphocytes. J. Immunol. 192:2109-2119. 195. Barron, L., B. Knoechel, J. Lohr, and A. K. Abbas. 2008. Cutting Edge: Contributions of Apoptosis and Anergy to Systemic T Cell Tolerance. J. Immunol. 180:2762-2766. 196. Getts, D. R., D. P. McCarthy, and S. D. Miller. 2013. Exploiting apoptosis for therapeutic tolerance induction. J. Immunol. 191:5341-5346. 197. Mahic, M., K. Henjum, S. Yaqub, B. A. Bjornbeth, K. M. Torgersen, K. Tasken, and E. M. Aandahl. 2008. Generation of highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) regulatory T cells by continuous antigen stimulation. Eur. J. Immunol. 38:640-646. 198. Chemnitz, J. M., R. V. Parry, K. E. Nichols, C. H. June, and J. L. Riley. 2004. SHP-1 and SHP-2 Associate with Immunoreceptor Tyrosine-Based Switch Motif of Programmed Death 1 upon Primary Human T Cell Stimulation, but Only Receptor Ligation Prevents T Cell Activation. J. Immunol. 173:945-954. 199. Chikuma, S., S. Terawaki, T. Hayashi, R. Nabeshima, T. Yoshida, S. Shibayama, T. Okazaki, and T. Honjo. 2009. PD-1-mediated suppression of IL-2 production induces CD8+ T cell anergy in vivo. J. Immunol. 182:6682-6689.

138

200. Keir, M. E., M. J. Butte, G. J. Freeman, and A. H. Sharpe. 2008. PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 26:677-704. 201. Sanchez-Fueyo, A., J. Tian, D. Picarella, C. Domenig, X. X. Zheng, C. A. Sabatos, N. Manlongat, O. Bender, T. Kamradt, V. K. Kuchroo, J. C. Gutierrez- Ramos, A. J. Coyle, and T. B. Strom. 2003. Tim-3 inhibits T helper type 1- mediated auto- and alloimmune responses and promotes immunological tolerance. Nat. Immunol. 4:1093-1101. 202. Tsushima, F., S. Yao, T. Shin, A. Flies, S. Flies, H. Xu, K. Tamada, D. M. Pardoll, and L. Chen. 2007. Interaction between B7-H1 and PD-1 determines initiation and reversal of T-cell anergy. Blood 110:180-185. 203. Lineberry, N. B., L. L. Su, J. T. Lin, G. P. Coffey, C. M. Seroogy, and C. G. Fathman. 2008. Cutting Edge: The Transmembrane E3 Ligase GRAIL Ubiquitinates the Costimulatory Molecule CD40 Ligand during the Induction of T Cell Anergy. J. Immunol. 181:1622-1626. 204. Mustafa, T., S. Phyu, R. Nilsen, R. Jonsson, and G. Bjune. 1999. A Mouse Model for Slowly Progressive Primary Tuberculosis. Scand. J. Immunol. 50:127-136. 205. Glatman-Feedman, A., A. J. Mednick, N. Lendvai, and A. Casadevall. 2000. Clearance and Organ Distribution of Mycobacterium tuberculosis Lipoarabinomannan (LAM) in the Presence and Absence of LAM-Binding Immunoglobulin M. Infect. Immun. 68:335-341. 206. Afonso-Barroso, A., S. O. Clark, A. Williams, G. T. Rosa, C. Nobrega, S. Silva- Gomes, S. Vale-Costa, R. Ummels, N. Stoker, F. Movahedzadeh, P. van der Ley, A. Sloots, M. Cot, B. J. Appelmelk, G. Puzo, J. Nigou, J. Geurtsen, and R. Appelberg. 2013. Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses. Cell. Microbiol. 15:660-674. 207. Vassiliki, A. B., E. Y. Tsai, E. J. Yunis, S. Thim, J. C. Delgado, C. C. Dascher, A. Berezovskaya, D. Rousset, R. Jean-Marc, and A. E. Goldfeld. 2000. IL-10– producing T cells suppress immune responses in anergic tuberculosis patients. J. Clin. Invest. 105:1317-1325. 208. Schorey, J. S., Y. Cheng, P. P. Singh, and V. L. Smith. 2015. Exosomes and other extracellular vesicles in host-pathogen interactions. EMBO rep. 16:24-43. 209. Nitza, A. G., J. O. Poreviato, B. Zaingales, L. Mendonca-Previato, and G. A. DosReis. 1996. Down-Regulation of T Lymphocyte Activation In Vitro and In Vivo Induced by Glycoinositolphospholipids from Trypanosoma cruzi. J. Immunol. 156:628-635. 210. Vang, T., H. Abrahamsen, S. Myklebust, J. Enserink, H. Prydz, T. Mustelin, M. Amarzguioui, and K. Tasken. 2004. Knockdown of C-terminal Src kinase by siRNA-mediated RNA interference augments T cell receptor signaling in mature T cells. European journal of immunology 34:2191-2199. 211. Reba, S. M., Q. Li, S. Onwuzulike, X. Ding, A. F. Karim, Y. Hernandez, S. A. Fulton, C. V. Harding, C. L. Lancioni, N. Nagy, M. E. Rodriguez, P. A. Wearsch, and R. E. Rojas. 2014. TLR2 engagement on CD4(+) T cells enhances effector functions and protective responses to Mycobacterium tuberculosis. European journal of immunology 44:1410-1421.

139

212. Cho, N. H., P. Feng, S. H. Lee, B. S. Lee, X. Liang, H. Chang, and J. U. Jung. 2004. Inhibition of T cell receptor signal transduction by tyrosine kinase- interacting protein of Herpesvirus saimiri. J Exp Med 200:681-687. 213. Lee, H. S., I. C. Boulton, K. Reddin, H. Wong, D. Halliwell, O. Mandelboim, A. R. Gorringe, and S. D. Gray-Owen. 2007. Neisserial outer membrane vesicles bind the coinhibitory receptor carcinoembryonic antigen-related cellular adhesion molecule 1 and suppress CD4+ T lymphocyte function. Infection and immunity 75:4449-4455. 214. Comer, J. E., A. K. Chopra, J. W. Peterson, and R. Konig. 2005. Direct inhibition of T-lymphocyte activation by anthrax toxins in vivo. Infection and immunity 73:8275-8281. 215. Fang, H., R. Cordoba-Rodriguez, C. S. R. Lankford, and D. M. Frucht. 2005. Anthrax Lethal Toxin Blocks MAPK Kinase-Dependent IL-2 Production in CD4+ T Cells. The Journal of Immunology 174:4966-4971. 216. Paccani, S. R., F. Tonello, R. Ghittoni, M. Natale, L. Muraro, M. M. D'Elios, W. J. Tang, C. Montecucco, and C. T. Baldari. 2005. Anthrax toxins suppress T lymphocyte activation by disrupting antigen receptor signaling. J Exp Med 201:325-331. 217. Bettina Gebert., Wolfgang Fischer, R. H. Evelyn Weiss, and Rainer Haas. 2003. Helicobacter pylori Vacuolating Cytotoxin Inhibits T Lymphocyte Activation. Science 301. 218. van der Velden, A. W. M., J. T. Dougherty, and M. N. Starnbach. 2008. Down- Modulation of TCR Expression by Salmonella enterica serovar Typhimurium. The Journal of Immunology 180:5569-5574. 219. Gerke, C., S. Falkow, and Y. H. Chien. 2005. The adaptor molecules LAT and SLP-76 are specifically targeted by Yersinia to inhibit T cell activation. J Exp Med 201:361-371. 220. Garside, P., and A. M. Mowat. 2001. Oral tolerance. Seminars in immunology 13:177-185. 221. Danelishvili, L., J. McGarvey, Y.-j. Li, and L. E. Bermudez. 2003. Mycobacterium tuberculosisinfection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells. Cellular microbiology 5:649-660. 222. Marchetti, M. C., B. Di Marco, G. Cifone, G. Migliorati, and C. Riccardi. 2003. Dexamethasone-induced apoptosis of thymocytes: role of glucocorticoid receptor- associated Src kinase and caspase-8 activation. Blood 101:585-593. 223. Araki, K., B. Youngblood, and R. Ahmed. 2014. Programmed Cell Death 1- Directed Immunotherapy for Enhancing T-Cell Function. Cold Spring Harbor symposia on quantitative biology. 224. Lineberry, N. B., L. L. Su, J. T. Lin, G. P. Coffey, C. M. Seroogy, and C. G. Fathman. 2008. Cutting Edge: The Transmembrane E3 Ligase GRAIL Ubiquitinates the Costimulatory Molecule CD40 Ligand during the Induction of T Cell Anergy. J Immunol 181:1622-1626. 225. Dure, M., and F. Macian. 2009. IL-2 signaling prevents T cell anergy by inhibiting the expression of anergy-inducing genes. Molecular immunology 46:999-1006.

140

226. Klatzmann, D., and A. K. Abbas. 2015. The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases. Nat Rev Immunol 15:283- 294. 227. Barbara J. Johnson., Iris Estrada, Zhu Shen, Stan Ress, Paul Willcox, M. Joseph Colsoton, and G. Kaplan. June 1998. Differential Gene Expression in Response to Adjunctive Recombinant Human Interleukin-2 Immunotherapy in Multidrug- Resistant Tuberculosis Patients. Infection and Immunity:2426–2433. 228. Zhang, Y., J. Liu, Y. Wang, Q. Xian, L. Shao, Z. Yang, and X. Wang. 2012. Immunotherapy using IL-2 and GM-CSF is a potential treatment for multidrug- resistant Mycobacterium tuberculosis. Science China. Life sciences 55:800-806. 229. Jiang, X., and Z. J. Chen. 2012. The role of ubiquitylation in immune defence and pathogen evasion. Nat Rev Immunol 12:35-48. 230. Vadim Makarov., Giulia Manina, Stewart T. Cole, and Giovanna Riccardi. 8 MAY 2009. Benzothiazinones Kill Mycobacterium tuberculosis by Blocking Arabinan Synthesis. Science 324.

141