Type I interferons and T regulatory cells : effects on development, homeostasis and function

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development, homeostasis and function

Amina Metidji

To cite this version:

Amina Metidji. Type I interferons and T regulatory cells : effects on development, homeostasis and function. Immunology. Université Pierre et Marie Curie - Paris VI; National institute of health (Etats-Unis), 2015. English. �NNT : 2015PA066058�. �tel-01147283�

Université Pierre et Marie Curie

National Institute of Health /

National Institute of Allergy and Infectious Diseases

Ecole doctorale de Physiologie et Physiopathologie

Laboratoire of Immunology / Cellular Immunology

Type I Interferons and T Regulatory Cells

Effects on Development, Homeostasis and Function

Par Amina Metidji

Thèse de doctorat d’Immunologie

Dirigée par Dr. Ethan M. Shevach

Présentée et soutenue publiquement le 24 Mars 2015 Devant un jury composé de :

Pr. François Lemoine (PU-PH) Président

Dr. Bruno Lucas (DR2-CNRS, HDR) Rapporteur 1

Dr. Eric Tartour (PU-PH, HDR) Rapporteur 2

Dr. Benoit Salomon (DR2-INSERM-HDR) Examinateur 1

Dr. Lars Rogge (CL-IP, HDR) Examinateur 2

Dr. Ethan M. Shevach Directeur de thèse

DEDICATION

I would like to dedicate this thesis to the people that I love the most in the world: My Mom, My Dad, my dearest brothers, and dearest sisters. I hope that this achievement will

complete the dream that you had for me when you chose to give me this education. Of course, to my best buddy Rocky.

TABLE OF CONTENTS

Type I IFN production ………... 14

Type IFN signaling ………... 17

B. Effects of Type I IFNs on Cells of the Immune System ……….. 19

Effects of type I IFNs on CD4+ T cells ……….. 19

Effects of type I IFN on effector and memory CD8+ T cells ………. 22

C. Effects of Type I IFNs on T Regulatory T Cells (Treg) ……….. 24

D. Type I IFNs and Lymphocytic Choriomeningitis Virus (LCMV) Infection ………... 28

Immune Responses to Acute LCMV infection ………... 29

Immune Responses to Chronic LCMV infection ………... 30

The Role of type I IFN During LCMV infection ………... 32

Role of Tregs in LCMV infection ……….. 33

E. Type I IFNs in Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE) ……… 35

Tregs in MS ………... 37

Experimental Autoimmune Encephalomyelitis (EAE) ……….. 37

Tregs in EAE ………. 38

Role of Type I IFNs in MS/EAE ……… 39

II. MATERIALS AND METHODS ………. 41

III. EXPERIMENTAL RESULTS ………... 46

IV. FIGURES ………... 62

V. DISCUSSION ………. 91

ACKNOWLEDGMENTS

I would like first to start by thanking my mentor, Dr. Ethan M. Shevach, who gave me this amazing opportunity to accomplish my PhD in his laboratory. Dr. Ethan M. Shevach

provided me with vast opportunities, helped me grow as a scientist, and supported me during my thesis. He also helped me to become a better writer, and equipped me with necessary research skills. I am honored to be his student. I thank Dr. Ethan M. Shevach from the bottom of my heart for everything that he has done for me.

I also would like to thank Dr. Isabelle Cremer, who allowed me to registered at the doctoral school and be able to do my training at the National Institute of Health.

I also must express my deep appreciation to all the lab members, past and present that have trained and helped me along the way. I especially need to thank Dr. Sadiye Rieder, Mathiew Sebastian, Dr. Michael Holt, Deborah Glass, Dr. Justin Edwards, and Patricia Korty who have been amazing colleagues and friends and without whom I would never have been able to turn my observations into a compelling scientific work.

A special thanks goes to the members of my thesis committee, Drs. Francois Lemoine, Bruno Lucas, Lars Rogge, Benoit Saloman, and Eric Tartour.

Finally, and most importantly, I thank my family. My mother and my father who always believed in me, and I cannot thank them enough for their support and love. I always worked harder to make them proud. I would also like to thank my brothers and my sisters to always being present for me.

ABREVIATIONS

ARM: Armstrong B

BBB: Blood brain barrier

BDNF: brain-derived neurotrophic factor BM: bone marrow

C

C13: Clone 13 cDCs: myeloid DCs

CNS: central nervous system CTL: cytotoxic T lymphocyte

CTLA-4: cytotoxic T-lymphocyte-associated protein 4 D

DCs: dendritic cells

DNA: deoxyribonucleic acid dsRNA: Double-stranded RNA E

EAE: Experimental Autoimmune Encephalomyelitis F

Foxp3: forkhead box P3 G

GITR: glucocorticoid-induced tumor necrosis factor receptor GP: glycoprotein polyprotein

I

IBD: inflammatory bowl disease IFN: Interferon

IFNAR: IFN receptor IL: interleukin

IRF-9: IFN regulatory factor 9 ISGF3: IFN-stimulated gene factor 3 ISRE: IFN-stimulated response elements J

JAK: Janus protein tyrosine kinases K

KO: knock out L

LAG-3: Lymphocyte-activation gene 3 LCMV: lymphocytic choriomeningitis virus LFA: lymphocyte function-associated antigen LIF: leukemia inhibitory factor

LPS: lipopolysaccharide M

mAb: Monoclonal antibody

Mda-5: melanoma differentiation-associated gene 5 MHC: major histocompatibility complex

MMP-9: matrix metalloproteinase 9 MOG: myelin oligodendrocyte protein MS: multiple sclerosis

Mtv: Mammary tumor virus

MYD88: myeloid differentiation primary-response protein 88 N

NIPC: natural IFN-producing cells NK: natural killer

NOD: nucleotide-binding oligomerization domain NP: nucleoprotein

P

PAMPs: pathogen-associated molecular patterns PD-1: programmed death-1

pDCs: plasmacytoid DCs PLP: proteolipid protein

PRRs: pattern-recognition receptors PTX: pertussis toxin

R

RIG: retinoic acid inducible gene RNA: ribonucleic acid

RRMS: relapsing–remitting multiple sclerosis S

Sag: superantigens

SBEs: STAT3-binding element SP: single positive

STAT: signal transducers and activators of transcription T

TCR: T cell receptor Teff: T effector

TGF-β: transforming growth factor beta Th: T helper cells

TLRs: Toll-like receptor TNF: Tumor necrosis factors Treg: T regulatory cells

TRIF: TIR-domain-containing adaptor protein inducing IFNβ TYK2: tyrosine kinase 2

V

VLA-4: Very Late Antigen-4 W

RESUME EN FRANÇAIS

L’interféron de type I (IFN) est une famille de cytokine avec des propriétés antivirales et immunomodulatrices. Alors que les effets antiviraux de l'IFN sont bien caractérisés, leurs propriétés immunomodulatrices le sont moins. Un certain nombre d'études récentes ont examiné le rôle de l’IFN de type I sur les cellules T régulatrices (Treg) en utilisant différents modèles expérimentaux de l'auto-immunité et de l'inflammation. Ces études ont abouti à des conclusions contradictoires. Nous avons réexaminé en profondeur les effets de l’IFN de type I sur le

développement, l'homéostasie, et la fonction des cellules Treg. Un certain nombre d'outils uniques ont été développés pour répondre à ces questions, y compris le développement de souris chimériques reconstituées avec une mixture de moelle osseuse de souris normale (WT) et de souris sans le récepteur de l’IFNα/β (IFNAR) KO, et des souris femelles hétérozygotes exprimant une délétion d’IFNAR spécifiquement sur les Treg. Dans ces deux modèles, la signalisation d’IFNAR favorise le développement de la lignée Treg dans le thymus et leur survie dans la périphérie. IFNAR KO Treg avait une expression élevée du gène pro-apoptotique Bim et une fréquence plus élevée de cellules positives pour les caspases. IFNAR KO Tregs de souris

chimérique affichent un phénotype plus naïve, accompagné par des niveaux inférieurs de CD25 et de phospho-STAT5. Par conséquent, dans les Tregs, IFNAR peut affecter directement ou

indirectement la phosphorylation de STAT5. Nous avons également généré des souris

chimériques en utilisant le foie fœtal de souris scurfy, les Treg dérivés de IFNAR KO ont été incapables de contrôler l'activation des cellules T effectrices et l'inflammation des tissus.

Nous avons aussi examiné les effets potentiels de l’IFN de type I sur la fonction des Tregs dans l’infection avec le virus chorioméningite lymphocytaire chronique (LCMV). L'infection des

souris WT avec LCMV clone 13 conduits à une augmentation spectaculaire du pourcentage de Treg exprimant le TCR Vβ5. Nous avons démontré que le pourcentage de Vβ5+ Treg était significativement réduit chez les souris IFNAR KO. IFNAR induit l´expansion des Tregs de manière intrinsèque dans ce modèle.

Encéphalomyélite auto-immune expérimentale (EAE) représente le modèle animal le plus largement étudié pour la sclérose en plaques (SP). Comme l’IFN est largement utilisé pour traiter la SP, il est essentiel de mieux comprendre ses mécanismes de protection. Des études antérieures ont montré que l’IFN de type I agis principalement sur les cellules myéloïdes, et non les

lymphocytes T au cours de l'EAE. Toutefois, suite à l'induction de l'EAE chez les souris

chimériques WT / IFNAR KO conduit au développement dune maladie plus sévère que les souris WT / WT. Nous montrons aussi que les souris avec une délétion conditionnelle de IFNAR dans les Tregs développent une forme très grave de l'EAE. Ces résultats démontrent que la

signalisation via IFNAR est nécessaire pour la fonction de suppressive des Treg dans l'EAE. En conclusion, ces études démontrent que sous certaines conditions, notamment le stress, une infection chronique, maladie auto-immune, la signalisation via IFNAR est essentiel pour maintenir le développement, l'homéostasie, et la fonction des Tregs.

ABSTRACT

Type I Interferons (IFNs) are a family of cytokines with antiviral and immunomodulatory properties. While the antiviral effects of IFNs are well characterized, their immunomodulatory properties are less clear. A number of recent studies have examined the role of Type I IFNs on Foxp3+ _{T regulatory (Treg) cells in different experimental models of autoimmunity and}

inflammation and reached conflicting conclusions. We re-examined in depth the effects of type I IFN on development, homeostasis, and function of Treg cells. A number of unique tools were developed to address these questions including mixed bone marrow (BM) chimeras between wild type (WT) and IFNα/β receptor (IFNAR)knockout(KO) mice, and heterozygous female mice expressing a Treg-specific deletion of the IFNAR. In these two models, IFNAR signaling promoted the development of the Treg lineage in the thymus and their survival in the periphery. IFNAR KO Treg had a higher expression of the pro-apoptotic gene Bim and higher frequency of active caspase positive cells. IFNARKOTreg from chimeric mice displayed a more naïve phenotype, accompanied by lower levels of CD25 and phosphorylated STAT5. Therefore, in Treg, IFNAR signaling may directly or indirectly affect phosphorylation of STAT5. In mixed chimeras with Scurfy fetal liver, Treg derived from IFNARKO BM were unable to control T effector cell activation and tissue inflammation.

It was also of interest to directly test whether type I IFN signaling in Treg was critical for their functions in infectious disease and autoimmunity. We examined the potential effects of type I IFN on Treg function in Chronic Lymphocytic Choriomeningitis Virus (LCMV) infection. Infection of WT mice with LCMV clone 13 leads to a dramatic increase in the percentage of Treg expressing the TCR Vβ5 subunit. We demonstrated that the percentage of Vβ5+ Treg was

manner to mediate expansion of Treg in this model. Experimental autoimmune encephalomyelitis (EAE) represents the most widely studied animal model for Multiple Sclerosis (MS). As IFN is widely used to treat MS, it is critical to better understand its protective mechanisms. Previous studies have demonstrated that Type I IFNs primarily act on myeloid cells, and not T

lymphocytes during the course of EAE. However, following induction of EAE in mixed bone marrow chimeras between WT and IFNAR KO mice, the decreased ratio of IFNAR KO to WT T cells is reversed and the mixed chimeras develop more severe disease than the WT/WT chimeras. Mice with a conditional deletion of the IFNAR in Treg rapidly developed a very severe form of EAE. These results demonstrate that signaling via the IFNAR is required for Treg suppressor function in EAE.

Collectively, these studies demonstrate that under certain condition including stress, chronic infection, and autoimmune disease, IFNAR signaling is essential to maintain Treg development, homeostasis, and function.

LIST OF FIGURES

FIGURE 2. Type I IFN regulates CD4+ T helper cell development.

FIGURE 3. IFNα/β supports CD8+ T-cell priming through direct effects on both antigen-presenting cells and the T cells.

FIGURE 4. Type I IFN during viral infection. FIGURE 5. WT Treg respond to Type I IFN in vivo.

FIGURE 6. Treg from WT, IFNARKO and IFNARfl/fl x Foxp3YFP-Cre/YFP-Cre are similar in terms of phenotype and number.

FIGURE 7. IFNARKO Tregs are at a competitive disadvantage in a mixed BM chimera. FIGURE 8. IFNAR KO Treg in the mixed chimeras exhibit enhanced apoptosis.

FIGURE 9. IFNAR KO Tregs in the chimeric environment exhibit a partial naïve phenotype. FIGURE 10. IFNARfl/fl x Foxp3YFP-cre/wt Treg display the same development and survival defect as IFNAR KO Treg from the chimeric mice.

FIGURE 11. Suppressive function of IFNAR KO Treg.

FIGURE 12. IFNARKOTreg fail to control T cell activation and tissue inflammation in a Scurfy disease model.

FIGURE 13. IFNARKO Tregs have diminished pSTAT5 and CD25 expression in the Scurfy disease model.

FIGURE 14. WT Treg phosphorylate STAT5 following exposure to poly (I:C).

FIGURE 15. IFNARKOTreg cells expand following IL-2 immune complex treatment in the thymus, but not in the spleen.

FIGURE 17. eSag-mediated Treg expansion requires intrinsic IFNAR signaling. FIGURE 18. WT/IFNARKOchimeras develop severe EAE.

FIGURE 19. The defect in reconstitution of WT/IFNAR KO mixed chimeras is reversed after immunization.

FIGURE 20. Cytokine production by cells from the spinal cord of WT/IFNAR KO chimeras. FIGURE 21. IFNAR KOT cells express higher levels of α4 integrin.

FIGURE 22. Mice with a Treg-specific deletion of the IFNAR develop severe EAE. Figure 23. Summary.

I. INTRODUCTION

A. Type I Interferons

Type I interferon (IFN) was first discovered by Isaacs and Lindenmann in 1957 and derives its name from a function to “interfere,” more precisely the ability to interfere with the replication of diverse types of viruses in vitro and in vivo. (1). The IFN family can be classified into three main types of cytokines: type I, type II and type III IFNs. There are many types I IFNs, and all share considerable structural homology. In humans and in mice, the type I IFN family is composed of 16 members, namely 12 IFN-α subtypes, IFN-β, IFN-ε, IFN-κ and IFN-ω (2, 3). The genes that encode type I IFNs are clustered on chromosome 9 in human (4) and on

chromosome 4 in mice (5). All members of the IFN I family bind to a common receptor, known as the IFNα/β receptor (IFNAR), which is expressed on most cell types (6) (Fig.1). By contrast, there is only a single type II IFN: IFN-γ (7). The gene that encodes this cytokines is located on chromosome 12 in humans and chromosome 10 in mice, and there is no structural homology with type I IFNs (8). The third type of IFN is the IFN-λ family, which includes IFN-λ1 (also known as IL-29), IFN-λ2 (also known as IL-28A) and IFN-λ3 (also known as IL-28B) (9, 10). These are distinct from the other types of IFNs and bind to a different receptor, which is composed of two chains, IFNLR1 (also known as IL-28Rα) and IL-10Rβ (9).

Type I IFN Production

Most cell-types can produce type I IFNs under certain conditions. In general, non-immune cells, such as epithelial cells and fibroblasts, infected with viruses or stimulated with double-stranded RNA (dsRNA), produce mainly IFN-β, while monocytes/macrophages and dendritic cells (DCs) produce also IFN-α upon activation (11-15). The major stimuli for the production of

type I IFNs by monocytes/macrophages are different RNA viruses, lipopolysaccharide (LPS) and dsRNA. The IFNs produced are of both the IFN-α and IFN-β subtype, but the amount produced from each monocyte is low. However, because these cells are present in high frequency in blood, a large total amount of type I IFN can be produced (16-18).

Circulating DCs are divided into two main subgroups, the plasmacytoid DCs (pDCs) and the myeloid DCs (cDCs). pDCs have been shown to be identical to the natural IFN-producing cells (NIPC), a cell type that have been known for a long time and was characterized as a rare cell type lacking lineage markers, but with an extraordinary ability to produce huge amounts of type I IFNs in response to a number of different stimuli (15, 19, 20). Furthermore, pDC can be induced to produce type I IFN. The inducers range include viruses, bacteria to a number of microbe-derived products such as bacterial or viral DNA, viral ssRNA or dsRNA and possibly viral glycoproteins, as well as synthetic compounds such as imiquimod (21, 22) (Table. 1). cDCs are high producers of IL-12, but have also been reported to produce type I IFNs under certain

conditions. However, compared to pDC, the cDC produce type I IFN in smaller amounts (mainly IFN-β) and respond to a more narrow range of inducers, such as certain viruses and dsRNA (15, 21, 22).

The major mechanisms utilized by viruses, bacteria and their products to induce type I IFN production involve activation of the germ-line encoded, innate immune receptors denominated pattern-recognition receptors (PRRs) (23-26). The PRRs bind special structures, pathogen-associated molecular patterns (PAMPs), and trigger the innate immune system, by inducing the production of different cytokines. The PAMPs are molecular structures that are found in products derived from microbes, for example double-stranded viral RNA and unmethylated, CpG-rich bacterial/viral DNA (23, 27).

proteins, the nucleotide-binding oligomerization domain (NOD)-like receptors and the Toll-like receptor TLRs (28, 29). The RIG-I-like proteins and the TLRs are involved in the induction of type I IFN as well as the induction of many other inflammatory mediators.

The RNA helicases retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (Mda-5) are the prevalent cytoplasmic receptors responsible for triggering type I IFN. The RIG-I and Mda-5 are constitutively expressed in low levels by most cells, but are highly up regulated upon type I IFN stimulation. (30, 31). These receptors transmits signals through the mitochondria-localized adaptor molecule IPS-1 or MAVS and the kinases TBK1 and IKKi to activate IRF3 and induce transcription of the type I IFN genes (32).

TLRs are the key sensors of microbial invasion in mammals (33), and they activate an innate defense that is crucial for host survival. The TLR family contains 10 members in the human and 13 members in the mouse. The TLRs implicated in the induction of type I IFN include TLR3, TLR4, TLR7, TLR8 and TLR9 (34, 35). Signaling through TLR3 and TLR4 induces type I IFN in multiple cells types in a manner dependent on TIR-domain-containing adaptor protein inducing IFNβ (TRIF) (32, 36, 37). By contrast, TLR7, TLR8 and TLR9 induce type I IFN production in DCs, mainly pDCs, via a pathway dependent on myeloid differentiation primary-response protein 88 (MYD88) (38, 39).

Table 1: Inducers of type I IFNs and responding cells (40)

Type I IFN Signaling

The IFNAR consists of two subunits, IFNAR 1 and IFNAR 2 (41), and is associated with Janus protein tyrosine kinases (JAK) family (42, 43). The IFNAR1 subunit is associated with the tyrosine kinase 2 (TYK2), whereas IFNAR2 is associated with JAK1 (44, 45). The JAK-STAT pathway was the first signaling pathway to be activated by IFNs (46). This pathway is required for the induction of many of the effects of type I IFNs. However, it is now clear that the

activation of JAK-STAT pathways alone is not sufficient for the generation of all IFNs biological activities (Fig. 1).

The binding of IFNα or other type I IFNs to IFNAR induces a cascade of protein phosphorylation and activation of several signal transducers and activators of transcription (STAT) family members (47, 48). In most cells, the STATs that are activated include STAT1, STAT2, STAT3 and STAT5 (49-51). STAT4 and STAT6 can also be activated by IFN-α, but

only in lymphocytes (47, 48, 52). After phosphorylation, the activated STATs form homodimers or heterodimers that translocate to the nucleus and initiate transcription (42, 53).

The phosphorylation of STAT1 and STAT2 induces the recruitment of the IFN regulatory factor 9 (IRF-9) to form the heterotrimeric complex, IFN-stimulated gene factor 3 (ISGF3) (54). This complex translocates to the nucleus and binds to IFN-stimulated response elements (ISRE) in the promoters of ISGs define to initiate the transcription of IFN responsive genes. IFNAR can also signal by inducing the activation and nuclear translocation of STAT1 homodimers that bind to IFNγ-activated site (GAS) enhancer elements in the promoters of ISGs (50, 55, 56). In addition, type I IFN can also induce the recruitment and phosphorylation of STAT3 (44, 57). Following its phosphorylation, STAT3 forms homodimers that translocate to the nucleus, where they bind to STAT3-binding element (SBEs) (Fig. 1).

Although most studies demonstrate that type I IFNs signal via the STAT pathways described above, some studies have suggested that the CRK, a family of adaptor proteins

composed of three members: CRKL, CRKI and CRKII may also play a role in signaling by type IFNs (58). The first evidence for an involvement of CRK proteins in IFN-mediated signaling was the finding that CRKL interacts with TYK2. In response to treatment of cells with IFNα or IFNβ, the SH2 domain of CRKL bind to the IFN-activated form STAT5, resulting in the formation of a CRKL-STAT5 complex. This complex translocates to the nucleus and binds a GAS element that is present in the promoter of certain ISGs (59, 60) (Fig. 1). Alternative signaling pathway for type I IFN exists such as the PI3K and NF-κB pathways and The MAPK pathway (Fig. 1).

B. Effects of Type I IFNs on Cells of the Immune System

Type I IFNs were initially defined by their antiviral properties, but are also potent immunomodulators that can act directly on components of the innate and adaptive immune systems. Type I IFNs can exert their effects on immune cells either directly, through IFNAR triggering, or indirectly by (a) induction of chemokines, which allow the recruitment of immune cells to the site of infection, by (b) inducing a second wave of cytokines, which could further regulate cell numbers and activities (e.g, IL-15, which plays a critical role in proliferation and maintenance of natural killer (NK) cells and memory CD8 T cells (61, 62) and (c) by stimulating other cell types critical for the activation of certain immune cells, such as DC for the activation of naive T cells.

Effects of type I IFNs on CD4

T cells

Type I IFN plays a broad role in CD4+ T cell functions by regulating the development and stability of long-lived memory cells. Its major effect is to protect activated cells from undergoing acute activation-induced cell death and thereby promoting the development of long-lived central memory cells (63). Recently, another study demonstrated a direct role for IFNα/β in promoting the development of human central memory-like CD4+ T cells and preserving elevated IL-2 expression preferentially in these cells versus their effector cell counterparts (64). Hence, IFNα/β acts to prevent terminal differentiation of effector CD4+ T cells by selectively regulating IL-2 expression at the expense of driving inflammatory cytokine secretion. Finally, CD4 T cells are also direct targets of type I IFN in the enhancement of antibody responses (65).

Type I IFNs also play are role in T helper 1 (Th1) cells development, but the results are complex. In the mouse, STAT4 activation was not detected in response to IFNα/β compared with

IL-12 (66), yet studies with human cells reported just the opposite, suggesting a species difference in the phosphorylation of STAT4 by type I IFN (52, 67, 68). The apparent species difference in STAT4 activation was found to involve STAT2 (52). Studies have shown that the human STAT2 C-terminus was critical in regulating IFNα/β dependent STAT4 activation in human cells. In contrast in the mouse, the STAT2 C-terminus is unique, as it failed to mediate STAT4 recruitment (69, 70). In order to determine the significance of the species-specific difference in STAT2, a chimeric mouse/human STAT2 molecule was generated (71). Treatment of mice expressing this hybrid molecule with IFNα/β resulted in normal ISGF3 formation and ISG expression. However, IFNα/β did not promote STAT4 phosphorylation or IFNγ expression in CD4+ T cells. Hence, the C-terminus of human STAT2 was required in human cells to promote efficient STAT4 phosphorylation in response to IFNα/β, but was not sufficient to restore this pathway in mouse cells. Indeed, recent studies have highlighted the importance of the STAT4 N-terminus in IFN-α/β-dependent STAT4 activation through specific contacts made in human cells, but not in mouse cells (72). However, these studies have revealed additional levels of complexity of cytokine receptors and their underlying molecular interactions that coordinate STAT

activation. For example, the duration of phosphorylation of STAT4 by IFNα/β signaling in human CD4+ T cells (73) may be insufficient to maintain STAT4 activation and Th1

development. Furthermore, STAT4 was rapidly dephosphorylated within 6 hours of IFNα/β stimulation (74). Hence, while IFNα/β may be more efficient at promoting acute STAT4

phosphorylation in human cells than in mouse cells, it cannot sustain STAT4 phosphorylation and is therefore not sufficient to drive Th1 commitment in either species (71, 75-77). However, when assessed in vivo, IFNα/β signaling may still contribute to Th1 development (78, 79). Likewise, mice deficient in IL-12 were still able to generate Th1 cells, demonstrating that multiple pathway were involved in Th1 development (80, 81). Alternatively, IFNα/β may promote the expression

of IL-21 and the IL-21R in T cells (82). As IL-21 induces Th1-associated genes, possibly in synergy with IL-18, type I IFN may contribute to Th1 development via this pathway (83, 84). All these data suggest that type I IFN is not sufficient to drive Th1 commitment via direct STAT4 activation, but it contributes by collaborating with other cytokines (Fig. 2).

In contrast to its positive effects on induction of Th1 cells, a recent study showed that type I IFN potently and specifically inhibited the ability of IL-4 to drive Th2 differentiation in human cells, but not murine cells (85). Moreover, type I IFN destabilized pre-committed Th2 cells and blocked Th2 cytokine expression. It also reduced the expression of the Th2 marker, CRTH2. The negative effects of type I IFN on Th2 responses appear, at least in part, to be mediated via

suppression of mRNA and protein levels of GATA3, which is critical for expression of CRTH2 as well as Th2-associated cytokines. The underlying mechanism of GATA3 suppression is not yet clear. The effect is not likely to be mediated by STAT4 or STAT1, as neither IL-12 nor IFN-γ inhibits Th2 commitment. However, recent studies demonstrated that type III IFN (IFN-λ) can suppress Th2 responses in a manner similar to type I IFN (86). Since both type I IFN and type III IFN can activate STAT2 and drive ISGF3 complex formation (87), STAT2 may play a crucial role in suppressing human Th2 development.

Type I IFNs contribute to the suppression of the human allergic T cell response (Fig. 2). A comparison of pDCs from healthy donors and asthma patients demonstrated that pDCs from

asthma patients secrete less type I IFN than healthy donor pDCs in response to viral infections and TLR ligand stimulation (88-90). Considering that IFNα blocks Th2 development and stability (85), it was proposed that the defect in IFN-α production in pDCs from asthma patients might skew T-cell priming toward Th2 development. It has also been suggested that the reduction in IFNα/β secretion during upper respiratory virus infections may lead to exacerbated lung

immune response to control of viral replication in the lungs (90).

Type I IFN was also shown to negatively regulate Th17 development in mice (91). Similar suppressive effects of type I IFN on human Th17 development have also been recently reported (92). Given the use of IFNβ clinically for the treatment of multiple sclerosis, a disease associated with increased inflammation and IL-17 levels in the central nervous system (93), the ability of IFNα/β to limit Th17 cells may explain the effectiveness of this treatment (94). Furthermore, the ability of IFNα/β to inhibit Th2 and Th17 cells suggests that it may play a key role in controlling allergic responses (Fig. 2).

Effects of type I IFN on effector and memory CD8

T cells

Several studies suggested that type I IFN might exert a direct effect on CD8+ T cells. Type I IFNs promote both IFN-γ secretion and expression of perforin and granzymes in murine CD8+ cells. Furthermore, type I IFNs were found to markedly enhance cytolytic activity by CD8+ T cells; this effect was dependent upon STAT4 (95). Type I IFNs also promote survival of CD8+ _T cells from WT but not IFNAR KO mice (63). The definitive report showing that CD8+ T cells represent direct targets of IFNα/β–mediated stimulation in vivo came from Kolumam and

colleagues (96). By adoptively transferring virus-specific naive CD8+ T cells from IFNAR KO or IFNAR-sufficient mice into WT hosts, type I IFN were shown to act directly on murine CD8+ T cells, facilitating their clonal expansion and differentiation into memory cells. Other studies confirmed this work (97-99). Several experiments in mice (95) have shown that, in addition to signals via TCR and CD28, naive CD8+ T cells required a third signal to differentiate into

effector cells. cDNA microarray analyses showed that IFN-α could regulate several critical genes involved in CTL functions (100), providing evidence that IFN-α promoted activation and

differentiation of CD8+ _{T cells by sustaining the expression of T-bet and Eomes through} chromatin remodeling. Recently, it was shown that IFN-α provides a strong and direct signal to human CD8+ T cells, thereby resulting in up regulation of genes critical for cytotoxic T-cell activity (cytolysis and IFN-γ secretion) and for the production of chemokines that attract other effector T lymphocyte subpopulations. Thus, IFN-α is absolutely critical for the differentiation of naïve human CD8+ _{T cells into effector cells (101) (Fig. 3).}

The effects of Type I IFN on priming effector responses and maintaining pools of memory cells may also be mediated indirectly by induction of other cytokines and by

enhancement of antigen presentation (102). Both IFNα/β and IFN-γ can promote homeostatic maintenance of CD8+ memory cells, but with differing requirements for synergy with other cytokines such as IL-12, IL-18 and IL-15 (103). Initial studies demonstrated that the induction of IFNα/β by CpG stimulation led to antigen-presenting cell-dependent T-cell proliferation, which also required IFNα/β signaling to the responding T cells (104). However, not all primary CD8+ T cells responses to pathogens, require IFNα/β signals. Thus, IFNα/β has ben shown to be were required for CD8+ expansion in response to lymphocytic choriomeningitis virus (LCMV), but minimally contributes to responses to vaccinia virus or Listeria monocytogenes infections (79). Based on this observation, it was postulated that antigenic load might contribute to CD8+

dependence on IFNα/β for full expansion, as LCMV viral titers are much higher during the peak of the infection than vaccinia (Fig. 3).

C. Effects of Type I IFNs on T Regulatory T Cells (Treg)

During the last decade, it has become clear that regulatory T (Treg) cells, which constitute approximately 10% of peripheral CD4+ T cells, are required for the maintenance of immune homeostasis (105, 106). Although CD4+ Tregs were originally identified (106) by their expression of high levels of the α subunit of the IL-2 receptor (CD25), it is now clear that the transcription factor, the forkhead box P3 (Foxp3), is most optimal marker for this population (107).

The foxp3 gene was originally identified as the defective gene in the mutant mouse strain, scurfy, and in children with the severe autoimmune disease IPEX (immunodysregulation,

polyendocrinopathy and enteropathy linked syndrome) (108-111). Scurfy mice succumb to X-linked recessive autoimmune and inflammatory disorders as a result of uncontrolled activation of CD4+ _{T cells. In a similar way children with IPEX succumb to several organ-specific}

autoimmune diseases, food allergy, severe dermatitis, high levels of IgE and sometimes eosinophilia (105, 112). The clinical and immunological similarities between Scurfy/IPEX in mice/humans, and the autoimmune disorders observed following depletion of Treg from mice have now been shown to be secondary to requirement for Foxp3 gene expression for the function of Treg cells (105, 113, 114). Traditionally, Treg cells in both mice and humans have been

characterized as CD4+ T cells with a high expression CD25 (IL-2Rα) (106, 115). However, CD25 expression is up regulated when T cells are activated and can therefore not be used to distinguish Treg from activated/effector T cells, especially not after in vitro stimulation or in patients with ongoing immune activation. Treg cells typically also express CTLA-4 (CD152) and GITR (glucocortocoid induced TNF family-related gene/receptor) but these markers have also been shown to be expressed by activated non-regulatory T cells (116-119).

The majority of Foxp3+ _{Tregs is generated in the thymus, and we use the term of} thymus-derived Treg (tTregs). Similarly, an unknown percentage of Treg can be generated from Foxp3- T conventional (Tconv) cells in peripheral sites. This population is named peripherally derived Tregs (pTregs) (120). Lastly, at least in mouse models, Foxp3+ Tregs can be generated in vitro by stimulation of T conventional cells in the presence of TGF-β, and these are termed in

vitro-induced Tregs (iTregs) (121). The role of Tregs in the control of organ-specific autoimmune diseases was initially defined by studies in mice thymectomized on day 3 of life (d3Tx). D3Tx mice develop organ-specific autoimmunity that can be prevented by reconstitution of the mice with CD4+Foxp3+ Tregs between days 10 and 12 of life (106). Similarly, immunodeficient mice reconstituted with CD4+Foxp3- T cells develop organ-specific autoimmunity and co-transfer of Foxp3+ _{cells prevents the development of disease. Polyclonal Tregs have also been shown to} prevent graft versus host disease (122), and prevent the immune response to tumors (123).

The cytokine IL-2 was first identified as a potent growth factor for T cells (124) and has more recently been demonstrated to be essential for the survival and function of Treg (125). Treg cells do not secrete IL-2, but do express all three subunits required for a functional high affinity IL-2 receptor: the α-chain (IL-2Rα/CD25), the β-chain (IL-2Rβ/CD122) and the common cytokine receptor γ-chain (IL-2Rγc/CD132) (126). Mice deficient in IL- 2, CD25, CD122 or the signal transducer and activator of transcription 5 (STAT5), develop autoimmune-like disorders and have reduced frequencies of Treg (127-131). Similarly, humans who lack CD25 succumb to disorders that are similar to IPEX (132). Transfer of wild-type CD4+CD25+ T cells into IL-2- and CD25-deficient mice have been shown to prevent the lymphoproliferative disease (126, 133, 134). This suggests that these mice have insufficient numbers of Treg or dysfunctional Treg and that IL-2 is crucial in order for Treg to function properly. In line with this it has been shown that

reduced numbers of Foxp3+_CD4+_CD25+ _{T cells in the periphery and elicits autoimmune gastritis} in mice (135), while treatment with IL-2 seems to up-regulate the expression of Foxp3 via mechanisms dependent on STAT-5, both in vitro and in vivo (136-138). In summary, IL-2 signaling is critical both for the development and the maintenance of Treg cells and it acts via STAT5 dependent mechanisms (131, 139).

Several studies have shown that the suppressive function of Treg is dependent on cell contact with their target cells (115, 119, 140). These in vitro studies have also demonstrated that the suppression is cytokine independent, as neutralization of IL-10 and TGF-β does not alter the suppressive activity. However, in vivo studies in mice in mouse models of both inflammatory bowels disease and allergic disease in the lung (141-143) have shown that have shown that both IL-10 and TGF-β contribute significantly to Treg suppression. However, they appear to play no role for in vivo suppression of autoimmune gastritis (144, 145).

A number of recent studies have examined the role of Type I IFNs on Foxp3+ T regulatory (Treg) cells in different experimental models of autoimmunity and inflammation and reached conflicting conclusions. In the classic adoptive transfer model of inflammatory bowl disease (IBD), one study (146) demonstrated that signaling via the IFNAR was essential for maintenance of Foxp3 expression and Treg suppressor function, while a second study (147) demonstrated that IFNAR knockout (KO) Treg were fully competent suppressor cells. Similarly, the transfer of the combination of wild-type (WT) CD45RBhi and WT Treg, but not IFNAR KO CD45RBhi and IFNAR KO Treg, induced colitis in RAGKO Trex 1 KO mice that express high levels of endogenous cytoplasmic DNAs that trigger type I IFN production. Development of disease depended on expression of the IFNAR on the WT effector cells, and not on the Treg, as IFNAR KO Teff cells did not cause disease (148). In contrast, in the tumor microenvironment, signaling via the IFNAR was required for the activation of tumor infiltrating Tregs to produce IL-10

resulting in suppression of angiogenesis and lymphoangiogenesis induced by tumor infiltrating Th17 cells (149). Lastly, a recent study (150) have demonstrated that type I IFNs directly inhibit Treg cell activation, proliferation, and function in vivo during acute infection with LCMV and that a failure of this inhibitory effect results in impaired function of virus specific CD4+ and CD8+ T cells and defective viral clearance.

Articles Type I IFNs enhance Tregs Type I IFNs inhibit Tregs Lee, S. E. et al.

Gastroenterology (146) X

Kole, A., Journal of

immunology (147) X

Srivastava, S., The Journal

of experimental medicine (150)

X Stewart, C. A., The

Journal of clinical investigation (149)

X Metidji, A., Journal of

immunology X

Table 2: The role of type I IFN on Tregs in different studies.

D. Type I IFNs and Lymphocytic Choriomeningitis Virus (LCMV)

Infection

LCMV is a member of the Arenaviridae family of viruses and contains a bi-segmented single-stranded ambisense RNA genome. The genome contains two genes on each of the two segments, termed L (7.2 kb) and S (3.4 kb), separated by a unique hairpin structure. The L

segment contains the viral RNA dependent RNA polymerase (L) and a small RING-finger protein (Z) which acts a matrix protein linking the viral nucleoprotein and membrane proteins and is the major protein involved in viral budding (151-153). The S segment contains the nucleoprotein (NP) and glycoprotein polyprotein (GP) that becomes cleaved post-translationally into SSP (signal peptide), GP1 and GP2 (154-156). Following cleavage, the GP complex is responsible for the initial binding of virions to cells and affecting their entry into cells.

LCMV, as well as several other arenavirus family members, uses the ubiquitously

expressed cell surface molecule α-dystroglycan for viral entry (157, 158). Differences in binding affinity of various strains of LCMV for α-dystroglycan has been correlated with changes in tissue tropism and disease outcome, high affinity binding leads to higher levels of replication, increased infection of antigen-presenting cells, and overall higher levels of immunosupression. Less

pathogenic strains of LCMV are more likely to use alternative receptors for cell entry, but these receptors remain less well characterized (159, 160).

LCMV is a natural pathogen in mice that has been used to elucidate many aspects of T cell immunity (161). LCMV is particularly useful as a model system because infection can lead to acute or chronic infections depending upon host and virus factors (159, 162). Peripheral

inoculation of adult mice with the acutely replicating strain LCMV Armstrong (ARM) induces a potent anti-viral CD8 T cell response that is capable of clearing virus within 7-10 days. Infection

with LCMV strain Clone 13 (C13), which is a genetic variant of the Armstrong (ARM) strain, leads to persistently high viremia for up to three months with some tissues never clearing virus (163-165). A study showed that type I IFN signals were required for CD8+ expansion in response to lymphocytic choriomeningitis virus (LCMV). The absence of type I IFN lead to the blockade CD8+ T cells during LCMV (96).

Immune Responses to Acute LCMV infection

The LCMV Armstrong (ARM) response is associated with a robust CD8+ T cell response that is composed of three distinct stages: naive, effector and memory. Following acute ARM infection, CD8+ T cell responses are directed at the immunodominant LCMV peptides NP396-404, GP33-41, and to a lesser extent GP276-286 in the context of MHC Class I H-2Db (166). CD8+ T cells reactive to these epitopes expand rapidly and reach a peak of expansion by day 8. These effector cells extravasate to peripheral tissues and control the infection, often by killing infected cells and producing effector cytokines Following this expansion and viral clearance >90% of these cells undergo apoptosis leaving a small population from which a stable pool of memory CD8+ T cells is generated (167, 168).

During the expansion phase of LCMV ARM infection, CD8+ T cells express effector proteins and begin to increase expression of the genes for IFN-γ, TNF-α, IL-2, perforin and granzyme B (166, 169, 170). T cell contraction following expansion is believed to limit the immunopathology that results from excessive cytolysis and cytokine production, and also resets the immune system following infection to become more able to respond to new infections (167). The memory CD8+ T cells that persist following acute infection are characterized by the ability to survive long-term in the absence of antigen, proliferate in response to homeostatic signals, and to

rapidly divide and produce effector cytokines upon re-challenge (171)

Although CD8+ T cells are essential for the clearance of LCMV ARM, there is also a robust CD4+ T cells response that is dispensable for the CD8+ cells response. Virus specific-CD4+ T cells expand early during infection, though in significantly lower numbers and with slower kinetics than CD8+ T cells and reach a peak expansion 1-2 days after CD8+ T cells (172, 173). The CD4+ _{T cell responses are primarily Th1 in nature, producing IFN-γ and IL-2 (174, 175).} Experiments in CD4+ deficient mice demonstrated that CD4+ T cells were dispensable to the clearance of acute infections, but in the absence of CD4+ T cell help, CD8+ T cells became functionally impaired and failed to maintain a stable memory population (176-178). The activation of CD4+ T cells in response to LCMV has been shown to have a much higher dependence on signaling through co-stimulatory molecules such as CD28, CD154, and OX40 (179, 180).

Immune Responses to Chronic LCMV infection

LCMV clone 13 (C13) was isolated from mice persistently infected with LCMV Armstrong virus (181). It has 2 silent mutations and 2 amino acid changes from the parental ARM strain. Infection of naïve mice with a high dose of the C13 strain of LCMV results in a protracted viremia that lasts approximately 2-3 months, with virus persisting in select tissues such as the kidney, brain and salivary glands (165). C13 infection does not lead to a robust CD8+ _and CD4+ T cell responses capable of clearing virus and protecting against future infection. During chronic LCMV infection, LCMV-specific CD8+ T cells also lose of their lytic capacity and the production of cytokines such as IL-2, TNF-α, and IFN-γ (165). The expansion of CD8+ T cells is diminished in C13 infected mice and the hierarchy of immunodominance is altered (182).

Although CD8+ _{T cells specific for NP}

396-404 are the immunodominant population during ARM infection, this CD8+ T cell population becomes nearly undetectable following C13 infection (165, 183, 184). Responses to GP epitopes are less affected and normally subdominant, but CD8+ clones specific for GP276-286 become immunodominant during C13 infection (183). It is widely accepted that chronic antigen exposure leads to diminished function and eventual deletion of cells that most recognize the most frequent viral-peptide: MHC Class I complexes (185-187). Because NP is the most abundantly expressed viral antigen, NP396-404-reactive clones are more likely to be stimulated and eventually exhausted, while clones recognizing the less abundant GP will be present and remain functional for longer periods of time (167, 176).

Recent studies analyzed the molecular mechanisms of T cell exhaustion during chronic infection. Expression of the inhibitory receptor, programmed death-1 (PD-1), is the hallmark of CD8+ T cell exhaustion. Blocking the PD-1/PD-L1 signaling pathway restores effector T cell function and leads to resolution of chronic LCMV infection (188). Further analysis of gene expression in exhausted T cells led to the identification of several other inhibitory molecules that mediate T cell exhaustion, including LAG-3, Tim-3 and 2B4 (189-191). These data revealed a complex pattern of regulation of CD8+ _{T cells by an array of co-expressed inhibitory receptors} during chronic viral infection.

LCMV-specific CD4+ T cells also undergo exhaustion and initially lose their lytic capacity, and their ability to make TNF-α. They retain low levels of IFN-γ production, but they failed to make IL-2 (165). Moreover, Mice lacking CD4+ T cells or depleted of CD4+ T cells prior to LCMV clone 13 infection cannot control viral replication and maintain high levels of persisting virus in their serum and tissues (192).

The Role of type I IFN During LCMV infection

While the administration of exogenous IFN strongly suggested that IFN was a valuable agent to treat viral infections (193), the availability of IFNAR KO mice has provided definitive proof that IFNα/β mediate potent protection against viruses in vivo (194). IFNAR KO mice were found to be susceptible to LCMV infection. During LCMV infection, IFNα/β promotes the ability of DCs to cross-present antigens (98, 195, 196), promotes the migration of DCs to lymph nodes by up-regulating chemokine receptor expression, ultimately resulting in enhanced T cell

activation (197, 198). DCs are potent producers of IL-12, which is crucial for driving Th1 responses during some viral infections, and important for induction IFNγ production by T cells and NK cells. However, high, but physiological levels, of IFNα/β strongly inhibit IL-12

production during LCMV infections (199, 200). The suppression of IL-12 production by IFNα/β may have developed to favor optimal cytotoxic responses by T cells and NK cells in response to virus, while limiting the pathological effects of excessive IL-12 production (200, 201). IFNα/β also enhanced the survival of CD4+ _{T cells, promoted their survival, and enhanced their ability to} help B cells (65) in response to LCMV.

In LCMV infection, the cytotoxic function of CD8+ T cell function, is positively regulated by IFNα/β (95, 100), and IFN-γ production is both positively (202, 203) and negatively (204) affected by IFNα/β. During LCMV infection, lymphocyte responses to type I IFNs may be reduced during viral infection, as type I IFNs have been shown to inhibit lymphocyte egress from lymphoid organs (205). In contrast, type I IFNs promote chemokine production for the correct trafficking of central memory T cells during recall responses to LCMV (206) and drive

inflammatory monocytes to produce factors such as IL-15 and IL-18, which support memory CD8+ T cell survival and function in infections (207). Activated CD8+ T cells ‘escape’ the

anti-proliferative effects of IFNα/β during viral LCMV infection by expressing lower total levels of STAT1 (208)

Two recent in vivo studies have identified a critical suppressive role for IFNα/β in chronic viral infection (209, 210). Treatment of mice persistently infected with clone 13 LCMV with a blocking mAb to the IFNAR reduced immune suppression, decreased expression of negative immune regulatory molecules, restored lymphoid architecture in mice, and ultimately facilitated clearance of the persistent infection. The control of persistent infection by blockade of type I IFN signaling required CD4+ T cells and was associated with enhanced IFN-γ production. It thus appears that in addition to its major role in neutralizing viral infection, IFNα/β plays a critical in the persistence of chronic LCMV infection by inhibiting T cells responses secondary to induction of immunosuppressive cytokines such as IL-10 and cell surface molecules such as PD-L1

(CD274) that inhibit T cell activation (Fig. 4). The cellular target (s) for the suppressive effects of IFNα/β remain to be elucidated.

Role of Tregs in LCMV infection

After an infection, the balance between Tconv and Treg is critical. Pathogen-specific CD4+ _{and CD8}+ _{T cells rapidly expand after infection and, in most cases, clear the infection. Treg} may play a role during acute infection, but their importance is still unclear (211). However, during chronic infection, Treg can play critical roles by limiting excessive immune activation and tissue damage, while at the same time facilitating pathogen persistence and maintenance of immunity (212). We have previously shown that expansion of Treg following chronic LCMV (C13) infection was most prominent amongst a subset of Treg expressing a particular TCR Vβ subunit(s) (Vβ5 in C57BL/6 mice and Vβ5 and 12 in BALB/c mice) (213). Furthermore, our

study demonstrated that Treg expansion in chronic LCMV infection was not directly mediated by LCMV, but was secondary to induction of Mammary tumor virus (Mtv)-superantigens (Sag) encoded in the mouse genome (eSag). A recent study has claimed that type I IFNs directly inhibit co-stimulation–dependent Treg cell activation and proliferation in vivo during acute infection with LCMV. In studies using mixed bone marrow chimeras between WT mice and IFNAR KO mice, loss of the IFNAR specifically in Treg cells resulted in functional impairment of virus-specific CD8+ and CD4+ T cells and inefficient viral clearance. The study claims that inhibition of Treg cells by IFNs is necessary for the generation of optimal antiviral T cell responses during acute LCMV infection (150).

E. Type I IFNs in Multiple Sclerosis (MS) and Experimental

Autoimmune Encephalomyelitis (EAE)

Given the diverse effects of IFNα/β in the innate and adaptive immune system, it is not surprising that these cytokines play a role in several autoimmune diseases. Psoriasis and systemic lupus erythematosus are improved by the inhibition of Type I IFNs (214, 215), while arthritis and multiple sclerosis benefit from the administration of Type I IFNs (216). Although the associations between Type I IFN and these diseases are established, the mechanisms responsible for the

differential effects of IFN have not yet been elucidated.

Multiple Sclerosis (MS)

Multiple sclerosis (MS) was first described by Charcot and is a chronic inflammatory disease of the central nervous system (CNS). About 2–3 million MS patients are mainly found in Europe and in countries with Caucasian immigration, including the USA, Australia and Northern Asia (217, 218). In the United States alone, an estimated 350,000 individuals are affected (219), and 50% of patients will need help walking within 15 years after the onset of the disease (220). MS strikes twice as many women than men worldwide. The cause of MS is unknown, but it appears to involve a combination of genetic susceptibility and a non-genetic trigger, such as a virus, bacterial infections, metabolism, or environmental factors, that together result in a self-sustaining autoimmune disorder that leads to recurrent immune attacks (221). Traditionally, MS is considered to be an autoimmune disease characterized by demyelination of the white matter via immune mechanisms. Th1 and Th17 CD4+ T cells play a role in the mediation of MS, along with B cells and macrophages that contribute to its pathogenesis (222, 223). Some of the most

common MS symptoms include paralysis, fatigue, walking and balancing problems, bladder dysfunction, and optic neuritis. Neurologists agree that patients may be grouped into four major categories based on the course of disease (224).

Although the pathogenesis of MS remains to be fully elucidated,a number of potential therapies have been developed that mainly function as immune modulators or inhibitors of immune cell migration. Some of these therapies reduce clinical disease activity and the

progression of lesion load. So far no therapy is available to cure MS or even halt its progression. The search for more effective approaches is therefore warranted. IFN-β is used in the treatment of MS and can reduce the frequency of clinical exacerbations (225). A number of other

immunotherapies have been developed for treatment of MS including Glatiramer acetate (GA/Copaxone®), a synthetic polymer of random sequences of four naturally occurring amino acids (L-tyrosine, L-glutamate, L-alanine, and L-lysine). Experimental work has demonstrated that GA suppresses EAE (226). It is believed that GA acts by binding to major histocompatibility complex class II molecules and out competes the binding of the putative MS (227). Natalizumab is a humanized anti- monoclonal antibody that targets the α4-chain of α4β1 integrin. The basis of “anti-adhesion therapy” for MS was derived from studies in animals with anti- α4β1 monoclonal antibody (228). Natalizumab reduces the number of T cells, macrophages, and B cells in the cerebrospinal fluid in treated patients and slows disease progression (229). Mitoxantrone is an anti-neoplastic agent interfering with DNA synthesis, but also has immune suppressive effects. It blocks myelin degradation by macrophages (230), reduces T cells activation and inhibits the proliferation of B cells. As well as a reduced ability of T cells to induce an immune response. Treated patients have reduced gadolinium-enhanced lesions on MRI as well as reduced relapse rate and disability progression (231). Fingolimod (FTY 720) is an oral sphingosine-1-phosphate (S1P) receptor modulator that functions by inhibiting lymphocyte exit from the lymph nodes

(232-234). Teriflunomide (Aubagio) (a derivative of leflunomide) is an oral drug, which binds to dihydro-orotate dehydrogenase (DHODH) and reversibly inhibits it. DHODH is a mitochondrial membrane protein, which is essential for pyrimidine synthesis (235, 236). It is believed that suppression of pyrimidine synthesis in rapidly proliferating cells, such as T and B lymphocytes, is responsible for the immunomodulatory effects of teriflunomide (237).

Tregs in MS

It is widely accepted that CD4+CD25hiFoxp3+Treg cells exert a protective role in MS. Various studies have demonstrated the presence of these cells within the CNS of MS

patients. Some studies have shown a comparable frequency of Treg in the periphery and the CNS of MS patients and healthy controls (238), while others have shown an increase in their

frequencies in MS patients (239, 240). The majority of studies demonstrate a decreased suppressive capacity of Tregscells from MS patients in vitro (241). More specifically, Tregs suppressive function is down regulated during relapses, when there is an increase in frequencies of CD4+ Th1 cells (242). The development of MS in these patients has also been linked to the decreased expression of Foxp3 in these Treg cells (239, 243).

Experimental Autoimmune Encephalomyelitis (EAE)

EAE is an animal model resembling MS in humans in its course and histopathology and serves as a primary animal model for elucidating MS (244, 245). As with MS, EAE is

characterized by inflammation, paralysis and destruction of the myelin sheath (246). EAE is induced by injection of proteins derived from myelin, including myelin oligodendrocyte protein

(MOG), MBP, or proteolipid protein (PLP), into susceptible strains of animals together with Freund’s adjuvant as an immunopotentiator (244, 246, 247). In addition, pertussis toxin (PTX) is injected, which leads to permeabilization of the blood brain barrier (BBB) (248). However, PTX has other biological effects that could contribute to its activity in EAE, such as breaking T cell tolerance and promoting clonal expansion and cytokine production by T cells (249). EAE is characterized by perivascular infiltrates, leading to neurological symptoms, beginning

approximately ten days after immunization. The cells, which predominantly initiate the disease, are CD4+T cells, as transfer of MOG-reactive CD4+T cells isolated from EAE-diseased animals results in appearance of disease in healthy recipient mice (250). It is known, that T cells of Th1 phenotype mediate CNS inflammation during EAE (251). However, during the last years several studies identified Th17 cells to be involved in the induction of CNS-autoimmunity in both animals with EAE and patients with MS (252, 253). The chemokine CCL20, expressed by epithelial cells of the choroid plexus, and its receptor CCR6, which is predominantly found on Th17 cells, are involved in T cell migration into the CNS (254, 255).

Tregs in EAE

Tregs appear to play a protective role in EAE. The transfer of Treg into the mice expressing a transgenic TCR specific for MBP was protective against EAE development (256-258). Varying results have been obtained for the exact mechanism of Treg cell action. Some studies implicate the amelioration of EAE through the production of anti-inflammatory cytokines such as IL-10 and/or down-regulation of IFN-γ, along with expression of molecules such as CTLA-4 and GITR (259-262). Some experiments have also shown that the presence of Tregs skews the immune response towards a Th2 dependent mechanism (263). Moreover the outcome

of disease has been shown to be dependent on a critical balance between the population of CD4+ Teffector cells and CD4+Tregcells (264).

Role of Type I IFNs in MS/EAE

IFNβ has been approved worldwide for the treatment of relapsing–remitting multiple sclerosis (RRMS). It reduces the relapse rate, the relapse severity, and the progression of

disability. Type I IFNs have both pro- and anti-inflammatory activities. Their anti-inflammatory activities are believed to dominate in the context of RR-MS (265), but the underlying

mechanisms of the drug are not yet fully understood (266). The mechanisms of action of type I IFN involve anti-proliferative and pro-apoptotic effects via a variety of molecular changes, including increases in both cyclin kinase inhibitors and several pro-apoptotic molecules (Fas/FasL, p53, Bax, Bak), as well as activation of pro-caspases 8 and 3 (267). Additionally, Type I IFNs inhibit the production of pro-inflammatory cytokines like IL-1 and TNF by

monocytes (268, 269). IFNβ induced STAT1 activation and repressed the activity of the NLRP1 and NLRP3 inflammasomes, resulting in the suppression of caspase-1-dependent IL-1β

maturation. Moreover, IFN induces production of the anti-inflammatory cytokine IL-10 that may signal via STAT3 to reduce the abundance of pro-IL-1α and pro-IL-1β (270). IFNβ suppressed IL-23 and increased IL-10 production by human dendritic cells (DC) activated with via TLR2 and dectin-1. It can also inhibit TLR9 agonist-specific secretion of chemokines (CCL3, CCL4, and CCL5) that act as ligands for CCR5-positive T helper 1 (Th1) cells (271). Furthermore, IFNβ impaired the ability of DC to promote IL-17 production by CD4+ T cells, and induced IL-27 expression by DC, which abrogated the generation of Th17 cells in vitro. This finding suggested an anti-inflammatory effect of IFN-β via the induction of IL-27 (272). In addition, DC migration

is negatively impacted by IFN-β through STAT-1-mediated transcriptional suppression of CCR7 and matrix metalloproteinase 9 (MMP-9) (273).

In MS, auto-reactive, activated T cells produce matrix metalloproteinases, whose

proteolytic activity breaks down the basement membrane of the endothelial barrier enabling cells to migrate into the brain parenchyma (274). IFN-β might directly or indirectly stabilize the BBB in RR-MS patients, as suggested by a number of in vitro studies (275, 276). Trafficking of activated T cells through fibronectin is reduced after IFN-β Treatment by a mechanism that decreases MMP-9 activity of T cells (277). Treating MS patients with IFN-β in vivo leads to an early reduction in the appearance of gadolinium-enhancing lesions. Thus far, the mechanism of action on the barrier-stabilizing effect of IFN-β has not been well understood. It may decrease T cell adhesion to the blood vessel wall by down-regulating the integrins lymphocyte function-associated antigen (LFA)-1 and Very Late Antigen-4 (VLA-4) on leukocytes (278). Finally, IFN-β therapy increases the expression of the protective neurotrophic factors, brain-derived

neurotrophic factor (BDNF), and leukemia inhibitory factor (LIF) (279, 280).

Several studies have suggested that IFN-β may induce both CD4+CD25+Foxp3+ Treg cells and CD8+ _{regulatory cells (281-283). IFN-β has been reported to induce the up-regulation of} GITRL on DCs and down-regulation of CTLA-4 on Tregs, resulting in their enhanced

proliferation in vivo. This result may provide a possible mechanistic explanation for the increased number of Tregs observed in MS patients treated with IFN-β (283).

II.

MATERIALS AND METHODS

Mice

WT C57BL/6, Mx1-Cre, Rosa/eYFP, Foxp3YFP-Cre and Scurfy mice were obtained from Jackson Laboratories (Bar Harbor, ME). Congenic C57BL/6 (CD45.1), IFNARKO, and RAG1 KO mice were obtained from Taconic Farms (Germantown, NY). IFNARfl/fl mice were kindly provided by (Ulrich Kalinke, Paul-Ehrlich Institut, Langen, Germany) and crossed to Foxp3 YFP-Cre_{. All other mice were bred in house. The Animal Care and Use Committee (ACUC) of the}

National Institute of Allergy and Infectious Diseases (NIAID) approved all experiments.

Bone marrow (BM) chimeras

Recipient mice were lethally irradiated with two doses of 550 Rads of total body

irradiation. Recipients were injected i.v on the same d with BM cells (1x106) or fetal liver cells (2 x 105) from donor mice and allowed to reconstitute for 6 to 8 weeks. The fetal liver cells were obtained at 15 d gestation; the cells were screened for the Scurfy mutation by PCR.

Flow cytometry and cell sorting

A single cell suspension was generated from spleens or thymus. Cell surface staining was performed with the following directly conjugated anti–murine antibodies (from BD Bioscience or eBioscience unless otherwise specified): antiCD4 (RM45), CD8 (536.7), CD19 (1D3), -CD25 (7D4), -CD44 (IM7), -CD45.1 (A20), -CD45.2 (104), -CD45RB (C363.16A), -CD62L (MEL-14), -CD69 (H1.2F3), -CD101 (IGSF2), -CD103 (2E7), -ICOS (7E.17G9). For

(eBioscience). Cells were then washed and stained with antibodies against Foxp3 (FJK16s), -GFP (life Technology). For IFNγ (XMG1.2) staining, cells were stimulated for 4 hours with cell stimulation cocktail plus protein transport inhibitor (eBioscience) and stained intracellularly. A kit to detect active pan-caspase was purchased from eBioscience. 1x106/ml splenocytes were stained with FITC-Z-VAD-FMK or APC-Z-VAD-FMK for 45 minutes in a 37°C incubator with 5% CO2. The MHC Class II tetramer that recognizes the LCMV GP66-77 epitope in conjunction with I-Ab was obtained from the NIAID tetramer core facility (Atlanta, GA). LCMV-specific MHC Class I tetramers were prepared and used as previously described (165). The data were acquired on an LSRII instrument (BD Bioscience) and analyzed using FlowJo software (TreeStar). For cell sorting experiments, cells were labeled with anti-CD4 (L3T4) beads and purified on the AutoMACS Cell Separator (Miltenyi Biotec), stained for desired cell surface markers, and sorted using a FACSAria flow cytometers (BD Biosciences).

In vitro suppression assay

CD4+ T cells were first enriched from a single cell suspension of lymph node (LN) cells using anti-CD4 microbeads (Miltenyi Biotec) and then FACS sorted on the basis of CD25 expression. Sorted CD4+CD25- cells were used as responders and CD4+CD25+ cells were used as Treg. Responder cells (5x104) were cultured in 96-well plates along with irradiated T-depleted spleen cells (5x104_{), 0.5 µg/ml anti-CD3, and the indicated number of Treg for 72 h in a humidified} incubator at 37°C/ 7% CO2. Cultures were pulsed with [3H] thymidine for the last 6h of culture (140).

Inflammatory Bowel Disease

CD4+CD25−CD45RBhi T cells were purified from WT mice by FACS sorting, and transferred (4 × 105) into RAG1KO mice in the presence or absence of CD4+CD25hiCD45RBlow Tregs (2 × 105_{) from WT or IFNAR KO mice. Mice were weighed just prior to T cell transfer (time 0)} and up to two times per week thereafter. Percent weight change was calculated as follows: (weight at time X – weight at time 0) / (weight at time 0). All mice in each experiment were sacrificed when any individual mouse showed clinical signs of severe disease or 20% weight loss (284).

Phospho STAT staining

To assess phosphor (p) STAT5 levels directly ex vivo, spleens were immediately disrupted and fixed with Cytofix/Cytoperm buffer (BD). After incubation for 12 min in a 37°C incubator with 5% CO2, the cells were washed, resuspended in 1ml Perm Buffer II (BD), and incubated on ice for 30 min. After an additional wash, cells were stained for surface and intracellular antigens, including pSTAT5 (pY694; Cell Signaling Technology) or pSTAT1 (pY701; CST), for 45 min on ice. In some experiments, cells were stimulated with different concentrations of mouse IFNβ (PBL Interferon Source) and mouse IFNα2 (eBioscience) for 15 min.

BrdU staining

Mice were injected with 1mg BrdU i.p. and sacrificed 24h later. BrdU incorporation was detected in splenocytes using BrdU Flow kit (BD Bioscience) and Ki-67 staining was performed with Flow kit (BD Bioscience).

ELISA

Mice were injected i.p. with 100mg of the synthetic double-stranded RNA homolog poly (I:C) (InvivoGen) and sacrificed 24h later. The IFNα level was measured in serum using a Mouse IFNα ELISA kit (PBL Interferon Source).

mRNA isolation, cDNA production, and real-time PCR

RNA extraction was performed using RNeasy columns (QIAGEN), and cDNA was generated using iScript cDNA synthesis kit (BIO-RAD) according to the manufacturer’s

instructions. Pre-synthesized Taqman Gene Expression Assays (Applied Biosystems) were used to amplify Bim (Mm00437796_m1), Bcl-2 (Mm00477631_m1), PUMA (Mm00519268_m1), Bax (Mm00432051_m1), Mcl-1 (Mm00725832_s1), Bcl-xL (Mm00437783_m1), Bad

(Mm00432042_m1), and Bak-1 (Mm00432045_m1). Actb was used as an internal control, and target gene values are expressed relative to Actb. Real-time PCR was conducted with the ABI Prism 7900HT, using TaqMan Universal PCR Master Mix (Applied Biosystems).

IL-2 immune complex treatment

WT + IFNARKO chimeras and IFNARfl/fl x Foxp3YFP-Cre/YFP-Cre mice were treated with IL-2 immune complexes, as previously described (285). Briefly, IL-2/anti–IL-2 mAb complexes were prepared by mixing recombinant murine IL-2 (1µg; PeproTech) with JES6-1 (5 µg) at the optimal 1:2 molar ratio and incubated for 10 min at room temperature. Immune complexes were then diluted in PBS and injected i.p. for 5 days consecutively. Expansion of T cells was assessed 2h after the last injection in the spleen and the thymus.