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Characterization of regulatory T cells induced by toxic shock syndrome toxin-1 and their potential application… Li, Haowei 2006

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C H A R A C T E R I Z A T I O N O F R E G U L A T O R Y T C E L L S I N D U C E D B Y T O X I C S H O C K S Y N D R O M E T O X I N - 1 A N D T H E I R P O T E N T I A L A P P L I C A T I O N I N A C U T E G R A F T - V E R S U S - H O S T D I S E A S E b y H A O W E I L I B . S c , JiNan U n i v e r s i t y , 1997 A T H E S I S S U B M I T T E D I N P A R T I A L F U L L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y i n T H E F A C L U L T I E S O F G R A D U A T E S T U D I E S ( E x p e r i m e n t a l M e d i c i n e ) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A M a r c h 2007 © Haowei Li , 2007 Abstract Repeated administration of a bacterial superantigen, toxic shock syndrome toxin-1 (TSST-1), has been shown to induce CD4+ regulatory T cells in mice. However, it is not clear i f the characteristics of TSST-l- induced Tregs differ from those of Tregs induced by other bacterial superantigens, such as staphylococcal enterotoxin A (SEA) . The mechanisms of TSST-l- induced Tregs are also not well characterized. Because in experimental settings Tregs can be used to effectively treat multiple diseases, the potential application of TSST-l- induced Tregs also needs to be determined. In this study, a side-by-side comparative study of Tregs induced by TSST-1 and S E A was conducted. Results showed that TSST-l- induced Tregs were different from those induced by S E A in suppressive function, cytokine profile and proliferative ability. Remarkably, TSST-l- induced Tregs were more potent than SEA-induced Tregs in suppressive activities and proliferative ability in vitro. The possible mechanism of TSST-l- induced Tregs was then investigated. TSST-l- induced Tregs did not induce death of target cells, inhibit the activation of target cells, or cause their target cells to acquire regulatory functions. Supernatants from TSST-l- induced Tregs were not suppressive and blockade of IL-10 by a monoclonal antibody did not reverse the suppression. In contrast, cell contact with target cells was required. In addition, TSST-l- induced Tregs were able to compete with their target cells for IL-2. Finally, the potential therapeutic application of TSST-l- induced Tregs was examined by determining their ability to control acute graft-versus-host disease i i ( a G V H D ) in a murine model. Data showed that TS ST-1-induced Tregs were able to mediate bystander suppression of a G V H D following re-activation upon administration of TSST-1 post transplant. The Tregs inhibited the production of proinflammatory cytokines triggered by alloresponses, but neither affected the engraftment or expansion of donor T cells nor enhanced the elimination of host A P C s . Blockade of IL-10 by in vivo administration of a monoclonal antibody did not reverse the suppression. Finally, although TSST-1-induced Tregs attenuated a G V H D , graft-versus-tumor ( G V T ) effects were preserved, suggesting that these cells differentially regulate a G V H D versus G V T effects. i i i Table of contents Abstract i i Table of contents iv List o f tables v i i i List o f figures ix List o f abbreviations x i Acknowledgements x i i i Chapter 1. Introduction 1 1.1. Regulatory T cells (Tregs) 1 1.1.1. Br ief history, definition and categories o f Tregs : 1 1.1.2. Phenotypes of Tregs 4 1.1.2.1 .Activation-induced proliferation and apoptosis 4 1.1.2.2. Cel l markers 8 1.1.2.3. Cytokine profiles 10 1.1.3. Generation of Tregs 12 1.1.3.1. Role of the thymus 12 1.1.3.2. Role of antigens 13 1.1.3.3. Role of cytokines 15 1.1.3.4. Role of costimulation 19 1.1.3.5. Role of other cell types.. 20 1.1.4. Mechanisms o f immunomodulation by Tregs 22 1.1.4.1. Targets of Tregs 22 1.1.4.2. Role of cytokines 24 1.1.4.3. Cytotoxic mechanisms 26 1.1.4.4.Inhibition of activation by Tregs 28 1.1.4.5.Infectious tolerance 29 1.1.4.6. Cytokine competition 31 1.1.4.7.In vivo actions 32 1.1.5. Role of Tregs in disease pathogenesis and potential 33 applications 1.1.5.1. Autoimmune diseases 34 1.1.5.2. Transplantation 36 1.1.5.3. Infections and malignant tumors 44 1.2. Superantigens 46 1.2.1. Definition and categories of superantigens 46 1.2.2. Biological effects of superantigens 47 1.2.3. Staphylococcal superantigens 48 1.2.3.1 .TSST-1 49 iv 1.2.3.2.SEA and other staphylococcal enterotoxins 50 1.2.4. Role of bacterial superantigens in diseases 51 1.3. Connections of bacterial superantigens and Tregs 54 Chapter 2. Hypothesis and specific aims 57 Chapter 3. Methods and materials 59 3.1. Mice , TSST-1 preparation and TSST-1 treatment 59 3.2. Ce l l isolation 59 3.3. Ce l l transfer 60 3.4. In vitro culture 60 3.5. C F S E staining and proliferation assay 61 3.6. Assay of cell death 62 3.7. Assay of cytokine production 62 3.8. Lethal shock induction 62 3.9. Antibodies and flow cytometry 63 3.10. V(3 analysis 64 3.11. Reisolation of naive splenocytes co-cultured with TSST-1- 66 induced Tregs and secondary co-culture 3.12. Culture of naive splenocytes with TSST-1-induced Tregs 66 conditioned medium 3.13. Culture of TSST-1-primed splenocytes with naive 67 splenocyte conditioned medium 3.14. Neutralization of IL-10 67 3.15. Tumor cells 68 3.16. M i x e d lymphocyte reaction 68 3.17. a G V H D induction and monitoring 69 3.18. Assay of endotoxin 69 3.19. Histological studies 70 3.20. Statistical analysis -. .70 Chapter 4. Generation and characterization of TSST-1 and S E A - 71 induced Tregs 4.1. Introduction 71 4.2. Experimental design 73 4.3. Results 74 4.3.1. Generation of superantigen-induced Tregs in C 5 7 B L / 6 mice ... 74 4.3.2. Cytokine production profile of TSST-1 and S E A - 76 induced Tregs 4.3.3. Proliferation profile of TSST-1 and SEA-induced-Tregs 81 4.3.4. Other phenotypic properties of TSST-1 and S E A - 84 induced Tregs 4.3.5. TSST-1-induced Tregs had broader suppressive 89 activities than SEA-induced Tregs v 4.3.6. VP usage by TSST-1 and SEA-induced Tregs 93 -Cross-reactive suppressive function of TSST-1-induced Tregs was not be due to shared VP families of T cells reactive to both TSST-1 and S E A 4.4. Discussion 98 Chapter 5. Possible Mechanism of suppressive properties of TSST-1 108 induced Tregs 5.1. Introduction 108 5.2. Experimental design 109 5.3. Results I l l 5.3.1. TSST-1-induced Tregs did not enhance cell death I l l of the target cells 5.3.2. TSST-1-induced Tregs enhanced the activation of I l l target cells 5.3.3. TSST-1-induced Tregs did not render their target 115 cells to acquire regulatory functions 5.3.4. Supernatant of stimulated TSST-1-induced Tregs 117 did not mediate suppressive activities 5.3.5. Suppressive activities of TSST-1-induced Tregs 117 were cell contact-dependent 5.3.6. Blockade of IL-10 with an anti-IL-lOR monoclonal 120 antibody did not reverse the suppressive functions of TSST-l- induced Tregs 5.3.7. Intracellular cytokines expression by naive splenocytes 122 was not suppressed in the presence of TSST-l - induced Tregs 5.3.8. TSST-l- induced Tregs competed for IL-2 with 126 naive splenocytes 5.3.9. Addition of exogenous IL-2 did not reverse the 131 suppression of TSST-l- induced Tregs 5.4. Discussion 133 Chapter 6. Potential application of TSST-l- induced Tregs in the 138 control acutegraft-vs-host disease ( aGVHD) 6.1. Introduction 138 6.2. Experimental design 138 6.3. Results 140 6.3.1. Development and optimization of an a G V H D animal model. . 140 6.3.2. Activation of TSST-l- induced Tregs led to suppression 144 of a G V H D 6.3.3. TSST-l- induced CD4+ Tregs mediated suppression of 148 a G V H D 6.3.4. Activation of TSST-l- induced Tregs did not affect 150 donor T cell expansion or enhance host antigen presenting cell v i elimination in vivo 6.3.5. Activation of TSST-1-induced Tregs modulated 152 cytokine production and serum endotoxin level 6.3.6. IL-10 blockade with IL-10 receptor monoclonal antibody 157 did not reverse the suppression by TSST-1-induced Tregs 6.3.7. Control of a G V H D by activation of TSST-1- 159 induced Tregs did not block graft-vs-tumor ( G V T ) response 6.4. Discussion 163 Chapter 7. General discussion, conclusions, and future directions 171 7.1. General discussion 171 7.2. Conclusions 174 7.3. Future directions 175 Bibliography 179 List of tables Table 4.1. Expression of Foxp3, C T L A - 4 , G I T R or CD25 on CD4+ 88 T cells of mice treated with TSST-1 , S E A or P B S Table 4.2. VP analysis of superantigen-induced Tregs 96 Table 6.1. Summary of cause of death in each group of mice 162 vni List of figures Figure 1.1. Summary of Tregs categories 5 Figure 1.2. Diagram of binding of superantigens to their ligands 52 Figure 4.1. Experimental design of studies described in Chapter 4 73 Figure 4.2. Initial studies to determine the suppressive activities 75 of superantigen-induced Tregs in vitro Figure 4.3. Cytokine levels in supernatant of TSST-1 or S E A - 77 induced Tregs following reactivation with TSST-1 or S E A Figure 4.4. Expression of cytokines by TSST-1 or SEA-induced Tregs 78 Figure 4.5. Proliferation response of superantigen-induced Tregs 82 Figure 4.6. Cel l death of superantigen-induced Tregs in vitro 85 Figure 4.7. Phenotype of superantigen-induced Tregs 88 Figure 4.8. Suppressive function of superantigen-induced Tregs 90 in vitro and in vivo Figure 4.9. VP analysis of superantigen-induced Tregs 95 Figure 4.10. Assay of V p l 5 expression by R T - P C R 97 Figure 5.1. Experimental design of studies described in Chapter 5 110 Figure 5.2. Effect of TSST-l- induced Tregs on the cell death of 112 target cells Figure 5.3. Effect of TSST-l- induced Tregs on the activation of target 113 cells Figure 5.4. TSST-l- induced Tregs did not render the target cells 116 to acquire regulatory functions Figure 5.5. The role of soluble factors in the suppressive function 118 of TSST-l- induced Tregs Figure 5.6. Cel l contact-dependency of TSST-l- induced Tregs 119 Figure 5.7. Effect of addition of anti-IL-10 receptor antibody 121 ix on the suppressive function of TSST-l- induced Tregs Figure 5.8. Expression of IL-2 by naive splenocytes in the presence 123 of TSST-l- induced Tregs Figure 5.9. Competition of IL-2 by TSST-l- induced Tregs 127 Figure 5.10. Effect of anti-CD25 antibody on the uptake of IL-2 129 by TSST-l- induced Tregs Figure 5.11. Expression of CD25 on TSST-l- induced Tregs in 130 the co-culture with naive splenocytes Figure 5.12. Effect of addition of exogenous IL-2 on the 132 suppression mediated by TSST-l- induced Tregs Figure 6.1. Experimental design of studies described in Chapter 6 139 Figure 6.2. a G V H D in B 6—•unconditioned B6D2F1 mouse model 142 Figure 6.3. a G V H D in B6—»cyclophosphamide-conditioned 143 B6D2F1 mouse model Figure 6.4. Histology of target organs of mice undergoing a G V H D 143 Figure 6.5. Activation of TSST-l- induced Tregs attenuated a G V H D 146 Figure 6.6. Activation of TSST-l- induced CD4+ Tregs mediated 149 suppression of a G V H D Figure 6.7. Effect of activation of TSST-l- induced Tregs on 151 donor T cells and host A P C s Figure 6.8. Effect of activated TSST-l- induced Tregs on cytokine 154 production in vivo and in vitro and serum endotoxin levels Figure 6.9. Role of IL-10 in the suppression mediated by 158 TSST-l- induced Tregs on a G V H D Figure 6.10. Effect of activated TSST-l- induced Tregs on G V T response... 160 Figure 6.11. Histological study of tumor infiltration of the 161 liver undergoing tumor challenge x List of abbreviations a G V H D Acute graft-vs-host disease A H S C T Allogeneic hematopoietic stem cell transplantation A P C s Antigen presenting cells B M T Bone marrow transplantation C D 4 0 L CD40 ligand C F S E Carboxy fluoroscein succinimidyl ester C T L A - 4 Cytotoxic T lymphocyte antigen 4 E A E Experimental autoimmune encephalomyelitis E L I S A Enzyme linked immunosorbant assay F B S Fetal bovine serum F C M Flow cytometry FITC Fluorescein isothiocyanate G - C S F Granulocyte colony stimulation factor G I T R Glucocorticoid-induced tumor necrosis factor receptor family-related gene G V T Graft-vs-tumor H S C T Hematopoietic stem cell transplantation IFN-y Interferon-gamma IL-2 Interleukin 2 IL-4 Interleukin 4 IL-10 Interleukin 10 M H C Major histocompatibility complex N O D Non-obese diabetic O V A Ovalbumin P B M C Peripheral blood mononuclear cells P B S Phosphate buffered saline P E Phycoerythrin PTSAgs Pyrogenic toxin superantigens R T - P C R Reverse transcription polymase chain reaction SCLD Severe combined inrmunodeficient S E A Staphylococcal enterotoxin A S E B Staphylococcal enterotoxin B XI s c Splenocytes TGF - P Transforming growth factor-beta T N F - a Tumor Necrosis Factor-alpha Tregs Regulatory T cells T r l cells T regulatory cells type 1 TSS Toxic shock syndrome TSST-1 Toxic shock syndrome toxin-1 7 - A A D 7-aminoactinomycin D Acknowledgements I would first like to express my foremost gratitude to my supervisor, Dr. Anthony W . Chow, for his guidance and support during this three-year doctoral training which I am sure is the cornerstone for my career and life in North America. I would also like to thank all my supervisory committee members, Dr. Paul Keown, Dr. K i r k Schultz and Dr. Megan Levings, for kindly attending my committee meetings despite their busy schedules and providing me extensive guidance and advice throughout this doctoral program. It is also a must to thank all the members of our laboratory who have always selflessly given me a hand whenever I needed. I would also acknowledge Dr. Lisa X u for her excellent instructions and help with the flow cytometry analysis. A n d finally, I wish to sincerely thank my family for their stalwart love and support for my decision to seek a career in science. They are always the source of my courage in face of difficulties. x i i i Chapter 1. Introduction 1.1. Regulatory T cells The main role of the immune system of the mammal is to protect the body from myriads of potentially harmful microbial pathogens. To this end, the immune system generates millions of T and B cells that recognize these microbial antigens by randomly arranging the genes of receptors that recognize these antigens. However, this random generation of T and B cells w i l l inevitably result in T and B cell clones that also recognize the antigens expressed by cells around the body. Therefore, the immune system has also to develop means to ensure the discrimination between the self and non-self antigens, inhibiting autoimmune responses while allowing effective immune responses against the non-self antigens. Over the past century, extensive research on immune tolerance has revealed several mechanisms by which the immune system establishes and maintains unresponsiveness to self antigens, including physical elimination (clonal deletion) or functional inactivation of self-reactive lymphocytes (clonal anergy), and regulation by subsets of T cells, also called dominant tolerance (Fehr and Sykes, 2004; Kurtz et al., 2004b). The later has been one o f the foci in immunology research in the past decade. 1.1.1. Br ie f history, definition and categories of regulatory T cells Gershon et al first discovered the regulation mediated by T cells about thirty years ago (Cantor, 2004). Following their findings, studies by many groups in different systems also pointed to the presence of subsets of T cells having the ability to suppress immune responses (Damle and Engleman, 1983; Jiang et al., 1992; Kumar and Sercarz, 1 1993; Qin et al., 1993; Taylor et al., 1983; Tsumfuji et al., 1983). Despite the evident experimental findings, the concept of regulation by T cells was still met with skepticism in the 80's mainly because at that time no specific marker for the T cells with regulatory functions has been found, making these cells rather elusive (Moller G , 1988). In 1995, after more than a decade's effort, Sakaguchi et al identified a subset of CD4+ T cells in the periphery expressing CD25 as a subset of naturally occurring regulatory T cells (Tregs, also called natural Tregs). Removal of this subset of Tregs by thymectomy 3 days after the mice were born induces autoimmune diseases in multiple organs which can be prevented by transfer of CD4+CD25+ Tregs (Sakaguchi et al., 1995). This paper thus solved one of the mysteries in this area. More importantly, the discovery of a subset of Tregs with a specific marker finally established the phenomenon of T cell-mediated immune regulation. Following these seminal studies, many groups also focused on the existence of antigen-induced Tregs. In contrast to the naturally occurring Tregs which are generated during the development of T cells in the thymus, it was found that Tregs can also be generated after antigen encounter in the periphery (Vigouroux et al., 2004). These Tregs were characterized by their ability to control immune responses in an antigen-specific manner or via bystander inhibition pathway (Chen et al., 1994; Groux et al., 1997; Jonuleit et al., 2000; McGui rk et al., 2002). Collectively, these Tregs were called antigen-induced Tregs or adaptive Tregs, to be distinguished from the naturally occurring Tregs (Bluestone and Abbas, 2003). In the past decade, many studies have shown that Tregs, both naturally occurring and antigen-induced, play a pivotal role in the control of immune responses to multiple antigens, including autoantigens, 2 alloantigens and microbial antigens. Research on Tregs not only provides insight into tolerance induction and maintenance by the immune system, but also holds great promise in the development of novel therapies for various conditions such as autoimmune diseases, organ transplantation and malignant tumors. The current consensus on the definition of Tregs is that this term refers to all subsets of T cells possessing the ability to suppress immune responses (Zou, 2005). While the CD4+CD25+ natural Tregs have been found to suppress immune responses triggered by any antigen, some Tregs may not always be suppressive. A case in point is the N K T cells, which were found to act as Tregs in some situations, such as in acute graft-vs-host disease (Lan et al., 2001), but were pathogenic in other systems (Zeng et al., 2003). Therefore, when the name of Tregs is used, one must be aware of the situation where this definition applies. Based on the lineage markers that define T cell subsets, Tregs have been found in CD4+ (Honey et al., 1999; Sakaguchi, 2005), CD8+ (Chang et al., 2002; Colovai et al., 2001; Jiang et al., 2003; Jiang et al., 2001), C D 4 - C D 8 - (Zhang et a l , 2000) and NK1.1+ apTCR+ T cell subsets (Sharif et al., 2002; Sharif et a l , 2001). CD4+CD25+ naturally occurring Tregs refer to a subset of CD4+ T cells that constitutively express CD25 and depend on the thymus for their generation. They have been found to play an important role in controlling immune responses triggered by multiple types of antigens, including autoantigens, alloantigens and microbial antigens (Sakaguchi et al., 2006). In contrast, antigen-induced CD4+ Tregs have been found in multiple systems after antigen priming (Barrat et al., 2002; Graca et a l , 2000; Jonuleit et al., 2000; Ploix et al., 1999). In many cases, natural Tregs were not depleted before antigen priming, thus it is 3 difficult to make conclusions related to the origin of the antigen-induced CD4+ Tregs. They might be derived from the natural Tregs, CD4+CD25- conventional T cells, or both (Kingsley et al., 2002; Zhang et al., 2001). However, in some reports, antigen priming followed the depletion of natural Tregs, and in this case, antigen-induced CD4+ Tregs were found to arise mainly from the conventional CD4+ T cell compartment (Karim et a l , 2004). A brief summary of different types of Tregs can be found in Figure 1.1. 1.1.2. Phenotypes of Tregs In addition to the suppressive activities of Tregs, they have other unique characteristics that can be used to differentiate them from naive T cells. These include activation-induced proliferation and apoptosis, cell markers and cytokine profile. 1.1.2.1. Activation-induced proliferation and apoptosis It has been well established that activation of antigen specific T cells leads to the proliferation of these T cells. Antigen-experienced memory T cells demonstrate enhanced proliferation in response to antigen stimulation. However, most of the studies show that both natural Tregs and antigen-induced Tregs exhibit an anergic state in response to antigen stimulation in terms of their proliferation response in vitro. Groux et al showed that the low degree of proliferation of T r l cells in response to cognate antigen stimulation was due to the high production of IL-10 and lack o f IL-2 by these cells. IL-10 has been shown to inhibit the proliferation of T cells, while IL-2 is a well known growth factor that supports T cell proliferation. Neutralization of IL-10 and 4 addition of IL-2 both could restore the proliferation of T r l cells (Groux et al., 1997). Similar to T r l cells, Tregs induced in other systems generally show a decreased proliferation in response to antigen stimulation (Jonuleit et a l , 2000; Levings et al., 2005). However, in some situations, antigen-induced Tregs might also exhibit an enhanced proliferation profile (Kemper et al., 2003), even though the mechanism for this response is unclear. r CD4+-Tregs ^CD4+: CD25+Foxp3+CTLA-4+GITR+: The so-called naturally occurring Tregs. Generation dependent on the thymus. Actions dependent on cell contact in vitro. T r l cells: Generation and actions are both dependent on IL-10. Can be induced in vitro and in vivo. Th3 cells: Induced by oral tolerance. Actions dependent on TGF-B. Other CD4+ Tregs: Induced by multiple measures and antigens with various mechanisms of actions. Qa-1 restricted: Induced by Qa-1-presented antigens. Exert functions via cytotoxic pathway. Other CD8+ Tregs: Induced by multiple measures and antigens with various mechanisms of actions. CD4-CD8-TCR+: The so-called double-negative Tregs. Induced by alloantigens. Exert functions via cytotoxic pathway. NKT cells: Generation dependent on C D 1 . Actions dependent on IL-4. CD8+-Figure 1.1. Summary of Tregs categories. 5 The ex vivo isolated natural Tregs proliferate poorly in response to anti-CD3 stimulation in vitro as compared to CD25- conventional T cells. Addit ion of exogenous IL-2 could restore their proliferative ability in vitro (Levings et al., 2001; Takahashi et al., 1998; Thornton and Shevach, 1998). This was also the basis of expanding natural Tregs for therapeutic purposes. In contrast, the proliferation o f natural Tregs in vivo was somewhat different. When ex vivo purified natural Tregs were transferred into lymphopenic hosts, such as SCID mice, these cells were able to undergo normal homeostatic proliferation and still maintained their suppressive activities (Gavin et al., 2002). Several groups further showed that natural Tregs could proliferate in normal mice and that this proliferation was dependent on the recognition of the cognate antigen of natural Tregs, demonstrating that the proliferation of natural Tregs also played an important role in their response to foreign antigens and in maintaining their peripheral homeostasis (Cozzo et al., 2003; Fisson et al., 2003; Walker et al., 2003). The proliferative state of Tregs has important implications for the development of effective immunotherapy. One critical issue in the clinical settings is the feasibility to obtain a sufficient number of Tregs for therapeutic purposes. In humans, only about 1% of CD4+CD25+ T cells actually possess suppressive activities (Baecher-Allan et al., 2001). Thus, it is extremely difficult to obtain a sufficient number of these cells for therapeutic application, where a very large number of Tregs are needed, such as in hematopoietic stem cell transplantation. Thus laborious in vitro expansion must be performed to harvest sufficient numbers of natural Tregs. Mult iple studies have shown that the expansion of natural Tregs without loss of function was possible both for human and murine natural Tregs (Beyersdorf et al., 2006; Karakhanova et al., 2006; 6 Levings et al., 2001; Masteller et al., 2005). In contrast to the proliferation profile of Tregs, the apoptotic profile of Tregs has been less well characterized. Although both murine and human natural Tregs are anergic in response to stimulation via the T cell receptor in vitro, their apoptotic state seems to be different. Studies by Papiernik et al showed that mouse natural Tregs were resistant to apoptosis as they could survive both virus-induced clonal deletion and Fas-induced apoptosis in vivo (Papiernik ef a l , 1998). However, different apoptotic properties of human natural Tregs were found. Taams et al reported that human natural Tregs isolated from human peripheral blood were prone to apoptosis in vitro, probably due to their low expression of the anti-apoptotic gene bcl-2. Exogenous cytokines, such as IL-2 or IFN-P, could prevent the apoptosis (Taams L S et al., 2001). Another report showed that human natural Tregs were more resistant to T C R induced apoptosis but more susceptible to CD95 induced apoptosis as compared to conventional T cells (Fritzsching et al., 2005). These data suggest that i f in vitro expanded natural Tregs are to be used for immunotherapy, more studies are required to characterize their apoptotic properties and determine their survival in vivo, since their ability to persist in vivo could be a crucial determinant of their therapeutic outcome. Even less information on the apoptotic properties of antigen-induced Tregs is available. Since antigen-induced Tregs are generated following antigen encounter, these Tregs are antigen-experienced cells. Thus it may be reasonable to assume that their ability to survive is somewhat similar to memory T cells. In one study, Zheng et al did show that Tregs generated in vitro in the presence of exogenous IL-2 and TGF - P were able to survive in vivo upon transfer to recipients and were able to suppress 7 chronic graft-vs-host disease (Zheng et al., 2004). However, more studies are necessary to characterize the apoptotic properties of antigen-induced Tregs in other systems. 1.1.2.2. Cel l markers Most studies of the surface markers of Tregs were conducted for natural Tregs. The first identified marker for natural Tregs, in addition to the lineage marker C D 4 , was CD25 (Sakaguchi et a l , 1995). Although under steady state, CD25 can be used to identify the Tregs population, this marker is not specific for Tregs due to several reasons. First, CD25 is also expressed by activated non-Tregs T cells. Thus this marker can not be used to identify natural Tregs from other conventional T cells under antigen stimulation. Second, it has been shown in human natural Tregs that only T cells expressing high level of CD25 had suppressive activities while those having low to medium level of CD25 expression were not suppressive (Baecher-Allan et al., 2001). Third, T cell clones generated from CD4+CD25+ Tregs showed that not all CD25+ clones were suppressive (Levings et al,, 2002). Fourth, further separation of the CD25+ population by other markers, such as C D 6 2 L identified that suppressive Tregs were in the CD62L+ or C D 6 2 h i g h compartment (Ermann et al., 2005; Taylor et a l , 2004). Finally, CD25- Tregs have been identified frequently (Graca et al., 2002; Oida et al., 2003; Ono et a l , 2006; Stephens and Mason, 2000; Unger et al., 2003; Uraushihara et al., 2003). Despite these drawbacks, CD25 is still a valuable marker for natural Tregs. This marker was also functionally linked to the suppressive activities of natural Tregs as the constitutive expression of this IL-2 receptor (CD25) allow them to engage IL-2 available in the microenvironment, and natural Tregs can compete for IL-2 produced 8 by naive T cells stimulated by antigens (de la Rosa M et a l , 2004). Following the identification of CD25 as a marker of natural Tregs, C T L A - 4 (Read et al., 2000; Takahashi et al., 2000) and G I T R (McHugh et al., 2002) were also found to be specifically expressed by natural Tregs. However, as for CD25 , these markers were also expressed on antigen stimulated T cells, thus they are not specific for Tregs. A major advance in the field came when several groups published their findings that the Foxp3 transcription factor was preferentially expressed on natural Tregs. Importantly, Foxp3 was found to be a critical factor in the differentiation of natural Tregs, as mice lacking the functional Foxp3 due to a mutation were deficient in natural Tregs and succumbed to autoimmune diseases which could be cured by transfer of natural Tregs. Additionally, over-expression of Foxp3 in mouse naive T cells caused them to acquire regulatory functions, suggesting this factor is not only necessary but also sufficient for the development of Tregs (Fontenot et al., 2003; Hor i et al., 2003; Khattri et al., 2003). Although these original findings created much excitement, later studies revealed several drawbacks of following expression of Foxp3 as an exclusive Treg marker. It was found that human natural Tregs also preferentially express Foxp3, but unlike in mouse cells, retroviral-mediated over-expression of this protein in human T cells did not result in acquisition of stable and potent regulatory function (Allan et a l , 2005). Moreover, since Foxp3 is a transcriptional factor and is not expressed on the cell surface, it can not serve as a marker for purifying natural Tregs for clinical application purposes. Recent studies have also identified other proteins that are preferentially expressed by natural Tregs (Baecher-Allan et a l , 2006; Bruder D et al., 2004; Seddiki et al., 2006), however, further studies are needed to confirm these 9 findings. Markers for antigen-induced Tregs are even scarcer. Antigen-induced Tregs often exhibit a stimulated phenotype with enhanced expression of CD25 or C T L A - 4 . One study did identify L A G - 3 as a marker for antigen-induced Tregs (Huang et al., 2004). More studies are required to confirm that L A G - 3 can serve as a marker for antigen-induced Tregs, and to identify more suitable markers for antigen-induced Tregs. In summary, there is no satisfactory or specific marker identifying Tregs in general, especially for antigen-induced Tregs. Thus the functional studies are the most reliable means for determining the presence of Tregs, especially for the antigen-induced Tregs. 1.1.2.3. Cytokine profile The cytokine production profile has been an important characteristic for identifying distinct subsets of effector T cells as exemplified in the T h l and Th2 cells (Mosmann et al., 1986).. The most classic study which showed a distinct profile for antigen-induced Tregs was the first report of T r l cells. In this study, the authors found that T r l cells, generated by stimulation o f T cells in the presence of exogenous IL-10, demonstrated a cytokine profile distinct from those of T h l and Th2 cells. These cells did not produce T h l cytokines, such as IL-2 and IFN-y, or typical Th2 cytokines, such as IL-4. Yet they produced high levels of IL-10 and medium levels of TGF-p\ and in humans low levels of LFN-y. Moreover, their cytokine profile was closely associated with their suppressive functions as IL-10 was necessary for the suppression mediated by T r l cells (Groux et al., 1997). Other grups also reported the existence of Tregs that 10 mainly produced TGF-p (Fukaura et al., 1996; Ochi et a l , 2006; Yoshida et al., 2000; Zheng et a l , 2002). In contrast, the cytokine profile of natural Tregs in vitro is characterized by a general lack of cytokines, although there is some evidence that they may produce TGF-p (Levings et al., 2002) and/or IL-10 (Barthlott et al., 2005) in vitro. It must be noted that in contrast to T r l cells, natural Tregs exert suppression in vitro via a contact-dependent and cytokine-independent manner. Thus although the production of these immunosuppressive cytokines was detected, it does not necessarily imply that these cytokines play a role in the suppression mediated by natural Tregs as in that mediated by antigen-induced Tregs. Generally speaking, the cytokine profile of Tregs is characterized by decreased production of proinflammatory T h l cytokines such as IL-2 and IFN-y and increased production of anti-inflammatory cytokines, such as IL-10 and TGF-p . However, more recent studies have shown that antigen-induced Tregs in different systems do not have a cytokine profile as typical as that of T r l cells. Jonuleit et al showed that Tregs induced via repeated stimulation by allogeneic immature dendritic cells did increase the production of IL-10. However, the suppression of these Tregs was cell contact dependent (Jonuleit et al., 2000). Several recent studies showed that antigen-induced Tregs actually produced IFN-y (Hong et al., 2005; Kemper et al., 2003; Riemekasten et al., 2004; Sawitzki et al., 2005; Stock et al., 2004). Some types of Tregs such as the C D 4 - C D 8 - Tregs, might not even have a distinct cytokine profile, and be capable of producing many proinflammatory cytokines, including IL-2, IFN-y and T N F - a , yet nevertheless exert immunosuppressive effects leading to long term tolerance of the allogeneic graft (Zhang et al., 2000) . Therefore, the presence of Tregs cannot be 11 determined only by the cytokine profile of the T cells, and a functional assay is still the best gold standard method to detect the presence of Tregs. 1.1.3. Generation of Tregs While natural Tregs depend on the thymus for their development, antigen-induced Tregs rely on antigen encounter in a specific environment for their generation. Multiple factors may be involved, and these are described below. 1.1.3.1. Role of the thymus The thymus is the primary lymphoid organ that mediates the maturation of T cells by positive and negative selection. The discovery of natural Tregs revealed its third function: generation of a subset of Tregs (Seddon and Mason, 2000). The importance of the thymus in the generation of natural Tregs was revealed by the absence of these Tregs in mice that received thymectomy on day 3 after birth. Natural Tregs were absent in these mice resulting in the occurrence of autoimmune diseases in multiple organs. Transfer of CD4+ T cells from normal mice could prevent the diseases (Shevach, 2000). The process by which these Tregs are generated in the thymus is not clear. Jordan et al used a transgenic mouse model to investigate the developmental process of natural Tregs in the thymus. The major finding was that thymocytes developing into natural Tregs required their T cell receptor (TCR) to have high affinity for autoantigen, but not too high to induce negative selection, because thymocytes in T C R transgenic mice that have low affinity for autoantigen failed to develop into natural Tregs (Jordan et al., 2001). Romagnoli et al recently showed that bone marrow 12 derived antigen presenting cells (APCs) , rather than thymic epithelial cells, were responsible for inducing selection of natural Tregs (Romagnoli et al., 2005). Definitely, more studies are required to elucidate the developmental process of natural Tregs in the thymus. In addition to the role of thymus in the generation of natural Tregs, recent studies provided evidence for the generation of CD4+CD25+Foxp3+ natural Tregs in the periphery, suggesting that the generation of natural Tregs involved both thymus-dependent and thymus-independent processes (Akbar et al., 2003). While the role of the thymus in generating natural Tregs is clearly defined, the role of the thymus in the generating antigen-induced Tregs is far less important than that of natural Tregs, as it has been clearly shown that antigen-induced Tregs could develop in athymic mice (Apostolou and von Boehmer, 2004). However, a recent paper by Deng et al showed that Tregs controlling transplant tolerance induced by ant i -CD45RB was dependent upon the thymus for their generation, suggesting that the thymus may also play an important role in generating antigen-induced Tregs in some systems (Deng et al., 2006). 1.1.3.2. Role of antigens Antigens play a more crucial role in the development of antigen-induced Tregs after their interactions with T cell subsets in the periphery. Several parameters affect the generation of antigen-induced Tregs, including the structure and dose of the antigen, and the method of antigen administration. Studies have shown that modified antigens, such as altered peptide ligands, preferentially induced Tregs. Chen et al found that peptides with low T cell stimulatory ability induce long term tolerance to a transplantation antigen via generation of Tregs (Chen et al., 2004). Some bacterial 13 antigens have an innate ability to induce Tregs, which may be an important mechanism for pathogenic organisms to evade the immune attack by the host (Lavelle et al., 2003; Marshall et al., 2003; McGui rk et al., 2002). The dose of antigen is also important in regulating the generation of Tregs. The most remarkable example has been shown in oral tolerance, where low doses of antigen administered orally induce Tregs, while high doses of the antigen actually induced deletion of the antigen-specific T cells (Weiner, 2001). The way of introducing antigens to the immune system is also critical for the generation o f Tregs. Firstly, the site of antigen encounter may determine the generation of Tregs vs. effector T cells. The most prominent example is mucosal tolerance. While subcutaneous immunization of antigen with adjuvant activated antigen-specific T cells to become T h l cells, oral administration of the same antigen induced antigen-specific T cells to become Tregs (Faria and Weiner, 2005). Secondly, repeated injection or chronic exposure to antigens was shown to be effective in inducing Tregs. For example, use of a pump to chronically release antigen in mice in which natural Tregs had been previously depleted by thymectomy and anti-CD25 resulted in the generation of readily detectable Tregs (Apostolou and von Boehmer, 2004). Mai le et al found that repeated stimulation of T cells with alloantigens could induce CD8+ Tregs (Maile R et al., 2006). The mechanisms by which antigens with altered structure or following chronic exposure induced Tregs are currently unclear. Garca et al proposed that the induction of Tregs by these two approaches is due to the delivery of a suboptimal activation signal to antigen specific T cells, which drove them to become Tregs (Graca et al., 2005). 14 1.1.3.3. Role of cytokines Cytokines are important players in deciding the T cell fate. Multiple cytokines have been shown to play important roles in the generation of both natural Tregs and antigen-induced Tregs, including interleukin-2 (IL-2), interleukin-10 (IL-10), transforming growth factor-(3 (TGF-P), and granulocyte-colony stimulating factor (G-CSF) . IL-2 has long been regarded as a crucial cytokine for generating effective T cell response. Thus, it was rather surprising when it was found that disruption of the IL-2 signaling pathway, by knockout of IL-2 or IL-2 receptor, resulted in autoimmune diseases of multiple organs in these knockout mice due to overreactive T cells to autoantigens (Sadlack B et al., 1995; Sadlack et al., 1993; Willerford et al., 1995). These findings demonstrated that IL-2 is not only a growth factor for antigen stimulated T cells, but may also be implicated in the maintenance of tolerance. However, it was not clear how IL-2 did this until the publication of several papers demonstrating that IL-2 was a crucial factor in the generation and maintenance of natural Tregs (Furtado et a l , 2002; Malek et al., 2002; W o l f M et al., 2001). These authors showed that disrupting IL-2 signaling pathway specifically led to the failure of development of natural Tregs. The absence of natural Tregs gave rise to the autoimmune diseases of multiple organs, which also occurred in mice lacking natural Tregs due to thymectomy early after they were born. Transfer of natural Tregs could cure the diseases in these mice. They also showed that IL-2 was not only required for T cell progenitor to develop into natural Tregs, but also required in the periphery where IL-2 maintained the homeostasis of natural Tregs. However, the role of IL-2 in the 15 generation of antigen-induced Tregs has not been wel l studied, and remains unclear at this time. Ever since IL-10 was discovered, its role in suppressing immune responses has been well-defined (Moore et al., 2001). It was the classic paper by Groux et al that further demonstrated its important role in the generation o f antigen-induced Tregs (Groux et al., 1997). These investigators stimulated T cells from transgenic mice exprssing T C R that is specific for O V A in the presence of exogenous IL-10 in vitro, and then generated T cell clones also in the presence o f exogenous IL-10. They then found that the generated T cell clones could shut down the production of T h l and Th2 cytokines but produced large amounts of IL-10 and low level of T G F - p in response to cognate antigen stimulation in vitro. These T cells suppressed the response of naive T cells to O V A in vitro via the production of soluble IL-10. These cells could also mediate suppression of colitis in vivo when they were activated. Neutralization of IL-10 in vivo could reverse their suppression, further confirming their effects observed in vitro. More importantly, stimulation of human T cells in the presence of exogenous IL-10 could also induce Tregs similar to mouse Tregs. This paper for the first time demonstrated the generation of an effector T cell subset distinct from T h l and Th2 cells, and established the relationship between immunosuppressive cytokines and the generation of Tregs, showing that a single cytokine in addition to antigen stimulation was able to induce Tregs. The authors called Tregs generated in this manner T regulatory cells type 1, or T r l cells. The induction of T r l cells is also a typical example of antigen-induced Tregs. It also suggests that during immune responses endogenous IL-10 is able to drive the generation of Tregs which in turn w i l l regulate the immune 16 responses. The generation of T r l cells has been confirmed in other systems (Akasaki et al., 2004; Ding et al., 2006; Lundqvist et al., 2005). More importantly, Massey et al showed that intranasal administration of antigens were not able to induce Tregs in IL-10 knockout mice as compared to wi ld type mice, further demonstrating the important role of IL-10 in the generation of antigen-induced Tregs (Massey et al., 2002). TGF - P is another major immunosuppressive cytokine. Its role in maintaining immune tolerance can also be revealed in TGF - P knockout mice which show autoimmune diseases in multiple organs (Shull et al., 1992). Studies showed that inducing Tregs is one of the ways by which TGF - P maintains immune tolerance. Chen et al and Park et al both demonstrated that stimulation of mouse T cells depleted of the natural Tregs population (CD4+CD25-) could induce them to become Tregs in the presence of TGF - P (Chen et al., 2003a; Park et al., 2004). Additionally, studies by Horwitz et al further expanded this finding in human T cells (Zheng et al., 2002). Thus it is clear that like IL-10, TGF - P has the ability to drive the generation of antigen-induced Tregs. Moreover, it has been shown that IL-10 and TGF - P have synergistic effects in inducing Tregs. Blazar's group studied the effects of IL-10 and TGF - P in inducing tolerance to alloantigens. In mixed lymphocyte reactions, they added exogenous IL-10, TGF - P or both to the cultures. Then they reisolated the donor T cells to test their response to alloantigens. They found that while IL-10 or TGF - P alone decreased responses to alloantigens by donor T cells, as shown by a reduced ability to mediate graft-vs-host disease in vivo, donor T cells stimulated by alloantigens in the presence of both IL-10 and TGF - P in fact lost the ability to mediate graft-vs-host disease. Moreover, they became Tregs as they suppressed acute graft-vs-host disease 17 ( aGVHD) mediated by naive T cells. Additionally, Tregs generated in the presence of both IL-10 and TGF - P have more potent suppressive functions than those generated in IL-10 or TGF - P alone (Chen et al., 2003b; Zeller et al., 1999). On the other hand, recent studies have also demonstrated that TGF - P is critical for maintaining the suppressive function and homeostasis of natural Tregs in the periphery (Huber et al., 2004; Marie et a l , 2005). G - C S F is a cytokine that directs the differentiation of neutrophils. It is used to mobilize hematopoietic stem cells into the peripheral blood which can be collected for hematopoietic stem cell transplantation (Rutella et al., 2005). Although a G - C S F mobilized graft contains about 10 times more mature T cells as compared to a graft collected from the bone marrow, it does not significantly increase the incidence of a G V H D , suggesting that this cytokine itself may have immune modulating effects. One study clearly demonstrated that the G - C S F mobilized graft contained dendritic cells that preferentially induced a Th2 response (Arpinati et al., 2000), suggesting that Th2 cells in response to alloantigen might inhibit a T h l response. A more recent study further showed that a structurally modified G - C S F could induce a unique subset of antigen presenting cells (APCs) in vivo. Co-transplant of this subset of A P C s into an allogeneic host could suppress acute graft-vs-host disease via induction of IL-10-producing Tregs (Morris et a l , 2004). Moreover, it has been demonstrated that G - C S F can also induce the generation of human T r l cells in response to alloantigen stimulation (Rutella et al., 2002). 18 1.1.3.4. Role of costimulation Effective T cell activation requires two signals, one delivered by the T cell receptor engaging its ligand, the MHC-peptide complex, and the other by costimulatory signals which can be delivered by multiple pathways, including CD28-CD80/86 or CD40 /CD40L. The importance of costimulation in T cell responses can be shown by the development of T cell tolerance following costimulation blockade. Disruption of the costimulation pathways has been shown to effectively induce transplantation tolerance and prevent autoimmune diseases. However, its role in generating natural Tregs was only recognized a few years ago. Salomon et al crossed the N O D mice with CD28 knockout mice, and surprisingly these mice showed a more dramatic decrease of CD4+CD25+ natural Tregs accompanied by accelerated disease progression. Thus, for the first time it was shown that the generation or maintenance o f natural Tregs required an intact costimulation system (Salomon et al., 2000). Kumanogoh et al later showed that CD40 and C D 4 0 L interaction was also required for the generation of natural Tregs (Kumanogoh et a l , 2001). It was thought that the costimulation might not be required for the development of natural Tregs in the thymus, but was essential for the homeostasis of natural Tregs in the periphery (Liang et al., 2005). Although it is clear that costimulation blockade could induce tolerance, the mechanism might not only be the inhibition o f T cell activation. Sykes's group showed that induction of tolerance by blockade of CD40 and C D 4 0 L interaction mainly involved clonal deletion (Kurtz et al., 2004a). However, it was frequently seen that blockade of costimulation actually led to the development of dominant regulation mediated by Tregs. Waaga et al induced tolerance to MHC-mismatch kidney graft by 19 using C L T A - 4 I g and they isolated alloreactive T cell clones from these tolerant rats. A s compared to the control rats which rejected the graft, T cell clones from the tolerant rats showed a Th2 phenotype and were able to suppress the responses of naive T cells to alloantigens (Waaga et al., 2001). Blazar's group had similar finding studying the role of CD40 and C D 4 0 L costimulation in a G V H D . They stimulated the donor T cells with alloantigens in vitro in the presence of anti-CD40L antibody to block this pathway. The stimulated T cells not only showed dramatically decreased ability to mediate a G V H D , but also became Tregs as they suppressed a G V H D when cotransferred with na'ive T cells (Blazar et al., 1998; Taylor et al., 2002a). However, the mechanisms of Tregs induction by costimulation blockade are not clear currently. In contrast to the induction of Tregs via costimulation blockade, activation of certain costimulation pathways could lead to the induction of Tregs (Akbari et al., 2002; Masuyama et a l , 2002; Watanabe et a l , 2006). 1.1.3.5. Role of other cell types A t the cellular level, the outcome of T cell fate depends on their interactions with other cell types. For the generation of Tregs, especially antigen-induced Tregs, other cell types have great impact. T cells require A P C s to be activated and their ultimate development is dependent very much on their interaction with A P C s . Dendritic cells are the only type of professional A P C s that are able to prime naive T cells. This unique ability enables them to play an important role in determining the cell fate of T cells with which they are interacting. Dendritic cells are a heterogeneous population which consists of 20 different subsets. Based on the activation status, dendritic cells can be divided into mature and immature dendritic cells. While mature dendritic cells are characterized by high expression of M H C and costimulation molecules and ability to induce the development of effector T cells, immature dendritic cells express low levels of M H C and costimulation molecules and the ability to induce tolerance of T cells (Banchereau et al., 2000). The induction of T cell tolerance by immature dendritic cells includes T cell anergy and generation of Tregs. Several studies have shown that priming of nai've T cells with immature dendritic cells led to the generation of Tregs (Charbonnier et al., 2006; Dhodapkar and Steinman, 2002; Gilliet and L i u , 2002; Jonuleit et al., 2000; Mahnke et al., 2003). Thus immature dendritic cells have also been called tolerogenic dendritic cells. Immunosuppressive cytokines in some cases are the critical mediator for the effects of dendritic cells on the generation of Tregs. Steinbrink et al showed that IL-10-treated dendritic cells had an immature dendritic cell phenotype and induced Tregs (Steinbrink et al., 2002). Dendritic cells residing in some anatomical sites preferentially show ability to induce Tregs. For example, dendritic cells isolated from the respiratory tract produce high levels of IL-10 in response to antigen stimulation and induce responding T cells to become Tregs. The induction of Tregs by dendritic cells at such sites is an important mechanism by which tolerance to harmless antigens is maintained (Akbari et al., 2001). Although the ability of immature dendritic cells to induce Tregs has been attributed to their low expression of M H C and costimulation molecules and secretion of immunosuppressive cytokines, other mechanisms may also exist.. In addition to mediating direct suppression on their target cells, natural Tregs 21 may function to allow other tolerogenic measures to induce Tregs. Blazar et al developed an in vitro system using anti-CD40L antibody to induce tolerance of alloantigens. The donor T cells became Tregs when stimulated by alloantigens in the presence of anti-CD40L antibodies as these donor T cells could suppress a G V H D when co-transferred with naive T cells. However, i f natural Tregs were depleted from the donor T cell population before alloantigen stimulation, blockade of C D 4 0 / C D 4 0 L did not lead to generation of Tregs (Taylor et al., 2002a). However, this effect of natural Tregs might not apply to other system for Tregs induction. In the absence of natural Tregs, TGF-p\ IL-10 or repeated stimulation by antigens are still able to effectively induce Tregs. For example, Levings et al showed that human allogeneic specific T r l cells were still able to be generated in the absence of natural Tregs, while neutralization of IL-10 led to the failure to generate these cells (Levings et al., 2005). Other than natural Tregs, N K T cells and mast cells have also been found to be essential for the generation of antigen-induced Tregs in some systems ( K i m et al., 2006; L u et a l , 2006). 1.1.4. Mechanisms of immunomodulation by Tregs 1.1.4.1. Targets of Tregs Studies revealed that natural Tregs could exert their suppression over a wide range of target cells. The most studied target is the CD4+ T cell. Thornton et al developed the in vitro suppression system where natural Tregs and C D 2 5 - conventional CD4+ T cells were co-cultured in the presence of anti-CD3 stimulation. The proliferation of these T cells was used as the readout of suppression. While natural 22 Tregs showed very poor proliferation in response to anti-CD3 stimulation, they suppressed the proliferation of conventional CD4+ T cells (Thornton and Shevach, 1998). In vivo suppression by natural Tregs on the CD4+ T cells was demonstrated in several disease models where transfer of conventional CD4+ T cells caused diseases and co-transfer of natural Tregs attenuated or prevented the disease (Annacker et a l , 2001; Mottet et al., 2003; Suri-Payer et al., 1998). Later on Picciri l lo et al first found in mice that natural Tregs could also directly suppress the functions of CD8+ T cells, including the activation of and IFN-y production by CD8+ T cells (Piccirillo and Shevach, 2001). Kursar et al demonstrated a role for natural Tregs in vivo in an infection model. These studies implied that natural Tregs could act like a two edged-sword. On the one hand, they might regulate the CD8+ T cell response to limit their harmful effects during immune responses. However, on the other hand, their presence might also blunt the favorable response mediated by CD8+ T cells, such as tumor immunity (Kursar et al., 2002). Subsequent studies in a tumor model did prove the latter. Antony et al demonstrated that natural Tregs suppressed the tumor immunity mediated by CD8+ T cells (Antony et al., 2005). Studies have also demonstrated that human natural Tregs can suppress CD8+ T cell functions similarly (Robertson et al., 2006). A P C s , especially dendritic cells, play an important role in initiating and regulating immune responses. Thus it is not surprising that Tregs could exert their effects on this component. Cederbom et al first showed that mouse natural Tregs down-regulated the costimulatory molecules on A P C s (Cederbom L et al., 2000). Prevention of maturation of dendritic cell by human natural Tregs was also demonstrated. Similar 23 to suppression of T cells, the effects of natural Tregs on A P C s were also cell-contact dependent (Misra et al., 2004). Houot et al recently demonstrated that natural Tregs were only able to suppress the activation of myeloid dendritic cells, but not plasmacytoid dendritic cells (Houot et al., 2006). In addition, natural Tregs have also been found to regulate the functions of B cells (Janssens et al., 2003; L i m et al., 2004; L i m et al., 2005; Seo et al., 2002; Zhao et al., 2006). In vitro studies showed that they mainly exert their effects on B cells via cytotoxic mechanisms. Moreover, they were also able to inhibit the functions of N K and N K T cells (Azuma et al., 2003; Ghiringhelli et al., 2005; Smyth et al., 2006; Trzonkowski et al., 2006). 1.1.4.2. Role of cytokines Immunosuppressive cytokines, mainly IL-10 and TGF-p \ are crucial in the suppression mediated by antigen-induced Tregs. IL-10 has been found to act as the main soluble factor that mediated antigen-induced Tregs. T r l cells, generated in the presence of exogenous IL-10, relied on releasing IL-10 for their actions. Thus in vitro, they were still suppressive even when they were separated from the target cells by a permeable membrane (Groux et al., 1997; McGui rk et al., 2002). IL-10 could act on multiple cell types, including T cells and A P C s . It could down-regulate the expression of M H C and costimulatory molecules by A P C s , and suppress the production of proinflammatory cytokines (Moore et al., 2001). Thus, by releasing this cytokine, IL-10-producing Tregs were able to affect multiple targets, leading to down-regulation of immune responses. Moreover, IL-10-treated A P C s were able to induce Tregs themselves, thus amplifying the effect of IL-10 in addition to its direct suppressive 24 effects (Steinbrink et al., 2002). TGF - P is another important immunosuppressive cytokine implicated in the suppressive function of Tregs. Chen et al first found that oral tolerance was associated with the generation of TGF-P-producing T cells with regulatory function. A t that time, the concept of Tregs was still not well established, and they called this cell type Th3 cells to distinguish them from the T h l and Th2 cells that had been described several years before (Chen et al., 1994). Other groups using different systems also demonstrated a critical role played by TGF-P-producing Tregs (Fukaura et al., 1996; Ochi et a l , 2006; Yoshida et al., 2000; Zheng et al., 2002). TGF - P was also able to induce naive T cells to develop into Tregs (Chen et al., 2003a; Park et al., 2004), or to confer dendritic cells with tolerogenic ability which in turn induced the T cells interacting with them to develop into Tregs (Kosiewicz et al., 2004). Interleukin-4 (IL-4) is a cytokine that drives the Th2 response and attenuates the T h l response (Cher and Mosmann, 1987; Krenger et al., 1995; Mosmann and Sad, 1996). IL-4-dependent suppression is prominent in N K T cell-mediated inhibition. Co-transfer of donor bone marrow derived N K T cells effectively inhibited a G V H D in an IL-4-dependent manner, as N K T cells from IL-4 knockout mice were not protective (Zeng et al., 1999). Activation of host N K T cells after bone marrow transplantation or by conditioning the host with multiple doses of total lymphoid irradiation and antibody-mediated T cell depletion to enrich the host N K T cells, suppressed a G V H D , and this effect was also IL-4-dependent (Hashimoto et al., 2005; Lan et al., 2001; Lan et al., 2003). The mechanism for this IL-4-dependent suppression was thought to be mediated by a skewed Th2 response, as donor T cells showed a Th2 cytokine profile. 25 In the case of natural Tregs, the role of cytokines in their suppression is controversial. On the one hand, the suppression mediated by natural Tregs in co-culture systems in vitro was found to be cell-contact dependent and cytokine-independent (Thornton and Shevach, 1998). This seems to rule out the role of soluble immunosuppressive factors. However, in vivo studies found that IL-10 was required for the suppression mediated by natural Tregs in some systems. The studies conducted by Dieckmann et al and Jonuleit et al provided a possible answer for the observation. They found that natural Tregs were able to induce their target cells to develop into IL-10-producing Tregs which in turn exerted suppressive functions (Dieckmann et al., 2002; Jonuleit et al., 2002). A s to the role of TGF-p , more conflicting results have been reported. Kullberg et al and Picciri l lo et al showed by using Smad3 knockout mice that natural Tregs did not require TGF - P to suppress (Kullberg M C et al., 2006; Picciril lo et al., 2002). Levings et al generated natural Tregs clones and showed that they produced TGF - P as compared to T r l cell clones which produced IL-10 (Levings et al., 2002), and showed that TGF-p was not required for suppression. In contrast, other groups showed that T G F - p was definitely required for the suppression by natural Tregs (Fahlen et al., 2005; Green et al., 2003; Nakamura et al., 2004). The discrepancy in these results could probably be due to the different strains of knockout mice used and/or the fact that the role of TGF - P is different depending on the model system. 1.1.4.3. Cytotoxic mechanisms Apoptosis is an essential mechanism for immune tolerance and regulating immune responses. For example, apoptosis mediated by Fas and FasL interaction is 26 one of the main ways to ensure tolerance to antigens in immune privileged sites (Ferguson and Griffith, 2006). Activation induced cell death is an important mechanism for controlling the expansion of effector T cells in the late phase of the immune response (Schluns and Lefrancois, 2003; Wong and Pamer, 2003). The importance of the cytotoxic pathway in immune regulation can also be shown in mice that have mutations in molecules mediating cytotoxic effects. For example, the Ipr mice have uncontrolled lymphoproliferative diseases due to the mutated Fas molecule (Adachi et al., 1996). The cytotoxic effects of Tregs were first found in antigen-induced Tregs. In the experimental allergic encephalitis (EAE) model, it was found that T cell immunization attenuated E A E , which was associated with the generation of CD8+ cytotoxic T cells restricted by Qa-1 molecule. These CD8+ cytotoxic T cells were able to recognize and k i l l the T cells activated by autoantigens which expressed Qa-1, thus attenuating E A E (Jiang et al., 1995; Jiang et al., 1998). Another example of cytotoxicity mediated by Tregs was the C D 4 - C D 8 - double negative Tregs. Donor-specific transfusion-induced double negative Tregs conferred long term tolerance to M H C mismatched heart graft. Zhang et al showed that these cells were able to acquire MHC-peptide complex from the host cells and interacted with the pathogenic T cells v ia these molecules, triggering the cytotoxic process mediated by the double negative Tregs (Ford et al., 2002; Zhang et al., 2000). Cytotoxicity could also be mediated by soluble factors. Q in et al recently identified granzyme B and perforin to be the soluble factors that mediated the suppressive effects of a Treg clone. They also showed that the granzyme B induced apoptosis was perforin dependent (Qin et al., 2006). 27 The cytotoxic effects mediated by natural Tregs were recognized several years ago. Janssens et al and Zhao et al both found that mouse natural Tregs were able to induce the death of B cells, which might be a major pathway to regulate B cell responses (Janssens et al., 2003; Zhao et al., 2006). Grossman et al showed that human antigen-induced Tregs expressed mainly granzyme A while natural Tregs mainly expressed granzyme B and were able to mediate cytotoxic effects via these molecules (Grossman et al., 2004). However, the in vivo significance of cytotoxic effects mediated by natural Tregs was not established and more studies are needed to further investigate this issue. 1.1.4.4. Inhibition of activation by Tregs Activation of antigen-specific CD4+ or CD8+ T cells is a critical step in generating productive immune responses. Activation of antigen-reactive T cells was associated with the expression of activation markers, such as CD25 and CD69. Several studies using in vitro systems showed that natural Tregs co-cultured with conventional CD4+ T cells or CD8+ T cells inhibited the expression of CD25 on target cells (Barthlott et al., 2005; de la Rosa M et al., 2004; George T C et al., 2003; Picciri l lo et a l , 2002), indicating that the natural Tregs could directly suppress the activation of target cells via T cell to T cell interactions. Two studies showed that natural Tregs suppressed the activation of CD4+ conventional T cells by deprivation of IL-2 produced by the activated target cells, but it was not known i f natural Tregs suppressed the activation of CD8+ T cells in the same way (Barthlott et al., 2005; de la Rosa M et al., 2004). In vivo studies showed that natural Tregs were able to inhibit the 28 upregulation of CD25 by naive T cells when they were co-transferred to lymphopenic recipient mice (Barthlott et a l , 2005). Trenado et al demonstrated similar effects in an a G V H D model. They isolated natural Tregs and then expanded them by stimulation with allogeneic A P C s in the presence of IL-2. These allogeneic specific natural Tregs were then co-transferred with naive T cells to irradiated allogeneic host to study their effects on naive T cells. They found that naive T cells co-transferred with ex vivo expanded natural Tregs showed a decreased expression of activation markers CD25 and CD69 (Trenado et al., 2006), thus further confirming the results from in vitro studies. However, more studies are needed to determine i f this mechanism also applies to human Tregs. 1.1.4.5. Infectious tolerance The term "infectious tolerance" was first coined by Gershon to describe the regulation by T cells. Infectious tolerance was further demonstrated by Waldmann's group in a series of studies using antibody to induce transplantation tolerance. They found that injection of a non-depleting anti-CD4 antibody was able to induce transplantation tolerance which was due to the induction of dominant regulation mediated by CD4+ Tregs. Transfer of CD4+ Tregs generated by anti-CD4 antibody to other naive hosts prevented rejection of allogeneic graft. Importantly, CD4+ T cells of the naive hosts developed regulatory functions and were able to confer regulatory functions to other naive hosts. Thus the original CD4+ Tregs induced by anti-CD4 antibody were able not only to directly suppress the responses of target T cells interacting with them, but also to convey the ability to induce other T cells to become 29 Tregs to the target T cells. Thus, the term "infectious" was used to describe this property (Qin et al., 1993). Their studies established that non-depleting antibodies to C D 4 and C D 8 , and an antibody that blocks C D 4 0 / C D 4 0 L interactions, all were able to induce infectious tolerance (Cobbold and Waldmann, 1998; Graca et al., 2000). This system was the best characterized model where infectious tolerance mediated by antigen-induced Tregs could be generated. Recent studies also revealed that infectious tolerance was involved in the regulation mediated by Tregs induced via mucosal tolerance (Alpan et a l , 2004; Unger et al., 2003). Alpan et al further showed a critical role that might be played by A P C s . Using a unique mouse model, they showed that oral tolerance-induced Tregs were able to induce other T cells to acquire a cytokine profile similar to Tregs. They demonstrated that A P C s were critical mediators o f this effect, as A P C s co-cultured with oral tolerance-induced Tregs were able to induce other T cells to become Tregs. IL-4 and IL-10 both played a role in transferring the Tregs-inducing effects from oral tolerance-induced Tregs to A P C s . However, other unidentified molecules may also be involved. Subsequent studies demonstrated that infectious tolerance might be one important mechanism for the suppression by natural Tregs. Several studies found that human natural Tregs were able induce their target cells to become Tregs in vitro (Dieckrnann et a l , 2002; Dieckmann et al., 2005; Jonuleit et al., 2002). Dieckmann et al showed that human CD4+CD25- conventional T cells co-cultured with natural Tregs in the presence of anti-CD3 stimulation not only became anergic and but also acquired suppressive functions mediated by the production of IL-10. The Treg-induction ability of natural Tregs required cell-cell contact and was mediated by molecules expressed 30 after activation as paraformaledhyde-fixed ex vivo isolated natural Tregs did not have this effect. However, the authors did not identify the molecules responsible for this effect (Dieckmann et al., 2002). Using a similar system, Jonuleit et al reported similar results. However, these author did demonstrate that TGF - P was in part responsible for the ability of natural Tregs to induce their target cells to become Tregs (Jonuleit et al., 2002). Collectively, these studies suggest that natural Tregs might exert their suppression by inducing the target cells to become Tregs. On the one hand, this could change the response of naive T cells to antigens; on the other hand, these induced Tregs could suppress the responses of naive T cells that had not been changed into Tregs, thus leading to the amplification of suppressive functions of natural Tregs. 1.1.4.6. Cytokine competition Cytokines are crucial for the development and regulation of immune responses. It has been found over two decades ago that competition of cytokines was an important way to regulate immune responses (Gunther J et al., 1982; Scheffold A et al., 2005). A previous study suggested that anergic T cells with regulatory functions were able to inhibit responses of target cells by competition for IL-2. Recent studies showed that cytokine competition was one of the main mechanisms by which natural Tregs exert their suppressive functions (Barthlott et al., 2005; de la Rosa M et al., 2004). de la Rosa et al, using an in vitro co-culture system, showed that human natural Tregs inhibited the activation of conventional T cells and suppressed their proliferation by depriving them of IL-2. Moreover, in the presence of activation and IL-2, the suppressive functions of natural Tregs were also enhanced (de la Rosa M et al., 2004). Barthlott et 31 al made similar observations using mouse natural Tregs in an in vitro system. They further showed that cytokine competition also applied to the suppression by natural Tregs in vivo. Moreover, they observed that upon consuming the IL-2 produced by target cells, IL-10 production by natural Tregs was increased, and in their system, IL-10 was required for the suppression mediated by natural Tregs in vivo (Barthlott et al., 2005). Cytokine competition mediated by Tregs is of great importance to tumor immunotherapy. Recently, it has been shown that adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes to non-lethally irradiated recipients induced potent anti-tumor effects and tumor regression (Dudley et al., 2002). In a mouse tumor model, Overwijk et al showed that administration of IL-2 and transfer of tumor specific T cells was also able to induce tumor regression (Overwijk et al., 2003). These studies suggested that the presence of natural Tregs deprived effector T cells of cytokines which are important for the generation of effective tumor immunity. That was why removal of natural Tregs by irradiation or addition of exogenous IL-2 to saturate the consumption by natural Tregs was able to boost the anti-tumor immunity (Gattinoni et al., 2006). 1.1.4.7. In vivo actions Most of the studies concerning the mechanisms o f Tregs were performed using in vitro systems, and not much is known about the in vivo behavior of Tregs in terms of how they exert their effects on their targets. Recent studies have begun to focus on the in vivo behavior of Tregs, mainly on that of the natural Tregs. For the natural Tregs to 32 mediate their suppression in vivo, the first important issue is how these cells go to the sites where they are needed. Some recent studies demonstrated that natural Tregs might have a unique migration pattern made possible by their expression of molecules facilitating their migration (Huehn and Hamann, 2005; Siegmund et al., 2005; Wei et al., 2006; Wysocki et al., 2005; Yurchenko et a l , 2006). While in vitro co-culture systems are able to reveal the outcome of suppression, advances of in vivo imaging techniques have made it possible to dissect the in vivo interactions of natural Tregs with their target cells in more detail. Although studies using in vitro systems showed that natural Tregs are able to directly suppress the responses of CD4+ T cells, in vivo studies revealed that natural Tregs did not directly make contact with pathogenic CD4+ T cells and suggested that their suppressive effects were mainly exerted via the interactions with dendritic cells (Tadokoro et al., 2006; Tang et al., 2006). However, natural Tregs could directly inhibit the functions of CD8+ cytotoxic T lymphocytes in vivo (Mempel et al., 2006). In addition, in vivo studies also suggested that antigen-induced Tregs might exert a suppressive effect on target cells in ways that differ from those of natural Tregs (Tischner et al., 2006). 1.1.5. Role of Tregs in disease pathogenesis and their potential applications Research on Tregs has generated insightful understandings in multiple diseases, and has also suggested novel therapeutic strategies for them. Three categories of diseases are selected for discussion as they are the most representative examples in this regard. 33 1.1.5.1. Autoimmune diseases The discovery of natural Tregs has been closely linked to the pathogenesis of autoimmune diseases since it was the lack of this subset of Tregs that induced the generation o f autoimmune diseases in animal models. Thus, in addition to neagtive selection, the generation of Tregs by the thymus is an important regulatory pathway used by the immune system to maintain tolerance to autoantigens in the periphery (Itoh et al., 1999; Sakaguchi et al., 1995; Shevach, 2000). The presence of a counterpart to mouse natural Tregs in humans is a further indication that this is an evolutionally conserved mechanism (Baecher-Allan et al., 2001). The studies of murine natural Tregs suggested that the malfunction of natural Tregs might lead to the loss of tolerance to autoantigens, and in turn cause autoimmune diseases, although this may not be the only factor in inducing autoimmune diseases (McHugh and Shevach, 2002). Mult iple recent studies in human subjects with autoimmune diseases support the importance of Tregs in their pathogenesis as functional defects have been detected in the natural Tregs of these patients (Bacchetta et a l , 2006; Balandina et al., 2005; Kriegel et al., 2004; Lawson et al., 2006; Lindley et al., 2005; Viglietta et al., 2004). Ehrenstein et al showed that effective treatment of autoimmune disease was associated with the recovery of suppressive functions of natural Tregs, further demonstrating a link between the function of natural Tregs and the pathogenesis of autoimmune diseases (Ehrenstein et al., 2004). The studies of natural Tregs in autoimmune diseases not only shed lights on the pathogenesis of autoimmune diseases, but also provided novel strategies for the treatment of autoimmune diseases. In multiple diseases, transfer of ex vivo purified 34 natural Tregs before disease induction was able to prevent autoimmune diseases (Kohm et al., 2002). For example, transfer of natural Tregs to mice with established colitis could prevent disease progression and lead to cure of the disease (Mottet et al., 2003). Although transfer of natural Tregs is an effective therapy for autoimmune diseases experimentally, there are obstacles for this to be applied clinically. First, as mentioned above, since the functions of the natural Tregs in autoimmune diseases are defective, infusion of autologous natural Tregs may not be effective in controlling the diseases. Second, the truly functional natural Tregs consist of only about 1-2% of the peripheral CD4+ T cells. This small number of cells can be of any therapeutic value only when they are expanded in vitro, a labor-intensive task. Thus, in the face of obstacles for using natural Tregs to treat autoimmune diseases, the use of antigen-induced Tregs seems to be more attractive. Oral tolerance and T cell vaccination, which'are known to induce TGF-P-producing Th3 cells (Chen et al., 1994) and Qa-1-restricted C D 8 Tregs respectively, have been shown to control autoimmune diseases experimentally (Jiang et a l , 1998). These studies did lead to clinical trials with some success (Achiron et al., 2004; Correale et al., 2000). Although natural Tregs isolated from patients with autoimmune disease may not be of therapeutic value, Tregs generated against other model antigens from the whole T cell repertoire in vitro still can suppress autoimmune disease via bystander inhibition as long as these Tregs are activated in vivo by their cognate antigens (Groux et al., 1997; L i et al., 2006; Stohlman et a l , 1999). A typical example is the transfer of T r l cells raised against O V A which suppressed colitis when the mice were fed O V A to activate these T r l cells in vivo (Groux et al., 1997). Therefore, use of Tregs to control 35 autoimmune diseases has potential therapeutic value, although a lot of work is still required. 1.1.5.2. Transplantation Solid organ transplantation and hematopoietic stem cell transplantation are used to treat multiple diseases. However, graft rejection and G V H D remain major obstacles. Although the use of powerful immunosuppressive drugs has partially solved the problem, induction of tolerance to transplantation has been regarded as a more desirable solution. Research in Tregs has generated great impact on the field of transplantation in recent years. First, studies have shown that alloantigen-specific Tregs can be induced to mediate transplantation tolerance in experimental models, such as anti-CD4 antibody infusion (Qin et al., 1993), co-stimulation blockade (Taylor et al., 2002a) and transfer of dendritic cells with regulatory functions (Morris et al., 2004). In patients who have received transplanted organs and withdrawn from immunosuppressive drugs, a tolerant state was associated with the generation of donor-specific Tregs (Waaga et al., 2001). These studies highlighted the importance of Tregs in the control of immune tolerance to alloantigens. Second, studies on the effects of immunosuppressive drugs on Tregs revealed important clinically relevant findings. Certain immunosuppressive drugs may have differential effects on Tregs. Although cyclosporine A is the most used immunosuppressive drug, it works by interfering with the IL-2 pathway, which is also required by natural Tregs. Given the role of natural Tregs in transplantation, use of cyclosporine A may not have favorable effects in some circumstances (Segundo et al., 2006; Zeiser et al., 2006). Thus, there is a need to 36 consider how to optimize the use of this powerful drug. On the other hand, it was also found that certain types of immunosuppressive drug might preferentially preserve the functions of Tregs, and these drugs should be further explored to maximize their effects (Battaglia et al., 2005; Coenen et al., 2006; Segundo et al., 2006; Zeiser et al., 2006). Third, the application of Tregs in the control of transplantation rejection may revolutionize the therapy for this disease. In addition to these use of Tregs from the whole T cell repertoire without in vitro manipulation, the use of natural Tregs also provides further opportunities for treatment. It has been shown that alloantigen-specific Tregs can be generated in vitro by stimulating isolated natural Tregs with alloantigens and further expanded using cytokines favoring their growth, such as IL-2. Transfer of these alloantigen-specific natural Tregs could lead to permanent graft acceptance (Nishimura et al., 2004). More importantly, unlike the effects of immunosuppressive drugs which are non-specific and may also blunt favorable immune responses, such as immune responses to infections and tumors, regulation by Tregs allows more favorable immune responses to occur. Several studies showed that transfer of donor-derived natural Tregs to recipients suppressed a G V H D while allowing the graft-vs-tumor ( G V T ) effects to be preserved in murine allogeneic hematopoietic stem cell transplantation models. This w i l l definitely enable allogeneic hematopoietic stem cell transplantation to be more widely used to treat solid or hematopoietic malignancies (Edinger et al., 2003; Jones et al., 2003; Trenado et al., 2003). Furthermore, Kar im et al showed that natural Tregs primed by a model antigen could mediate active suppression of transplantation rejection of skin grafts via bystander inhibition, providing another novel strategy for using Tregs to control transplantation rejection (Karim et al., 2005). 37 Hematopoietic stem cell transplantation (HSCT) was first used as a rescue following high dose chemotherapy and irradiation therapy for hematopoietic malignancies since these treatments cause hematopoietic failure. However, after about three decades of research, it was found that the curative effects of H S C T were not due to the ability to rescue hematopoietic failure. Experimentally, murine studies demonstrated that transfer of allogeneic immune cells could eradicate leukemia cells (Truitt, 2004). In clinical settings, it was observed that H S C T between twins and removal of T cells in the graft had higher recurrence rate than allogeneic H S C T ( A H S C T ) with T cell-replete graft, suggesting that the curative effects of A H S C T was due to the immune responses directed against alloantigens mediated by donor T cells (Horowitz et al., 1990). This notion was finally confirmed when donor lymphocytes infused into recipients with recurrent leukemia following H S C T successfully induced disease regression (Kolb et al., 1990; Kolb et al., 1995). Currently A H S C T has been regarded as the most powerful and well-characterized immunotherapy against hematopoietic malignancies, whose anti-tumor effects have been termed graft-vs-tumor ( G V T ) effects (Fowler, 2006; Ko lb et al., 2004) . Realizing its potency against hematopoietic malignancies, A H S C T has been successfully used to treat various solid tumors in recent years (Childs et al., 2000; Talmadge, 2003). However, the great benefits of G V T effects have been seriously undermined by concurrent graft-vs-host disease ( G V H D ) (Kolb et al., 2004; Reddy and Ferrara, 2003). The occurrence of G V H D requires the presence of three conditions: 1) inability of the host to reject the donor graft; 2) a donor graft with immune competent cells; and 3) mismatched transplantation antigens between the donor and the host. These 38 conditions are frequently seen in conditioned recipients of A H S C T (Devetten and Vose, 2004). Thus, G V H D occurs most frequently in the setting of A H S C T . Based on the pathophysiology and clinical manifestation, there are two types graft-vs-host diseases, acute G V H D and chronic G V H D . While chronic G V H D happens generally 100 days after bone marrow transplantation and manifests as an autoimmune disease-like syndrome, acute G V H D happens within 100 days after transplantation and causes a very high mortality rate. a G V H D manifests as an inflammatory process with acute onset that affects multiple target organs, including the skin, liver, gut and lungs. Studies in the past decade have gained insightful understandings of this life-threatening disease. Before transplantation, the potent conditioning measures, such as total body irradiation and high dose chemotherapy, cause tissue damage, to the gut in particular, and lead to endotoxin translocation from the gut. After transfusion of a donor graft, T cells in the graft are activated by alloantigens presented by the host A P C s . This response can be greatly potentiated by the presence of endotoxin released into the systemic circulation. Then, activated T cells in turn develop into a T h l response and induce high levels of proinflammatory cytokines, including I L - l p Y L F N - y and T N F - a , leading to the so-called "cytokine storm". This further results in damage of the gut that leads to more endotoxin translocation, forming a positive feedback amplification loop. The activated T cells w i l l then home to and infiltrate multiple target organs, inducing tissue damage that finally gives rise to failure of the target organs (Devetten and Vose, 2004; Jaksch and Mattsson, 2005; Reddy and Ferrara, 2003). During the priming of donor T cells, memory T cells w i l l be generated and are responsible for the persistence 39 of the disease (Zhang et al., 2005a; Zhang et al., 2005b). The use of powerful immunosuppressants has been effective to some degree in preventing or treating a G V H D . However, this treatment can also lead to serious problems such as disease recurrence, infection and toxicity. After it was demonstrated that T cells were responsible for a G V H D , T cell depletion via antibody was used for the prevention of a G V H D , and has been highly successful. However, due to the loss of T cells that mediate graft-vs-tumor and anti-infection effects, disease recurrence and serious infections make this treatment less useful (Goker et al., 2001). A s the conditioning-induced damage plays a critical role in the pathogenesis of a G V H D , conditioning measures o f reduced intensity have been developed to prevent a G V H D . However, a G V H D still remains the most serious complication despite some success (Slavin et al., 2002). The identification of natural Tregs has prompted the development of novel approaches against a G V H D . Several groups investigated the use of donor derived natural Tregs to prevent a G V H D . They found that mouse natural Tregs not only showed low responses to alloantigen stimulation, but also suppressed the responses of naive T cells to alloantigens in vitro. More importantly, they did not mediate a G V H D in vivo, but suppressed the ability of naive T cells to mediate a G V H D when co-transferred with naive T cells to irradiated recipients (Cohen et al., 2002; Edinger et al., 2003; Hoffmann et a l , 2002; Jones et al., 2003; Taylor et al., 2001; Taylor et al., 2002b; Trenado et al., 2003; Trenado et al., 2006). In addition, several groups found that suppression of a G V H D by natural Tregs did not affect the G V T responses, making it a potentially very attractive form of therapy clinically (Edinger et al., 2003; Jones et al., 40 2003; Trenado et a l , 2003). Based on the report that the suppressive functions of natural Tregs were enhanced when these Tregs were activated (Thornton and Shevach, 1998), Taylor et al showed that natural Tregs stimulated and expanded in vitro still retained their suppressive activities on a G V H D in vivo (Taylor et al., 2002b). Trenado et al also developed an in vitro expansion system, where they stimulated natural Tregs with allogeneic A P C s in the presence of exogenous IL-2 to generate alloantigen-specific natural Tregs. They reported that this approach could expand natural Tregs by 100-fold in 30 days. These expanded alloantigen-specific natural Tregs had more superior suppressive activities over a G V H D as compared to the natural Tregs polyclonally expanded with anti-CD3 and anti-CD28 microbeads, and lead to long term survival of the recipients. Furthermore, co-transplant of these alloantigen-primed natural Tregs preserved G V T effects and led to accelerated immune reconstitution (Trenado et al., 2003; Trenado et al., 2006). These studies have important implications for the use of natural Tregs clinically. For natural Tregs present in human peripheral blood, only 1% of the CD4+CD25+ T cells are truly suppressive (Baecher-Allan et al., 2001). Based on the murine studies above, suppression of a G V H D in vivo by natural Tregs was observed when the ratio of naive T cells to natural Tregs was 2:1 or 1:1, meaning that the number of natural Tregs should be at least half of that of the naive T cells. This is a great challenge due to the scarcity of the truly suppressive natural Tregs. However, the studies mentioned above did show the feasibility of expanding natural Tregs in vitro to the desired number. Moreover, it has also been shown that human natural Tregs indeed can be expanded in large scale (Hoffmann et al., 2004). Currently, there is an ongoing clinical trial to 41 investigate i f expanded human natural Tregs can be used to suppress a G V H D in A H S C T (Lechler et al., 2005). Tregs generated in other systems were also shown to inhibit a G V H D in murine studies. Blazar's group demonstrated that natural Tregs-depleted C D 2 5 - conventional T cells became hyporesponsive to alloantigens and had a greatly decreased ability to mediate a G V H D in vivo after they were stimulated with alloantigens in the presence of exogenous IL-10 and TGF - P in vitro. More importantly, these cells became Tregs as they suppressed a G V H D mediated by naive T cells when they were co-transferred with naive T cells (Chen et al., 2003b; Zeller et al., 1999). They also showed in an in vitro mixed lymophocyte reaction system that blockade of CD40 and C D 4 0 L interactions could also lead to hyporesponsiveness to alloantigens by donor T cells with suppressive activities on a G V H D in vivo (Taylor et al., 2002a). However, these studies did not lead to a clinical trial. One possible reason is the anergic state of the generated Tregs which makes it extremely difficult to obtain sufficient number of Tregs to be used clinically. Th2 cells have been known to suppress the responses mediated by T h l cells. Thus Th2 cells were also studied for their ability to suppress a G V H D which was regarded as a pathogenic T h l response. E x vivo generated Th2 cells were demonstrated to suppress a G V H D in vivo while not affecting the G V T responses (Foley et al., 2005). A clinical trial has been performed in a very small cohort o f patients, making it difficult to draw any conclusions at the present time and its efficacy still needs to be further tested. Furthermore, it was reported that C D 4 - C D 8 - T cells were also able to inhibit a G V H D (Young et al., 2003). However, due to the scarcity of this cell type, 42 these cells may be of very little clinical importance. In addition to the suppression mediated by conventional NKl.rapT cells, N K l . l + a p T cells were also shown to inhibit a G V H D . Strober et al identified a cell population in the mouse bone marrow that was able to suppress mixed lympohocyte reaction in vivo about two decades ago (Strober et al., 1987). Later, Strober's group further demonstrated that donor derived bone marrow N K T cells were able to suppress a G V H D when co-transferred with naive donor T cells in an IL-4 dependent manner (Zeng et al., 1999). Despite their suppressive activities on a G V H D , it is not feasible to enrich and harvest donor derived bone marrow N K T cells for transplantation. On the other hand, the host N K T cells were also shown to regulate a G V H D i f they were properly activated (Hashimoto et al., 2005). Thus Strober's group developed a conditioning approach that specifically enriched the host N K T cells (Lan et al., 2001; Lan et al., 2003). The approach included multiple doses of total lymphoid irradiation plus antibody-mediated T cell depletion. After this conditioning, the dominant cell population in the lymphoid organs was N K T cells. a G V H D was totally blocked when naive donor T cells were transferred to these recipients while the same number of naive T cells caused severe lethal a G V H D when transferred to recipients receiving total body irradiation as conditioning. They showed in a murine a G V H D model that one hundred times more donor T cells were required to mediate lethal a G V H D in recipients conditioned by multiple total lymphoid irradiation and antibody-mediated T cell depletion as compared to mice receiving conventional total body irradiation conditioning. The lack of a G V H D was due to the dominant suppression by N K T cells enriched by the conditioning approach since CD1 knockout mice recipients lacking 43 N K T cells were not protected against a G V H D . The suppression was dependent on IL-4 as IL-4 knockout mice recipients were not protected either. In addition, suppression of a G V H D did not interfere with the G V T responses. More importantly, the results of this approach was highly reproducible in humans and very promising clinical data have been reported using this approach to prevent a G V H D (Lowsky et al., 2005). Collectively, these studies show that suppression of a G V H D by Tregs may be a potential solution for a G V H D which remains the main barrier for the wide clinical use of A H S C T to treat multiple diseases. 1.1.5.3. Infections and malignant tumors Although the immune system has evolved to develop many powerful measures to successfully fight infections caused by myriads of microorganisms, immune evasion by pathogens is still common. Recently, much attention has been paid to the role of Tregs in the immune evasion by pathogens. Many pathogens can block the immune attack by inducing Tregs. Recent experimental studies have accumulated ample evidence showing that effective immune response against some pathogens could be blunted by pathogen-induced Tregs (Belkaid et a l , 2006; Lavelle et al., 2003; Marshall et al., 2003; McGui rk et al., 2002; Rouse et al., 2006). In support o f these experimental studies, virus specific Tregs have been found to be present clinically in patients infected with different viruses that suppressed the T h l responses to the viruses (Bolacchi et al., 2006; Franzese et al., 2005; Rushbrook et al., 2005; X u et al., 2006). The presence of natural Tregs is another important factor that favors the persistence of pathogens. Belkaid et al first demonstrated that natural Tregs were responsible for the 44 persistence of intracellular pathogens (Belkaid et al., 2002) and removal of natural Tregs could result in the enhanced immune response against these pathogens (Rad et al., 2006). Similar to microbial infections, Tregs also play a critical role in the immune evasion of malignant tumors. Malignant tumors have developed effective ways, such as production of immunosuppressive cytokines IL-10 or TGF - P , to induce tumor antigen-reactive T cells to become Tregs, favoring the persistence of tumor cells (Gajewski et al., 2006; Munn and Mellor , 2006). Furthermore, the presence of natural Tregs likely suppresses the overall responses to malignant tumors, as natural Tregs have also been shown to suppress C D 4 and C D 8 T cells, N K cells and N K T cells, which may play a role in mediating tumor immunity. The important role of natural Tregs in the control of tumor immunity is demonstrated by prolonged tumor survival in the absence of this population (Shimizu et al., 1999). Elucidating the roles of Tregs in the pathogenesis of infections and malignant tumors has great impact on developing more effective therapies against them. In contrast to their role in the therapy of autoimmune diseases and transplantation, Tregs should be deleted to allow more powerful anti-infection and anti-tumor effects in most cases. Depletion of Tregs has been shown to induce effective anti-tumor (Shimizu et al., 1999; Turk et al., 2004; Vieh l et al., 2006) and anti-infection immunity (Mendez et al., 2004; Taylor et a l , 2005) in experimental studies. Induction of lymphopenia was shown to greatly enhance tumor immunity. One of the mechanisms which has been proposed is the removal of natural Tregs (Gattinoni et al., 2006). Vaccination is an effective way to prevent infections. Multiple groups have shown that elimination of 45 natural Tregs prior to immunization effectively enhanced the efficacy of vaccination against multiple infectious pathogens (Moore et al., 2005; Stober et a l , 2005; Tabbara et al., 2005; Toka et al., 2004). O f note, although vaccination with tumor antigens works well in animal models with malignant tumors, to date it has not been remarkably effective in human subjects. Possibly, the presence of natural Tregs may limit the effectiveness of these strategies as their removal can greatly potentiate the efficacy of vaccination against malignant tumors (Antony et al., 2005; Kudo-Saito et al., 2005). 1.2. Superantigens 1.2.1. Definition and categories o f superantigens For nominal antigens to be recognized by T cells, they must be taken up and processed by A P C s . The antigen-derived peptide w i l l bind to M H C molecules and are presented to T cells v ia interactions with the T cell receptor. Generally, only one in 10 4-10 5 T cells w i l l be activated as a result. However, this process does not apply to superantigens which are defined by their unique interactions with T cells. The term superantigen was first coined by Marrack et al to define their ability to directly crosslink the M H C class II molecules and the T cell receptor in a V P specific manner and activate the T cells without the requirement of prior uptake and processing by A P C s . Thus, due to their relatively non-selective actions as compared to the nominal antigens, superantigens can activate 5-30% of the T cell population dependent on the size of a certain V P family of T cells (White J et al., 1989). According to the sources of superantigens, they can be divided into bacterial and viral superantigens. The gram-positive Staphylococcus aureus is one of the major 46 microorganisms producing bacterial superantigens. V i r a l superantigens derived from the endogenous mouse mammary tumor viruses were the best characterized viral superantigens (Leung et al., 1997). Additionally, based on the target cells, superantigens can also be divided into T cell superantigens and B cell superantigens. The definition above also applies to B cell superantigens since they are able to bind to the conserved region of the antibody molecule and activate polyclonal B cells (Goodyear and Silverman, 2003; Viau and Zouali, 2005). This action is different from the interactions of nominal antigens and antibodies, where antigen recognition involves the complementarity determining regions of the antibody. It should be noted that bacterial T cell superantigens are the main focus for discussion in this thesis. 1.2.2. Biological effects of superantigens A l l superantigens possess the ability to activate a large number of T cells or B cells. In vitro, stimulation of human or murine naive T cells with bacterial superantigens leads to massive proliferation and production of T h l cytokines, such as IL-2, T N F - a , IFN-y (Arad et a l , 2001; Dauwalder et a l , 2006; K u m et al., 2001). Costimulation is required for the activation of T cells by superantigens (Kum W W et al., 2006; Saha et a l , 1996a; Saha et al., 1996b). This activation process in turn results in the deletion via apoptosis or anergy of superantigen-reactive T cells (Kawabe and Ochi, 1991; MacDonald et al., 1991; Rellahan et a l , 1990; White et a l , 1989). CD4+ T cells are the main cell type responding to superantigen stimulation, although CD8+ T cells can also be activated by superantigens (Sabapathy et al., 1994). It is thought that the activation o f T cells by superantigens plays an important role in the pathogenesis of 47 toxic shock syndrome, both in human cases and in animal models of toxic shock syndrome. Suppression of antibodies against superantigens has been documented, which has been thought to be the result of a preferential T h l polarization effect o f superantigens (Leung et al., 1997). The second biological activity of superantigens is their ability to enhance endotoxin-induced lethal shock (Dinges et al., 2000; Leung et al., 1997). It has been shown in mice and rabbits that injection of bacterial superantigens could greatly enhance the response caused by endotoxin by up to 50,000 fold. This enhancement has been hypothesized to be the direct induction of hypersensitivity to endotoxin or indirect effects of decreasing the clearance of enodotoxin. Bacterial superantigens can also cause emesis in clinical settings, which is characteristic of S E A and S E B , the causative agents for food poisoning. However, the mechanism for this biological effect is unknown (Dinges et al., 2000; Leung et al., 1997). In addition, superantigens are able to directly interact with epithelial cells and activate them. This interaction involves receptors other than M H C Class II molecules and the T cell receptor, but the precise nature is elusive at the present time. The effects of superantigens on epithelial cells have also been implicated in the pathogenesis of toxic shock syndrome. 1.2.3. Staphylococcal superantigens Staphylococcus aureus produces various exoproteins that include toxic shock syndrome toxin-1 (TSST-1), the staphylococcal enterotoxins (SEA, S E B , S E C n , S E D , S E E , S E G , S E H , and SEI), the exfoliative toxin A and B ( E T A and E T B ) , and 48 leukocidin. These proteins play an important role in the pathogenesis of Staphylococcus aureus infections. TSST-1 and the staphylococcal enterotoxins are also called pyrogenic toxin superantigens (PTSAgs) due to their superantigenic properties (Dinges et a l , 2000). 1.2.3.1. TSST-1 Encoded by tstH present on the bacterial chromosome within a 15.2-kb mobile genetic element called staphylococcal pathogenicity island 1, the mature TSST-1 is a single polypeptide chain with a molecular weight of 22,000 daltons. TSST-1 is highly soluble in water, although it contains a high percentage of hydrophobic amino acids. TSST-1 is resistant to heat and proteolysis as can be shown by the fact that it can be boiled for more than 1 h without detectable loss of biological activity, and it is not cleaved after prolonged exposure to trypsin (Dinges et al., 2000). TSST-1 is antigenically distinct from other PTSAgs and does not have significant primary sequence homology to other known proteins, including other P T S A g s (Dinges et al., 2000). TSST-1 was first identified as the causative agent of toxic shock syndrome associated with menstruation and use of tampons. TSST-1 reacts with Vp2 of human T cells (Proft and Fraser, 2003). In B 1 0 . B R mice, it has been reported that TSST-1 primarily stimulated VP 15 (Callahan et al., 1990). A s compared to other PTSAgs , TSST-1 possesses the unique ability to cross vaginal mucosal surfaces and is the only PTSAgs able to reactivate bacterial cell-wall induced arthritis. However, unlike the staphylococcal enterotoxins, TSST-1 does not possess the ability to cause emesis 49 (Dinges et al., 2000). Structurally, TSST-1 consists of two adjacent domains. Domain A (residues 1 to 17 and 90-194) contains a long central a-helix (residue 125-140) surrounded by a five-strand P-sheet. Domain B (residue 18-89) consists of five P strands forming a barrel motif (Acharya et al., 1994; Prasad GS et al., 1993). One study demonstrated a zinc-binding site in the TSST-1 molecule which might potentiate superantigenicity at low toxin concentration (Prasad et al., 1997) although the binding of TSST-1 to M H C Class II molecules is Zinc-independent. Crosslinking of the T cell receptor and M H C Class II molecules is required for the superantigenic activities of TSST-1 , as demonstrated by mutational studies. Hurley et al first showed that the binding of TSST-1 to the T cell receptor required the central a-helix, and an a-helix at the N -terminal (Hurley et al., 1995). Studies in our laboratory identified several important binding sites of TSST-1 to the M H C Class II molecules (Kum W W et al., 1996; K u m W W et a l , 2000) . The interaction of TSST-1 with M H C Class II molecule and the T cell receptor is illustrated in Figure 1.2-A. 1.2.3.2. S E A and other staphylococcal enterotoxins Staphylococcal enterotoxins consist of a family of structurally related exotoxins produced by certain Staphylococcus aureus bacterial strains. They were first recognized as agents causing food poisoning and can be divided into serological types, S E A , S E B , SECi-3, S E D S E E , S E H and SEI. The mature form of S E A consists of 233 amino acids with a molecular weight of 2 3 K D (Dinges et a l , 2000). S E A stimulates a wide range of V P families of human T cells, including V p i . l , 5.3, 6.3, 6.4, 6.9, 7.3, 7.4, 50 9.1, and 18 (Proft and Fraser, 2003). In B 1 0 . B R mice, S E A was found to react with V p 1, 3, and 11 families o f T cells (Callahan, 1990) S E A is structurally similar to TSST-1 . However, differences are also noted. High affinity binding of S E A to M H C Class II molecules is Zinc-dependent (Schad et a l , 1995) and a number of studies suggested that S E A had two binding sites on M H C Class II molecules, as S E A competed with TSST-1 in binding H L A - D R 1 while TSST-I did not inhibit S E A binding (Hudson et al., 1995; Thibodeau et al., 1994). S E A is the most potent superantigen among the staphylococcal enterotoxins. Recent data suggested that this might be due to its stronger Zinc-dependent binding to M H C class II molecules (Pless et al., 2005). The inferred binding of S E A to M H C Class II molecule is shown in Figure 1.2-B. 1.2.4. Role of bacterial superantigens in diseases Superantigens have been implicated in multiple human diseases. Staphylococcal enterotoxins are involved in food poisoning due to their ability to cause emesis. Toxic shock syndrome (TSS) is characterized clinically by hypotension, diffuse erythematous rash, generalized edema and lymphocytopenia. TSST-1 is the dominant toxin implicated in TSS, menstrual cases in particular, and other PTSAgs are important in non-menstrual TSS. The superantigenic activities of these toxins cause massive polyclonal activation of T cells followed by massive production of proinflammatory cytokines that in turn result in severe hypotension, hyperthermia, and the devastating dysregulation of multiple physiological systems and organs, leading to life-threatening multi-organ failure. The pathogenicity of TSST-1 is also enhanced by 51 its unique ability to cross mucosal surfaces (Dinges et al., 2000; McCormick et al., 2001). Other contributing factors of superantigens to the initiation and progression of TSS include the direct activation of the innate immune system via their interactions with epithelial cells (Watson et al., 2005) and ability to potentiate the effects of endotoxin (Blank C et al., 1997). TSST-1 ___ Figure 1.2. Diagram of binding of superantigens to their ligands. A . A modeled tri-molecular complex composed of a TSST-1, M H C Class II molecule and T cell receptor (Dinges et al., 2000). B . Interactions of two S E A molecules with two M H C Class II molecules inferred from mutation studies (Fraser et al., 2000). 52 Due to their ability to activate polyclonal T cells which may include the autoantigen-reactive T cells, superantigens have long been implicated in the pathogenesis of autoimmune diseases. On the one hand, clinical and experimental data showed that selective expansion of VP family of T cells has been detected in patients with various autoimmune disease, suggesting the exposure of pathogen-derived superantigens, although direct identification of pathogen-derived superantigens in these studies was not successful (Conrad et al., 1994; Paliard et al., 1991). One study suggested that superantigens derived from human endogenous retroviruses could be a possible cause of type I diabetes (Conrad et al., 1997). Further evidence was accumulated by studies showing that timely injection of superantigens caused activation or acceleration of autoimmune diseases in multiple animal models (Brocke et al., 1993; Kageyama et al., 2001; Schiffenbauer et al., 1993; Schwab et al., 1993; Soos et al., 1995). In addition to the direct activation of autoantigen-reactive T cells, the proinflammatory responses elicited by superantigens may indirectly facilitate the activation of autoreactive T cells; thus leading to the initiation or progression of autoimmune diseases. On the other hand, the ability of superantigens to mediate deletion of T cells also provides a therapeutic strategy for the treatment of autoimmune diseases. A s the superantigen-reactive T cells overlapped with autoreactive T cell clones that mediate autoimmune diseases, it has been suggested that administration of superantigens could actually attenuate autoimmune diseases. This has also been well proven in various animal models of autoimmune disease (Kawamura et al., 1993; K i m et al., 1991; Prabhu Das et al., 1996; Soos et al., 1993). The timing o f superantigen administration was critical in generating the therapeutic effects. The same concept has 53 also been demonstrated using B cell superantigens in autoimmune disease models where B cells play an important role (Viau and Zouali, 2005). The superior ability of superantigens to induce activation of the immune system is also favorable for the treatment of malignant tumors where tolerance to the progressing tumors frequently occurs. Stimulation of T cells in draining lymph nodes of mice bearing tumors could overcome the deficient function of T cells, leading to tumor-specific effector T cells that were able to mediate tumor regression (Shu et al., 1994). Injection of superantigens to mice could confer protection against tumor challenge or induce regression of established tumors in mice (Torres et al., 2001). Similarly, superantigens were able to enhance the efficacy o f other immunotherapeutic measures against malignant tumors (Gidlof et al., 1997; Pulaski et al., 2000; Wahlsten et al., 1998). A recent clinical trial showed that injection of bacterial superantigen had favorable effects on pleural effusions caused by non-small cell .lung cancer and prolonged the survival of patients (Ren et al., 2004). More studies are required to further exploit the beneficial effects of superantigens on the therapy against malignant tumors. 1.3. Connections of bacterial superantigens and Tregs Research on superantigens related to their role in diseases and in particular their interactions with T cells, has contributed much to our understanding of the mechanisms of immune tolerance. After the intrathymic deletion of immature self-reactive T cells was conclusively shown in the 80's, the mechanisms of induction of peripheral tolerance in mature T cells were still poorly understood. Studies in the late 80's and 54 early 90's using superantigens as a paradigm for tolerizing antigens shed new light into this field. Exposure of T cells to bacterial superantigens, both in vivo and in vitro, could result in the clonal expansion of the responding T cells bearing specific VP segments followed by the selective clonal deletion of a large fraction o f the responding T cells mediated by apoptosis. The remaining T cells showed an anergic state in response to superantigen stimulation (Kawabe and Ochi, 1991; MacDonald et al., 1991; Rellahan et al., 1990; White et al., 1989). This was the first direct evidence showing recessive mechanisms played a role in the peripheral immune tolerance to antigens. These findings supported the hypothesis that peripheral tolerance to autoantigens that were not expressed in the thymus during negative selection was in part mediated by clonal deletion or induction of anergy of autoreactive T cell clones. Later, some studies showed in other experimental systems that anergic T cells actually could mediate active suppression of responses of naive T cells, suggesting that immune tolerance could also be induced or maintained by T cell-mediated dominant mechanisms. This concept at that time was still controversial, although the idea of regulating immune responses by T cells with suppressive functions had been first raised about two decades before (Diaz-Gallo et al., 1992; Lombardi G et a l , 1994). However, in the middle 90's, a series of studies conducted by Sakaguchi et al. convincingly demonstrated that peripheral tolerance to autoantigens indeed was maintained by a dominant mechanism mediated by a subset o f CD4+ T cells co-expressing the a unit of the IL-2 receptor CD25 (Asano et al., 1996; Sakaguchi et al., 1995). 55 Studies on the induction of Tregs by superantigens were first performed by Sundstedt et al who showed that repeated injection of S E A to mice induced high levels of IL-10 and low level of proinflammatory cytokines IL-2, IFN-y and T N F - a in the serum. Furthermore, enhanced IL-10 production was actually responsible for the decreased levels of IFN-y and T N F - a , but not IL-2. Depletion studies showed that CD4+ T cells were responsible for the high production of IL-10. Collectively, these data suggested that repeated administration of a bacterial superantigen could induce a subset of CD4+ T cells with regulatory functions (Sundstedt et al., 1997). Following this study, consecutive reports confirmed that repeated injection of low to moderate doses of superantigens induced Tregs. CD4+ T cells from mice treated with repeated injection of superantigens not only changed their cytokine production profile and proliferative state, but also developed active suppressive functions on the responses of naive cells to the superantigen stimulation (Feunou et al., 2003; Grundstrom et al., 2003; Noel et al., 2001). For example, T cells from mice injected repeatedly with S E A or S E B produced lower levels of IL-2 and IFN-y, but increased level of IL-10. When co-cultured with naive splenocytes in the presence of superantigen stimulation, these superantigen-primed T cells suppressed the ability of naive splenocytes to proliferate and to produce IL-2 and IFN-y (Noel et al., 2001). These studies collectively suggested that in addition to clonal deletion and anergy, a dominant mechanism mediated by superantigen-induced Tregs existed for the control of immune responses to superantigens. 56 Chapter 2: Hypothesis and specific aims M y studies focused on the study of Tregs induced by the staphylococcal superantigen known as toxin shock syndrome toxin-1 (TSST-1). Previously, studies in our laboratory showed that repeated injection of TSST-1 was able to induce Tregs in B A L B / c mice. However, most of the basic features of the TSST-l - induced Tregs were unclear, such as the specificity and mechanism of action of their suppressive activities, their capacity to proliferate and produce cytokines, and their susceptibility to apoptosis. The unifying hypothesis for the present project is: TSST-1 induces Tregs with distinctive properties Surrounding this hypothesis, the specific aims were: • To determine whether repeated injection of TSST-1 can induce regulatory T cells in C57BL/6 (B6) mice. Although previous studies in our laboratory showed that repeated injection of TSST-1 could induced Tregs in B A L B / c mice, this finding should be further confirm using B 6 mice. In vitro experiments using a co-culture system and in vivo studies using a lethal shock model would be performed to assay the suppressive functions of TSST-1-primed CD4+ T cells. • To elucidate the differences of Tregs induced by TSST-1 and S E A . It is not clear whether TSST-l- induced Tregs differ from those induced by S E A . A side-by-side comparison of Tregs induced by TSST-1 and S E A would be performed. These Tregs would be compared for their cytokine profile (cytokine secretion and intracellular expression), proliferation response, cell 57 death, markers (surface and intracellular), and suppressive activities (in vitro and in vivo). To elucidate the mechanisms of TSST-l- induced Tregs. The mechanisms of TSST-l- induced Tregs were unclear and required further investigation. Studies would be performed to determine whether TSST-l- induced Tregs could induce cell death or inhibit the activation of their target cells. The role of infectious tolerance, soluble factors and cytokine competition in the suppression mediated by TSST-l- induced Tregs would also be studied. To investigate the potential clinical application of TSST-l- induced Tregs. Acute graft-vs-host disease ( a G V H D ) is the major complication following allogeneic bone marrow transplantation or transplantation of organs containing lymphoid tissues, such as the small intestine. Triggered by the mismatched alloantigens expressed by the recipients, a G V H D is the major barrier for the wide use of allogeneic bone marrow transplantation, which is otherwise the curative therapy for a number of hematological diseases including leukemia (Goker et al., 2001). Multiple recent studies showed that immunotherapy, the use of Tregs in particular, may provide a promising potential solution (Blazar and Taylor, 2005; Goker et al., 2001). Elucidating the effects of TSST-1 induced Tregs on a G V H D w i l l provide a novel therapeutic strategy for a G V H D . The clinical application potential of these cells would be addressed by using a murine a G V H D model. 58 Chapter 3: Methods and materials 3.1. Mice , TSST-1 preparation and TSST-1 treatment Female C57BL/6 (H-2 b) and B6D2F1 (H-2 b / d ) mice, 6-8 weeks old, were purchased from Charles River Laboratories (Wilmington, M A ) and housed at Jack Be l l Research Centre. Recombinant TSST-1 was purified from culture supernatants of Staphylococcus aureus RN4220 containing plasmid pBS(tst) by a combination of preparative isoelectric focusing and chromatofocusing. Our laboratory has previously demonstrated that highly pure TSST-1 preparations can be obtained using this method (Kum et al., 1993). Purity of toxin preparations was demonstrated by silver staining after S D S - P A G E . S E A was purchased from Toxin Technology (Sarasota, F L ) and was further purified by chromato-focusing, using a p H 6-8 gradient polybuffer exchanger. M i c e were subcutaneously injected with 4 pg TSST-1 or S E A in 0.2 m L P B S for three times at the interval of 4 days. Control mice were subcutaneously given the same volume of P B S . Splenocytes or CD4+ T cells from mice treated with TSST-1 , S E A or P B S were isolated 2 hours after the 3 r d injection. 3.2. Ce l l isolation To isolate splenocytes, spleens from naive, TSST-1 , S E A or PBS-treated mice were removed and homogenized to single cell suspension on a cell strainer (BD Pharmingen). Cells were washed with R P M I 1640 culture medium (Stemcell Technologies, Vancouver, B C ) and red cells were removed by hypotonic lysis in Gey's solution for 1 minute. Splenocytes then were washed with R P M I 1640 culture medium twice and resuspended in complete R P M I 1640 culture medium at 8><106/mL. Ce l l 59 viability was determined by typan blue exclusion. C D 4 + T cells were further isolated from the splenocytes by using a magnetic isolation kit according to the product manual (Stemcell Technologies, Vancouver, B C ) and adjusted to 2 x i o 6 / m L for in vitro studies. For studies of TSST-l -pr imed C D 4 + T cells to suppress lethal shock, C D 4 + T cells were adjusted to 5 x l 0 7 /mL. For studies of TSST- l -pr imed C D 4 + T cells to suppress a G V H D , C D 4 + T cells were adjusted to 2 . 5 x l 0 7 / m L . The purity of C D 4 + T cells was greater than 90% as determined by flow cytometry. 3.3. Ce l l transfer To investigate the suppressive effects of superantigen-primed C D 4 + T cells on lethal shock, C D 4 + T cells were isolated from mice repeatedly injected with TSST -1 , S E A or P B S . 2 x l 0 7 o f C D 4 + T cells in 0.4 m L P B S were injected to each mouse via the lateral tail vein. Two hours later, mice were subjected to lethal shock induction and survival was monitored. In some groups, C D 4 + T cell-depleted, splenocytes were recovered and injected into mice at the dose of 8x10 /mouse. 3.4. In vitro culture For co-culture experiments, 4 x l 0 5 / w e l l naive splenocytes were co-cultured with titrated numbers of TSST -1 , S E A or PBS-primed C D 4 + T cells in flat-bottom, 96-well plates in the presence of TSST-1 or S E A (1 nM) . Cells were cultured at 37°C, 5% C 0 2 in 200 uL R P M I 1640 medium supplemented with 10% F B S (Hyclone, Logan, U T ) , 50 u M 2-mercaptoethanol ( 2 - M E ; Sigma, St Louis, M O ) , 10 m M H E P E S buffer, I m M sodium pyruvate, antibiotics [100 U / m L penicillin; 100 mg/mL streptomycin (Stemcell 60 Technologies, Vancouver, B C ) ; and 2 u,g/mL polymycin B (Sigma, St Louis, MO) ] . Supernatant was collected after 48 hours of culture. In some experiments, TSST -1 , S E A or P B S primed splenocytes ( 4 x l 0 5 /well) were also cultured under these conditions. Supernatant was collected at different time points post-stimulation. Supernatant was stored at -70°C until assay for various cytokines by E L I S A . 3.5. C F S E staining and proliferation assay To label cells with C F S E , splenocytes or C D 4 + T cells ( 5 x l 0 6 / m L ) were stained with 5 n M C F S E (Molecular Probes, Eugene, OR) in P B S at 37°C for 10 minutes. Cells were washed with cold R P M I 1640 medium for three times and then resuspended in completed R P M I 1640 medium containing 10% of F B S for 30 minute. To assay the proliferation o f superantigen-primed C D 4 + T cells, 4 x l 0 5 /well CFSE-labeled splenocytes from mice treated with TSST-1 or S E A were plated in flat-bottom, 96-well plates and cultured at 37°C, 5% CO2 in 200 uL complete R P M I 1640 culture medium in the absence or presence of TSST-1 or S E A . A t different time points, cells were harvested, washed and stained with ant i -CD4 antibody. Then, flow cytometry analysis was performed to determine the proliferation of C D 4 + T cells. Degree of proliferation of C D 4 + T cells was calculated as follows: [numbers of proliferated C F S E and C D 4 positive cells (low C F S E staining cells)] / [total C F S E and C D 4 positive cells] x 100%. To determine the effects of superantigen-primed C D 4 + T cells on the proliferation of naive splenocytes, 4 x 1 0 5 / w e l l CFSE-labeled nai've splenocytes were co-cultured with titrated numbers of TSST -1 , S E A or PBS-primed C D 4 + T cells in flat-bottom, 96-well plates in the presence of TSST-1 or S E A (1 nM) as described in the co-culture 61 experiments. 72 hours later, cells were harvested and washed twice with P B S followed by flow cytometry analysis of proliferation of the naive splenocytes. Degree of proliferation of naive splenocytes was calculated as follows: [numbers o f proliferated C F S E positive naive splenocytes (low C F S E staining cells)] / [total C F S E positive naive splenocytes] x 100%. 3.6. Assay of cell death To assay cell death, cells were suspended in P B S and stained with 7 - A A D (Sigma, St Louis, M O ) at the concentration of 4ug/mL for 10 minutes at 4°C. Cells then were washed twice with P B S and were analyzed by flow cytometry. 3.7. Assay of cytokine production Supernatants from in vitro cell cultures were tested for the presence of IL-2, IL-12 (p70) and IL-10 by specific E L I S A , according to instructions from the manufacturer (BD Pharmingen). T N F - a and IFN-y were measured by E L I S A also according to instructions from the manufacturer (eBioscience, San Diego, C A ) . 3.8. Lethal shock induction A two-hit lethal shock model was established based on a previous published study (Visvanathan et al., 2001). Each mouse was injected intraperitoneally with 20mg of D-galactosamine (Sigma-Aldrich, St Louis, M O ) , 0.01 pg of endotoxin (from E. coli 055:B5, Sigma-Aldrich, St Louis, M O ) and 4 pg of TSST-1 or S E A in 0.2 ml of P B S . The survival of mice was then monitored for 72 hours post-injection. In initial studies, 62 we confirmed that this regimen could induce 100% lethality in na'ive mice within 24 hours post-stimulation. 3.9. Antibodies and flow cytometry The following conjugated monoclonal antibodies were obtained from B D Pharmingen (San Diego, C A ) : F ITC or PE-anti-CD4, P E or APC-ant i-IL-10, F ITC or PE-anti-IFN-y, PE-anti-IL-4, PE-ant i-TNF-a and PE-an t i -CTLA-4 . APC-anti-IL-2 and APC-ant i -CD25 were purchased from Biolegend (San Diego, C A ) . FITC-anti-Foxp3 and biotin-anti-GITR antibody were purchased from eBioscience (San Diego, C A ) . For surface staining, cells were washed with P B S supplemented with 1% F B S and stained for 20 min at 4°C with optimal dilutions of each antibody, washed again, and analyzed. For intracellular staining of cytokines, Golgistop ( B D Pharmingen, San Diego, C A ) was added to the cell culture for the last 5 hours. Then, cells were harvested, washed, stained with anti-CD4 antibody and fixed with cytofix ( B D Pharmingen, San Diego, C A ) . The fixed cells were further peameabilized with Permwash ( B D Pharmingen, San Diego, C A ) and stained with various antibodies at 4°C for 30min. After staining, cells were washed and analyzed. Foxp3 and C T L A - 4 were stained using the intracellular staining methods mentioned above. For apoptosis detection, cells were incubated with 4 ug/mL of 7 - A A D (Sigma-Aldrich, St Louis, M O ) for 20 min at 4°C. To assay the T cell or antigen presenting cell chimerism of B6D2F1 that received B6 splenocytes, cells were washed with P B S supplemented with 1% F B S and stained for 20 min at 4°C with optimal dilutions of PE-ant i -H-2D b (BD Pharmingen, San Diego, C A ) and A P C -anti-mouse C D 4 , APC-anti-mouse C D 8 or APC-anti-mouse M H C class II antibodies. 63 Flow cytometry analysis was performed on a FACSCa l ibu r machine (Becton Dickinson, San Jose, C A ) and data were analyzed using W i n M D I 2.8. 3.10. V p analysis Splenocytes were first stimulated with I n M of TSST-1 or S E A for 3 days, and then they were washed with R P M I 1640 medium twice to remove the superantigens. Cells were then resuspended in complete R P M I 1640 medium at 37 °C, 5% CO2 overnight for the reexpression o f T cell receptor (Makida et al., 1996). The proportion of cells expressing V p 3, 8, 10, 11, 12 or 15 that reacted to either TSST-1 or S E A stimulation were analysed. Although V P 17 had been reported to respond to both TSST-1 and S E A in some mouse strains (Marrack and Kappler, 1990), it has been shown that C57B1/6 mice do not express functional V P 17 (Wade et al., 1988), and hence V P 17 expression was not analysed. Cells were stained with anti-CD4 antibody (BD Pharmingen) and antibody against Vp3 (BD Pharmingen, San Diego, C A ) , 8, 10, 11, or 12 (Cedarlane, Hornby, ON) and analyzed by flow cytometry. Since there was no commercially available antibody against V p i 5 , R T - P C R was used to analyze the VP15 expression. P B S , TSST-1 , or SEA-primed CD4+ T cells were first stimulated by TSST-1 or S E A for three days, then they were isolated magnetically as mentioned above. Total R N A was then isolated from 1><107 superantigen-stimulated CD4+ T cells using the RNeasy Total R N A isolation kit (Qiagen Inc., Mississauga, Ontario) according to the manufacturer's direction. R N A was reverse transcribed using Omniscript reverse transcriptase (Qiagen Inc., Mississauga, Ontario). V p i 5 specific primers were designed using PrimerQuest. software (http://www.idtdna.com/UBC-64 NAPS/Login.aspx) and sequences accessed from the Nucleotide database at N C B I (Accession numbers AE00663, AE00664 and AE00665). Primers were synthesized by Integrated D N A Technologies Inc. (Coralville, IA) and purified by standard desalting. Primer sequence for V(315 was: forward primer (5' to 3'): A G C T T G G T A T C G T C A A T C G C C T C A ; reverse primer (5' to 3'): A C T G T C A G C T T T G A G C C T T C A C C A . 4ul (~118ng) of c D N A was added to each lOOul P C R reaction containing 0.2 u M of each forward and reverse primer, 10X Buffer, 10 m M dNTPs and 2.5 U of Taq D N A polymerase (Qiagen Inc., Mississauga, Ontario). Samples were mixed on ice and a simple hot start was provided by an initial denaturation at 94°C for 3 minutes. This was followed by 4 cycles of 1 minute each at 94°C, 55°C and 72°C, with a final extension of 10 minutes at 72°C and then rest at 4°C. 22.5ul of P C R product, along with 2.5 ul of 10X gel loading solution (Ambion Inc., Austin, T X ) was run on a 2% agarose gel containing ethidium bromide at 100 V , along with a 100 bp D N A ladder (Invitrogen Corporation, Burlington, Ontario) to assess the presence of various V(3 segments. Gels were viewed using AlphaEase F C system (Alpha Innotech) and images were saved and the molecular weight of each band was determined using the same software. The 1-D Mul t i line densitometry analysis tool was used to measure the integrated area of each band, which is representative of the intensity of each band. 65 3.11. Reisolation of naive splenocytes co-cultured with TSST-l- induced Tregs and secondary co-culture To determine i f naive splenocytes would acquire regulatory functions after co-culture with TSST-l- induced Tregs, TSST-l- induced Tregs were first stained with C F S E as mentioned above. Then l x l 0 7 / w e l l na'ive splenocytes were co-cultured with the same number of CFSE-labeled TSST-l- induced Tregs in 6-well plates in 6mL of complete R P M I 1640 medium. In the control group, na'ive cells were co-cultured with CFSE-labeled PBS-primed CD4+ T cells. Cells were then cultured at 37°C, 5% C 0 2 in the presence of TSST-1 (1 nM). 40 hours later, cells were recovered and dead cells were removed by gradient centrifugation. The CFSE-negative naive splenocytes were then reisolated by F A C S . New na'ive splenocytes ( 2 x l 0 5 / w e l l ) were then co-cultured with re-isolated splenocytes at the ratio of 1:1 or 1:2 in 96-well flat-bottom plates in the presence of TSST-1 and cultured at 37°C, 5% C 0 2 in 200 uL complete R P M I 1640 culture medium. Concurrently, new na'ive splenocytes and re-isolated splenocytes were cultured alone in the presence of TSST-1 . 48 hours later, supernatant was collected and stored at -70 °C until assay by E L I S A . 3.12. Culture of na'ive splenocytes with TSST-l- induced Tregs conditioned medium 2 x l 0 6 / w e l l TSST-l-pr imed splenocytes were plated in 24-well plates in l m L of complete RPMI1640 medium and cultured at 37°C, 5% C 0 2 in the presence of TSST-1 (1 nM) for 48 hours. To imitate the na'ive splenocyte co-cultured with Tregs at high ratio (5:4), 1 .2xl0 6 magnetically isolated TSST-l- induced CD4+ Tregs were added to 2 x l 0 6 /well TSST-l -pr imed splenocytes and cells were cultured under the same 66 condition for 48 hours. Then the supernatant was recovered and residual cells were removed by centrifugation. 200 ul of supernatant was then used to culture 4><105/well naive splenocytes in 96-well flat-bottom plates for 48 hours, and supernatant was collected and stored at -70 °C until assay by E L I S A . TSST-1 was added to final concentration of l n M . 3.13. Culture of TSST-l-pr imed splenocytes with naive splenocyte conditioned medium 2 x l 0 6 / w e l l naive splenocytes were plated in 24-well plates in l m L of complete R P M I 1640 medium and cultured at 37°C, 5% C 0 2 in the presence of TSST-1 ( l n M ) for 48 hours. Supernatant was collected and centrifuged to remove residual cells. Concurrently, TSST-1 or PBS-primed splenocytes were prepared. 4 x l 0 5 / wel l TSST-1 or PBS-primed splenocytes were cultured in the naive splenocytes conditioned medium for 48 hours, and supernatant was collected and stored at -70°C until assay by E L I S A . In some groups, anti-CD25 antibody and control antibody (Biolegend, San Diego, C A ) were added to the culture at the concentration of 10 ug/mL. 3.14. Neutralization of IL-10 Blockade of IL-10 was achieved by the anti-IL-10 receptor monoclonal antibody (clone 1B1.2). This antibody has previously been shown to be able to neutralize actions of IL-10. Together with its control antibody (clone GL113), it is a generous gift from Dr. Al ice M u i (Department of Surgery, University of British Columbia) (O'Farrell A M et a l , 1998). For in vitro studies, anti-IL-10 receptor 67 antibody was added to the culture at the concentration up to 100 pg/mL. The control antibody was added at the same concentration. For in vivo blockade, l m g of antibody was given intraperitoneally at day 0 and 0.5 mg of antibody was given once a week 7 days after the first dose (Kingsley et al., 2002). 3.15. Tumor cells P815 cells were purchased from A T C C . They were frozen in liquid nitrogen upon being received. Three days before infusion, they were thawed and the cells were cultured in Dulbecco's modified Eagle's medium containing 1.5 g/L sodium bicarbonate and 4.5 g/L glucose (Stemcell Technologies, Vancouver, B C ) , 10% F B S (Hyclone, Logan, U T ) at the density of 1><105 / m L as recommended in 25mL tissue culture plates. After three days, cells reached confluence and they were detached by vigorous pipetting. They were then washed with cold D M E M medium and mixed with B6 or F l splenocytes to reach the dose of 10,000 cells/mouse in 0.2 m L R P M I 1640 medium. 3.16. M i x e d lymphocyte reaction For mixed lymphocyte reaction, recipients of B6 splenocytes were sacrificed at different time points after transplantation. Total splenic T cells were isolated. 2><105 /well splenic T cells were co-cultured with 2><105/well mitomycin C-treated B6 or F l splenocytes. Cells were cultured at 37°C, 5% C 0 2 in 200 uL R P M I 1640 medium supplemented with 10% F B S (Hyclone, Logan, U T ) , 50 m M 2-mercaptoethanol (2 -ME; Sigma, St Louis, M O ) , 10 m M H E P E S buffer, I m M sodium pyruvate, antibiotics [(100 68 U / m L penicillin; 100 mg/mL streptomycin (Stemcell Technologies, Vancouver, B C ) ; and 2 u.g/mL polymycin B (Sigma, St Louis, MO)) . Supernatant was collected after 5 days of co-culture and was stored at -70°C until assay by E L I S A . 3.17. a G V H D induction and monitoring a G V H D was induced as reported (Owens, Jr. and Santos, 1968). Briefly, recipient B6D2F1 mice were conditioned by intraperitoneal injection of cyclophosphamide (Sigma-Aldrich, St Louis, M O ) at the dose of 300 mg/kg. 24 hours later, each recipient mouse was injected with 4 X 1 0 7 B6 splenocytes via the lateral tail vein. The survival of recipient mice was monitored and mice were weighed once a week. In some experiments, TSST-1 was given to recipient mice at the dose of 4 ug/mouse/injection in 0.2 m L P B S subcutaneously. When two doses of TSST-1 were given, the interval was 4 days. M i c e in the control group were given P B S . In tumor challenge experiments, mice dying from tumor progression were determined by the lack of a G V H D clinical signs of a G V H D and the presence o f liver and spleen enlargement with obvious tumor nodules upon autopsy. 3.18. Assay of endotoxin Serum endotoxin level was determined by a chromogenic limulus amebocyte lysate endpoint assay using a kit purchased from Cambrex (Walkersiville, M D ) according to the instructions from the manufacturer. 69 3.19. Histological studies Tissues harvested from different groups of mice were fixed with P B S containing 10% formaldehyde and embedded in paraffin. 5 um sections were then cut and stained with hematoxylin and eosin for histological examination. Sections were prepared by Waxit Inc. (University of British Columbia, Vancouver, B C ) 3.20. Statistical analysis Descriptive data were expressed as [mean ± standard error o f mean] and analyzed with Prism 4.0. P values<0.05 were considered statistically significant. Group comparisons were made by two-tailed Student's t test. Kaplan-Meier survival analysis was used to determine the differences of survival in groups o f mice. 70 Chapter 4: Generation and characterization of regulatory T cells induced by repeated injection of toxic shock syndrome toxin-1 4.1. Introduction Staphylococcal exotoxins, including TSST-1 and staphylococcal enterotoxin A , B , C l , C2 , C3 , D , E , G , H , and I, are called superantigens because they are able to crosslink the M H C class II molecules on the A P C s and T cell receptors bearing specific VP segments without the need of prior antigen processing, thus activating large numbers o f T cells (Marrack and Kappler, 1990). The activated T cells then respond by potent proliferation and massive production of proinflammatory cytokines, such as IL-1, IL-2, IFN-y, IL-12 and T N F - a (Kum et al., 2001). S E A and S E B are among the most studied bacterial superantigens. Earlier studies using these two superantigens demonstrated that exposure o f T cells to bacterial superantigens, both in vivo and in vitro could result in the clonal expansion of the responding T cells bearing specific VP segments followed by the selective clonal deletion of a large fraction of the responding T cells mediated by apoptosis. The remaining T cells showed an anergic state in response to superantigen stimulation (Kawabe and Ochi, 1991; MacDonald et al., 1991; Rellahan et al., 1990; White et a l , 1989). Later studies further showed that repeated stimulation with these bacterial superantigens was also found to induce the toxin reactive T cells to become Tregs, as shown by the changed cytokine production profile, proliferative state, and the active suppressive effects on the responses of naive cells to the superantigen stimulation (Feunou et al., 2003; Grossman et a l , 2004; Noel et a l , 2001). For example, T cells from mice injected repeatedly with staphylococcal enterotoxin A (SEA) or 71 staphylococcal enterotoxin B (SEB) produced lower levels of IL-2 and IFN-y, but increased level of IL-10 (Noel et al., 2001). When co-cultured with naive splenocytes in the presence of superantigen stimulation, these superantigen-primed T cells suppressed the ability of naive splenocyte to proliferate and to produce IL-2 and IFN-y (Feunou et a l , 2003). TSST-1 is another well-characterized bacterial superantigen. Despite the structural differences with S E A or S E B , previous reported studies indicated that its biological activities mostly overlap with those of S E A or S E B , in terms of its ability to induce the cytokine and proliferation responses in T cells, and also the ability to induce lethal shock in mice. However, recent data in our laboratory suggest the presence of important differences in biological activities between them. For instance, our laboratory showed that TSST-1 did not induce apoptosis of human P B M C at low concentration as compared to S E B (Huang, 2004). Furthermore, until now, most of the data regarding superantigen-induced Tregs were accumulated using S E A or S E B , while the characteristics of TSST-l- induced Tregs are less clear. Therefore, the differences between Tregs induced by TSST-1 or other bacterial superantigens are also unknown. Thus to gain further understanding of TSST-l- induced Tregs, a side-by-side comparative study of Tregs induced by TSST-1 and other superantigens is warranted. In this study, the biologic properties of TSST-1 vs SEA-induced Tregs in C57BL/6 mice are directly compared. 72 4.2. Experimental Design Mice were injected subcutaneously with TSST-1 or S E A at the dose of 4 p.g/mouse /injection at the interval of 4 days. Mice in the control group were injected with P B S . Two hours after the 3 r d injection, splenocytes were isolated from these mice. Cel l surface or intracellular markers were analyzed by flow cytometry. Splenocytes were stimulated with TSST-1 or S E A and the cytokine, proliferation and apoptosis profiles were then analyzed by E L I S A or flow cytometry ( F C M ) . To assay the suppressive functions, CD4+ T cells were further enriched from the splenocytes. They were then co-cultured with naive splenocytes in the presence of TSST-1 or S E A , and their effects on cytokine and proliferation responses were determined by E L I S A and F C M respectively. To investigate the in vivo suppressive functions, CD4+ T cells were adoptively transferred to naive mice which then were subjected to lethal shock induction. Survival of the recipients was monitored. A flow diagram of the experimental approach is depicted below. TSST-1 , x3 S E A , x3 P B S , x 3 Splenocytes * >> C D 4 + T c e n s analysis by by E L I S A and / I f F C M F C M / V Effects on lethal Cytokine responses Proliferation shock assayed by E L I S A responses assayed by F C M Figure 4.1. Experimental design of studies described in Chapter 4. 73 4.3. Results 4.3.1. Generation of superantigen-induced Tregs in C57BL/6 mice Previous studies in our laboratory have established that repeated injection of TSST-1 in B A L B / c (H-2 d ) mice at the dose of 4pg/mouse/injection at the interval of 4 days induced CD4+ Tregs (Cameron, 2004). However, in the present study, which w i l l also examine the effects of Tregs in an a G V H D model, C 5 7 B L / 6 (B6, H-2 b ) mice were used as donor mice from which Tregs were derived. Thus in i t ia l studies were performed to confirm that the same protocol that generated CD4+ Tregs in B A L B / c mice can also do so in B6 mice despite differences in genetic background between these two strains of mice. To generate TSST-1 or SEA-induced Tregs in B6 mice, TSST-1 or S E A was administrated subcutaneously for three times at the interval of 4 days at the dose of 4pg/mouse/injection in 0.2mL P B S . In the control group, the same volume o f P B S was given in the same way. Two hours after the 3 r d injection, splenocytes from mice treated with TSST-1 , S E A or P B S were isolated and CD4+ T cells were further enriched by magnetic separation. Co-culture experiments were then performed to assay the suppressive activities of these superantigen-primed CD4+ T cells. Titrated numbers of superantigen-primed CD4+ T cells were co-cultured with fixed number of na'ive splenocytes in the presence of TSST-1 or S E A for 48 hours and then the supernatant was assayed for levels of various cytokines by E L I S A . A s shown in Figure 4.2, the addition of TSST-l -pr imed CD4+ T cells to naive splenocytes in the presence of TSST-1 led to decreased levels of IL-2, IL-12 and IFN-y. Similarly, co-culture of SEA-primed CD4+ T cells with na'ive splenocytes in the presence of S E A 74 20(H lOOH O-l Stimulation with TSST-1 IL-2 150-1 100H 50H — i i 5:0 5:0.06 5:0.25 5:1 5:2 5:4 A— o Stimulation with SEA IL-2 "s-0 5:0'.06 5:0'.25 5M~ 5:2 5:4 0 s 150-1 o ** 100' c o o 5o^ c (LI o s-4) OH 0 210-1 140H 70H ~sTo 5:0'.06 5:0'.25 iTl 5:2 5:4 250n i i i 5:0 5:0.06 5:0.25 5:1 5:2 5:4 IFN-y —r-5:0 IFN-y 5:0.06 5:0.25 5:1 5:2 5:4 5:0 5:0.06 5:0.25 • Ratio of naive splenocytes : CD4+ T cells 5:1 —I— 5:2 5:4 F i g u r e 4 . 2 . I n i t i a l s t u d i e s t o d e t e r m i n e t h e s u p p r e s s i v e a c t i v i t i e s o f s u p e r a n t i g e n -i n d u c e d T r e g s in vitro. T i t r a t e d n u m b e r s o f C D 4 + T c e l l s f r o m m i c e t r e a t e d w i t h r e p e a t e d i n j e c t i o n o f T S S T - 1 o r S E A w e r e c o - c u l t u r e d w i t h a fixed n u m b e r o f n a i v e s p l e n o c y t e s i n t h e p r e s e n c e o f T S S T - 1 o r S E A f o r 4 8 h o u r s , a n d t h e n s u p e r n a t a n t w a s a s s a y e d f o r v a r i o u s c y t o k i n e s b y E L I S A . A s s h o w n , T S S T - l - p r i m e d C D 4 + T c e l l s d o w n - r e g u l a t e d p r o i n f l a m m a t o r y c y t o k i n e l e v e l s i n d u c e d b y T S S T - 1 , w h i l e S E A - p r i m e d C D 4 + T c e l l s w e r e a b l e t o d o w n - r e g u l a t e t h o s e i n d u c e d b y S E A . D a t a w e r e e x p r e s s e d a s t h e p e r c e n t a g e o f c y t o k i n e p r o d u c t i o n b y n a i v e s p l e n o c y t e s a l o n e . • : N a i v e a l o n e ; A : N a i v e + T S S T - l - p r i m e d C D 4 + T c e l l s ; T : N a i v e + S E A -p r i m e d C D 4 + T c e l l s ; • : N a i v e + P B S - p r i m e d C D 4 + T c e l l s . D a t a a c c u m u l a t e d f r o m 2 i n d e p e n d e n t e x p e r i m e n t s . 7 5 resulted in decreased levels of IL-2, IL-12 and IFN-y. Thus these data confirmed that our protocol was able to generate superantigen-induced Tregs in B 6 mice and that the generated Tregs could down-regulate responses of naive splenocytes to their cognate antigens. Moreover, these initial studies also set up a co-culture system to assay and investigate the mechanisms of the suppressive functions of superantigen-induced Tregs described in Chapter 5. Following these experiments, a comparative study investigating the cytokine profile, proliferative ability, apoptosis, and suppressive activities of TSST-1 vs SEA-induced Tregs was then performed. 4.3.2. Cytokine production profile of TSST-1 and SEA-induced Tregs A s Tregs are associated with distinct cytokine production profiles, the cytokine production profile of TSST-1 or SEA-induced Tregs were compared. To study their cytokine profile, splenocytes from mice treated with repeated injection of TSST-1 or S E A were stimulated with TSST-1 or S E A for various periods o f time, then E L I S A was performed to quantify the cytokine levels in the supernatant and flow cytometry analysis was performed to further determine the intracellular cytokine expression by CD4+ T cells. i) PBS-primed CD4+ T cells: PBS-primed splenocytes stimulated with TSST-1 or S E A produced mainly proinflammatory cytokines, such as IL-2, IFN-y and T N F - a , while the level of anti-proinflammatory cytokine IL-10 was low. Their production was time-dependent, as the levels of these cytokines increased as the culture time increased (Figure 4.3). Flow cytometry analysis confirmed the expression of these cytokines at 40 hours post-stimulation. A s shown in Figure 4.4, no cytokine positive cell population 76 2500 IL-2 2000 1500 1000 500 0 -^S-Tregs+T -¥-S-Tregs+S IFN-y bt) C e '& SJ u e u C o U 12h 24h 48h 72h 96h 12h 24h 48h 72h 96h 1250-1 1000-750-500-250-0-_ 12h 24h 48h 72h 96h 1250-1 1000-750-500-250-12h 24h 48h 72h 96h 12h 24h 48h 72h 96h 0 L _ ^ . . . . 12h 24h 48h 72h 96h 400-1 12h 24h 48h 72h 96h 12h 24h 48h 72h 96h 12h 24h 48h 72h 96h 3000 IL-10 2 4 0 0 1800 1200 600 0 12h 24h 48h 72h 96h 3000 2400 1800 1200-1 600 0 12h 24h 48h 72h 96h 3000-1 2400-1800-1200-600-0-12h 24h 48h 72h 96h Figure 4.3. Cytokine levels in supernatant of TSST-1 or SEA-induced Tregs following reactivation with TSST-1 or SEA. Splenocytes from mice treated with TSST-1, SEA or PBS were stimulated with TSST-1 or SEA for various periods of time, then supernatant was harvested and assayed by ELISA for IL-2, IFN-y, IL-10, and TNF-a. T-Tregs: TSST-l-induced Tregs; S-Tregs: SEA-induced Tregs; P-CD4: PBS-primed CD4 T cells; T: TSST-1; S: SEA. Data were accumulated from four independent experiments, n=4. 77 P-CD4+R P-CD4+T P-CD4+S Q 3% 10° 10' 101 ID1 T-Tregs+R IK jfc.;20.1°/o ioD io' io1 io3 S-Tregs+R 8.4% io° io' Ho1 io1 IL-2-APC 14.5% 23.3% mm o o 10" 10' io1 io3 & 0' 0° o* io1 io3 T-Tregs+T T-Tregs+S 22.6% 10° 10* io1 io3 S-Tregs+T S-Tregs+S 32% IP'* 24.4% 1DD IL-2 Figure 4.4. Expression of cytokines by TSST-1 or SEA-induced Tregs. A . Representative raw data of expression of IL-2 by TSST-1 or SEA-induced Tregs assayed by flow cytometry. Splenocytes from mice treated with TSST-1 , S E A or P B S were cultured in the absence or presence of TSST-1 or S E A for 45 hours, then intracellular staining of IL-2 followed by flow cytometry analysis was performed. Data shown was representative of flow cytometry analysis of IL-2 expression by TSST-1 or SEA-induced Tregs, or PBS-primed CD4+ T cells from one of four independent experiments, n=4. T-Tregs: TSST-l- induced Tregs; S-Tregs: S E A -induced Tregs; P -CD4: PBS-primed C D 4 T cells; T: TSST-1 ; S: S E A ; R: R P M I 1640 medium. 78 B 45n 3(H 15H IL-2 X RPMI TSST-1 SEA RPMI TSST-1 SEA RPMI TSST-1 SEA 5(H 404 £ 301 = 20-1 10' + T i -ft U © a o fl 3 o s u PM I F N - Y X X X 60-40-2<H RPMI TSST-1 SEA RPMI TSST-1 SEA I L - 1 0 1 X TL RPMI TSST-1 SEA RPMI TSST-1 SEA RPMI TSST-1 SEA 35n 284 2 H 14H TNF-a X X RPMI TSST-1 SEA RPMI TSST-1 SEA RPMI TSST-1 SEA PBS -CD4 T TSST-l-Tregs SEA-Tregs Figure 4.4. B . Data presented were aggregated from four independent experiments, n=4. The bar graphsrepresented the percent of cytokine positive CD4+ T cells. *, P O . 0 5 , compared with SEA-Tregs+R, t-test. 79 was detected in PBS-primed CD4+ T cells cultured in the absence o f stimulation while cytokine positive populations were detected in TSST-1 or SEA-stimulated PBS-primed CD4+ T cells, showing that the cytokine expression by PBS-primed CD4+ T cells depended on the induction by superantigen stimulation. ii) TSST-l-induced Tregs: In response to TSST-1 stimulation, IL-2 was only detected at the 12 hour time point, but undetectable at other time points. IFN-y and T N F - a were detected in the supernatant at levels comparable to those in PBS-primed CD4+ T cells stimulated with TSST-1 , although the levels were lower at later time points. IL-10 production was greatly enhanced at all time points. When TSST-l- induced Tregs were stimulated with S E A , IL-2 was not detectable at any time point while IFN-y, T N F - a and IL-10 were detected, although their levels were generally lower than those in the supernatant of TSST-l- induced Tregs stimulated with TSST-1 (Figure 4.3). Unlike the PBS-primed CD4+ T cells and SEA-induced Tregs, whose cytokine expression was triggered by superantigen stimulation, flow cytometry analysis found that T S S T - l -induced Tregs expressed all cytokines assayed even in the absence of superantigen stimulation. A significantly higher percent of cytokine positive CD4+ T cells was found in TSST-l- induced Tregs than in SEA-induced Tregs in the absence of stimulation. When TSST-l- induced Tregs were stimulated with TSST-1 or S E A , the percentage of cytokine positive CD4+ T cells was only slightly increased as compared to that in the absence of stimulation (Figure 4.4). iii) SEA-induced Tregs: Levels of IL-2, IFN-y and T N F - a greatly decreased in the supernatant of SEA-induced Tregs stimulated with S E A as compared to PBS-primed CD4+ T cells stimulated with S E A , while the level of IL-10 was elevated. However, 80 when SEA-induced Tregs were stimulated with TSST-1 , the cytokine production profile was rather similar to that of the PBS-primed CD4+ T cells stimulated with S E A , which was high IL-2, IFN-y and T N F - a and low IL-10 (Figure 4.3). Interestingly, despite the low levels of IL-2, IFN-y and T N F - a in the supernatant of SEA-induced Tregs stimulated with S E A , flow cytometry analysis showed that these cytokines were actually expressed by SEA-induced Tregs, as cytokine positive CD4+ T cells were readily detectable in SEA-induced Tregs. Similar to PBS-primed CD4+ T cells, S E A -induced Tregs cells did not show marked expression of these cytokines in the absence of superantigen stimulation, although the percent of cytokine positive SEA-induced Tregs in the absence o f stimulation was slightly increased as compared to PBS-primed CD4+ T cells (Figure 4.4). These data show that cytokine expression by SEA-induced Tregs was also dependent on superantigen stimulation. 4.3.3. Proliferation profile of TSST-1 or SEA-induced Tregs It has been shown that superantigen stimulation resulted in T cell anergy, which was demonstrated by reduced incorporation of [ 3H]thymidine. Moreover, an anergic state has been shown to be a feature of Tregs. To study the proliferation profile of superantigen-primed CD4+ T cells, splenocytes from mice treated with repeated injection of TSST-1 or S E A were labeled with C F S E and cultured in the absence or presence of TSST-1 or S E A . The proliferation o f CD4+ T cells was determined by flow cytometry analysis at different time points. A s shown in Figure 4.5, PBS-primed CD4+ T cells and SEA-induced Tregs did not proliferate in the absence of superantigen 81 A P-CD4+R P-CD4+T P-CD4+S u 1.2% 10° in1 in 1 in 3 T-Tregs+R 64% 10 D ID' 10 3 IO 3 S-Tregs+R 10° i o ' 7.9% S<5 lif in 0 in' 35% n 3 '1 n 3 n* o" i o ' 46.6% o 1 i o 3 T-Tregs+T 10 10' 10° 10' 10 3 1 0 3 1 0' S-Tregs+S 54.4% ^ CD4 Figure 4.5. Proliferation response of superantigen-induced Tregs. A . Representative raw data of proliferation response of superantigen-induced Tregs. Splenocytes from mice treated with TSST-1 , S E A or P B S were first stained with C F S E and then cultured in the absence or presence of TSST-1 or S E A . A t different time points post-stimulation, cells were harvested and analyzed by flow cytometry for the proliferation of CD4+ T cells. Representative data shown were proliferation of CD4+ T cells stimulated in the absence or presence of l n M superantigens for 3 days. T-Tregs: TSST-l- induced Tregs; S-Tregs: SEA-induced Tregs; P -CD4: PBS-primed C D 4 T cells; T: TSST-1; S: S E A ; R: R P M I 1640 medium. PBS-primed CD4+ T cells cultured in the absence of superantigen (P-CD4+R) were used as control to determine the C F S E intensity of non-proliferating cells. 82 B Day 1 Day 2 Day 3 Day 5 Day 1 Day 2 Day 3 Day 5 Time of stimulation (day) Figure 4.5. B . Data presented are accumulated data from four independent experiments and show the proliferation of CD4+ Tcells in the absence of stimulation or in the presence of different concentration of superantigen stimulation. • : TSST-l- induced Tregs+RPMI; • : SEA-induced Tregs+RPMI; 0: PBS-primed C D 4 T cells +RPMI; A: TSST-l- induced Tregs+TSST-1; T : SEA-induced Tregs+SEA; o : PBS-primed C D 4 T cells+TSST-1; • : PBS-primed CD4 T cells+SEA. *, P O . 0 5 , compared with S-Tregs+S, t-test, n=4. 83 stimulation and proliferation was detected when they were stimulated with TSST-1 or S E A , showing that the proliferation of PBS-primed CD4+ T cells and SEA-induced Tregs was dependent on superantigen stimulation. Interestingly, in the absence o f superantigen stimulation, TSST-l- induced Tregs could undergo proliferation as compared to the control PBS-primed CD4+ T cells and SEA-induced Tregs over the 5-day period. The maximum degree of proliferation of TSST-l- induced Tregs in the absence of TSST-1 was comparable to that in the presence of TSST-1 at day 5. In the presence of cognate antigen stimulation, both TSST-1 and SEA-primed CD4+ T cells proliferated more potently than the control PBS-primed CD4+ T cells. However, the proliferation of TSST-l -pr imed CD4+ T cells at day 2 and day 3 was significantly higher than that of SEA-induced Tregs when cells were stimulated with different doses o f cognate antigen. A t day 5, difference o f proliferation between these two types o f superantigen-primed CD4+ T cells was only observed at 0.1 n M . Therefore, the proliferation of TSST-l- induced Tregs was antigen-independent, and in the presence of the cognate antigen stimulation, the proliferation response of TSST-l- induced Tregs was more sensitive than that of SEA-induced Tregs. 4.3.4. Other phenotypic properties of TSST-1 and SEA-induced Tregs Previous studies showed that superantigen stimulation o f T cells might lead to deletion of superantigen-reactive T cells via cell death. Thus the cell death of TSST-1 and SEA-induced Tregs in response to their cognate antigen stimulation in vitro was also investigated. A s showed in Figure 4.6, in the absence of antigen stimulation, P B S -primed CD4+ T cells and SEA-induced Tregs underwent increased cell death as the 84 P-CD4+R P-CD4+T P-CD4+S Q % U 8^ *v 21.2% ioa io' ia 1 io3 T-Tregs+R | | | t ! l M > . ^ 23% 10° in' in2 in3 1 S-Tregs+R 26.9% : 14.4% 1DD io' i o 3 i a 3 i T-Tregs+T 23.7% S-Tregs+S 19.7% io° io 1 i o z io 3 7-AAD 12.3%: - • 7-AAD Figure 4.6. Ce l l death of superantigen-induced Tregs in vitro. A . Representative raw data of cell death of superantigen-induced Tregs in vitro. Splenocytes from mice treated with TSST-1 , S E A or P B S were cultured in the absence or presence of TSST-1 or S E A . A t different time points post-stimulation, cells were harvested, stained with 7 - A A D and PE-anti-CD4 antibody and analyzed by flow cytometry for the cell death of CD4+ T cells. Representative data shown were the cell death of CD4+ T cells stimulated in the absence or presence of I n M superantigens after 1-day culture. T-Tregs: TSST-l- induced Tregs; S-Tregs: SEA-induced Tregs; P -CD4: PBS-primed C D 4 T cells; T: TSST-1 ; S: S E A ; R: R P M I 1640 medium. 85 6 s U H + o u + I t--B 75' 50-25-75n 50H 8 25-PH RPMI 75n 50H 25H 0 O.lnM Day 1 Day 3 Day 5 Day 1 Day 3 75n lnM 50H Day 5 25H 0' lOnM D a y l Day 3 Day 5 Day 1 Time of stimulation (days) Day 3 Day 5 Figure 4.6. B . Data presented are accumulated data from four independent experiments showing the cell death of CD4+ Tcells in the absence o f stimulation or in the presence of different concentration of superantigen stimulation. • : TSST-l- induced Tregs+RPMI; • : SEA-induced Tregs+RPMI; 0: PBS-primed C D 4 T cells +RPMI; A: TSST-l- induced Tregs+TSST-1; T : SEA-induced Tregs+SEA; o : PBS-primed C D 4 T cells+TSST-1; • : PBS-primed C D 4 T cells+SEA. *, P O . 0 5 , compared with S-Tregs+S, t-test, n=4. 86 culture time increased. In contrast, TSST-l- induced Tregs did not show this time dependent cell death, suggesting that TSST-1 is anti-apoptotic or pro-survival. A significantly lower percent of CD4+ T cells undergoing cell death was detected in TSST-l- induced Tregs at day 3 and day 5 than in SEA-induced Tregs. In the presence of l n M and 10 n M cognate superantigen stimulation, the survival of TSST-1 and S E A -induced Tregs was better than PBS-primed CD4+ T cells as shown by the lower percent of CD4+ T cells undergoing cell death at day 3 and day 5. N o significant difference in the percent of CD4+ T cells undergoing cell death was detected between TSST-1 and SEA-induced Tregs at any time point. In response to 0.1 n M superantigen stimulation, a significant difference was detected at day5 between TSST-1 and S E A -induced Tregs. Thus TSST-l- induced Tregs showed a better ability for survival in the absence of antigen stimulation or in the presence of low concentration of antigen stimulation. A s the expression of Foxp3, C T L A - 4 , CD25 or G I T R has been associated with the Treg phenotype, the expression of these molecules on TSST-1 or SEA-induced Tregs was investigated. Results showed that the percentages of Foxp3 and GITR positive CD4+ T cells were not significantly different between TSST-l -pr imed and SEA-primed CD4+ T cells. In contrast, the percentages of CD25 and C T L A - 4 positive cells were significantly higher in TSST-l -pr imed CD4+ T cells than in SEA-primed CD4+ T cells (Figure 4.7 and Table 4.1). The expression of CD25 or C T L A - 4 in superantigen-primed CD4+ T cells was also higher than that in the PBS-primed control CD4+ T cells. 87 TSST-l-Tregs SEA-Tregs PBS-CD4 U 3 9.4% IP"'-12.7% 11.3% I Foxp3 44.5% l"s 18.4% 13.1% CTLA-4 24% Ml 21.5% lift 17.8% GITR 35% I D " i 22% 11.3% CD25 Figure 4.7. Phenotype of superantigen-induced Tregs. Representative raw data of phenotypical analysis of superantigen-induced Tregs. F low cytometric analysis was performed to determine the expression of Foxp3, C T L A - 4 , G I T R and CD25 on splenic CD4+ T cells from mice injected with 3 times of TSST-1 or S E A . Representative data of four independent experiments were shown. Left column, TSST-l- induced Tregs. Middle column, SEA-induced Tregs. Right column, PBS-primed CD4+ T cells. Table 4.1. Expression of Foxp3, C T L A - 4 , G I T R or CD25 on CD4+ T cells of mice treated with TSST-1 , S E A or P B S . *, p < 0.05, compared with SEA-Tregs, t-test, n=4. TSST-l-Tregs SEA-Tregs P B S - C D 4 Foxp3 8.8=1=1.0 13.0±1.8 8.4±1.3 C T L A - 4 49.4±3.4* 25.9±3.8 12.6±1.8 G I T R 24.1±6.3 20.6±0.5 14±1.7 CD25 45.3±4.1* 22.2=bl 8.9±1 88 4.3.5. TSST-l- induced Tregs had broader suppressive activities than SEA-induced Tregs Tregs are defined by their suppressive activities over the immune responses; thus, elucidation of the differences between the suppressive activities of Tregs induced by TSST-1 or S E A was pivotal for the comparison. In vitro studies were first performed to determine the suppressive actions of superantigen-induced Tregs on cytokine production by co-culture experiments. To this end, a fixed number of naive splenocytes were co-cultured with a titrated number of purified CD4+ TSST-1 or S E A -induced Tregs in the presence of TSST-1 or S E A for 48 hours and cytokine levels in the supernatant were assayed by E L I S A . A s shown in Figure 4.8-A, the addition of TSST-l- induced CD4+ Tregs to na'ive splenocytes resulted in decreased levels of IL-2, IL-12 and IFN-y induced by either TSST-1 or S E A in a dose-dependent manner. In contrast, the addition of SEA-induced CD4+ Tregs to the culture lead to decreased levels of IL-2, IL-12 and LFN-y induced by S E A , but not by TSST-1 . Therefore, these in vitro data showed that the suppressive function of TSST-l - induced CD4+ Tregs was broader than that of SEA-primedCD4+ T cells. To further confirm the in vitro findings, a lethal shock model was used to test the in vivo suppressive function of TSST-1 or SEA-induced Tregs. CD4+ Tregs from mice treated with TSST-1 or S E A were adoptively transferred to naive recipient mice and then they were subjected to lethal shock challenge by TSST-1 or S E A . The results showed that mice without transfer of T cells all died quickly after challenge (Figure 4.8-B, mean survival TSST-1:9.5±1.3 h, SEA:8±0.9 h , n=5). Survival of recipient mice of TSST-l- induced Tregs was significantly prolonged in response to either TSST-89 Stimulation with TSST-1 2001 IL-2 150 100 o u c o u G a u s-w PH Stimulation with SEA I50 n IL-2 150n 1004 IL-12 150 100 5:0.06 5:0.25 IL-12 5:0.06 5:0.25 IFN-y — i — 5:1 5:2 5:0 5:0.06 5:0.25 5:0 5:0.06 5:0.25 5:1 5:2 5:4 Ratio of naive splenocytes : CD4+ T cells 5:1 — I — 5:2 5:4 — I — 5:4 Figure 4.8. Suppressive function of superantigen-induced Tregs in vitro and in vivo. A : Suppressive activities of superantigen-induced Tregs in vitro. Titrated numbers of CD4+ T cells from mice treated with repeated injection o f TSST-1 or S E A were co-cultured with a fixed number of naive splenocytes in the presence of TSST-1 or S E A for 48 hours, and then supernatant was assayed for various cytokines by E L I S A . A s shown, TSST-l- induced CD4+ Tregs down-regulated proinflammatory cytokine levels induced by TSST-1 or S E A , while SEA-induced CD4+ Tregs were only able to down-regulate those induced by S E A but not by TSST-1 . Data were expressed as the percentage of cytokine production by naive splenocytes alone. • : Naive alone; • : Na'ive + TSST-l- induced Tregs; T : Na'ive + SEA-induced Tregs;*: Na'ive + PBS-primed CD4+ T cells. *, P<0.05, compared with na'ive splenocytes alone, paired t-test, n=4. 90 B Challenge with TSST-1 0 S Navie Naive+T-Tregs Navie+S-Tregs Naive+T non-CD4 Naive+P-CD4 T © fl o u CL Challenge with SEA 0 Naive Naive+T-Tregs Naive+S-Tregs Naive+S non-CD4 Naive+P-CD4 T 10 20 30 Time post lethal shock induction (h) Figure 4.8. B . Suppressive effects of TSST-1 or SEA-induced Tregs in vivo. Mice were first transferred with CD4+ T cells (2><107/mouse) isolated from mice treated with repeated injection of TSST-1 , S E A or P B S . Two hours after cell transfer, they were challenge with lethal shock induced by TSST-1 or S E A . Survival was monitored for 72 hours post-challenge. *, P O . 0 5 , compared with naive mice (no cell transfer), n=5. 91 c Stimulation with TSST-1 Stimulation with SEA 45 T • Naive •A- Naive+T-Tregs • Naive 0 0 5:0 5:0.06 5:0.25 5:1 5:2 5:4 5:0 5:0.06 5:0.25 5:1 5:2 5:4 Ratio of naive splenocytes : CD4+ T cells Figure 4.8. C. Effects of superantigen-induced Tregs on the proliferation of naive splenocytes. To study the effects of superantigen-induced Tregs on the proliferation responses of na'ive splenocytes, na'ive splenocytes were first stained with C F S E and then were co-cultured with titrated number of magnetically isolated CD4+ T cells from mice treated with three injections of TSST-1 , S E A or P B S in the presence of TSST-1 or S E A for 7 2 hours. Then the proliferation of na'ive splenocytes was assayed by flow cytometry. A s shown, addition of TSST-1 or SEA-induced CD4+ Tregs did not lead to decreased proliferation of naive splenocytes, rather, they increased the proliferation of na'ive splenocytes. Thus, although TSST-1 or S E A -induced Tregs could down-regulate the cytokine responses of na'ive splenocytes, they did not suppress the proliferation responses of their target cells. Results shown were accumulated data from three independent experiments (n=3). Navie: naive splenocytes; T-Tregs: TSST-l- induced Tregs; S-Tregs: SEA-induced Tregs; P -CD4 T: PBS-primed CD4+ T cells. 9 2 1 (18±2.9 h) or S E A (16.5±2.7 h) challenge. In contrast, survival of mice that received SEA-induced Tregs was prolonged only when challenged with S E A (13.9±2.5 h), but not with TSST-1 (9.4±0.8 h). Transfer of CD4+ T cell-depleted TSST-1 or S E A -primed splenocytes, or PBS-primed CD4+ T cells did not confer protection, further confirming the suppressive effect of superantigen-induced CD4+ Tregs. Interestingly, the addition of TSST-1 or SEA-induced Tregs to naive splenocytes stimulated with TSST-1 or S E A did not lead to the inhibition of naive splenocytes in vitro. Instead, proliferation of naive splenocytes was increased in the presence of TSST-1 or SEA-induced Tregs in a dose-dependent manner. This effect was also more remarkable in TSST-l- induced Tregs (Figure 4.8-C). In summary, the data above further confirmed findings in the initial studies showing that in our repeated superantigen injection system, CD4+ Tregs could be induced. Moreover, T S S T - l -induced Tregs had broader suppressive abilities than SEA-induced Tregs. 4.3.6. VP usage by TSST-1 and S E A induced Tregs—Cross-reactive suppressive function of TSST-l- induced Tregs was not due to shared VP families of T cells reactive to both TSST-1 and S E A Previous studies have reported that in mice TSST-1 can react with T cells bearing VP 15 while S E A can react with T cells bearing VP3, 10, 11, 12. A s shown above, since TSST-l- inudced Tregs could suppress responses triggered by S E A , it was possible that TSST-l- induced Tregs contained Vp families of T cells that were also reactive to S E A . To exclude this possibility, Tregs induced by TSST-1 or S E A were analyzed for the T cell receptor VP repertoire. To this end, splenocytes from mice 93 treated with TSST-1 or S E A were stimulated with their cognate antigen in vitro and flow cytometry was performed to detect the expansion of specific VP family of T cells. A s shown in Figure 4.9 and Table 4.2, stimulation o f PBS-primed splenocytes with S E A resulted in the expansion of VP3 and V p i l CD4+ T cells which was demonstrated by the increased percent of Vp3 (40.4±3.4 vs 4.9±0.5) and VP 11 CD4+ T (25±3.4 vs 5.8±0.8) as compared to PBS-primed splenocytes in the absence of toxin stimulation. However, stimulation of PBS-primed splenocytes with TSST-1 resulted in the decreased percent of VP3 positive CD4+ T cells which was due to the expansion of other V P families, thus showing that Vp3 CD4+ T cells did not react to TSST-1 . Moreover, we could not detect the expansion of V p l O and V p i 2 CD4+ T cells as reported in PBS-primed splenocytes stimulated with S E A . A s expected, stimulation of SEA-induced Tregs led to the expansion of Vp3 and V p i l CD4+ T cells. In contrast, stimulation of TSST-l- induced Tregs with the cognate antigen did not lead to expansion of any V P family of CD4+ T cells that we analyzed. Since it was not possible to assay the V p l 5 expression due to lack of commercially available antibody against this segment, R T - P C R was used to determine the expression of V p i 5 of TSST-1 and SEA-induced Tregs. As shown in Figure 4.10, stimulation of PBS-primed CD4+ T cells or TSST-l- induced Tregs with TSST-1 led to increased expression of V p i 5 while stimulation of these cells with S E A did not. Thus, TSST-1 reacted with V p i 5 family T cells. Taken together, the data showed that SEA-induced Tregs consisted of VP3 and 11 families o f T cells while TSST-l- induced Tregs consisted of V p i 5 family o f T cells. Therefore, the data here demonstrated that the suppression of SEA-triggered 94 P-CD4+R P-CD4+T P-CD4+S 10° 10' ID 1 10 A 10 10" 10' I 0 D 10' 10* 10' T-Tregs+R T-Tregs+T U 8 IP 0.5% S-Tregs+R 20.8% m 0.8% 10" io' io2 io3 S-Tregs+S 36.5% Vp3 io" io" io^ io* io* io° io' io' itP io* Figure 4.9. VP analysis of superantigen-induced Tregs. Splenocytes from mice treated with TSST-1 , S E A or P B S was cultured in the absence or presence of TSST-1 or S E A for 3 days. Then cells were washed to remove the toxin and cultured in the absence of toxin overnight followed by flow cytometry analysis of VP family of the CD4+ T cells. Raw data shown was one of three experiments of Vp3 analysis. T-Tregs: TSST-l- induced Tregs; S-Tregs: SEA-induced Tregs; P -CD4: P B S -primed C D 4 T cells; T: TSST-1 ; S: S E A ; R: RPMI1640 medium. 95 Cells PBS-CD4 T S S T - -Tregs SEA-Tregs stimulant R P M I TSST -1 S E A R P M I TSST -1 R P M I S E A Vp3 5.0±0.5 2.4±0.4 40.4±3.4 1.9±0.9 1.5=1=0.5 14.9±3.3 36.1=1=2.9 Vp8 19.1=1=1.3 10.6±2.8 5.0=1=1.2 4.6=1=1.5 4.3=1=1.2 19.8±2.8 12.9±1.3 v p i o 5.3±0.3 3.4±0.6 3.9±0.3 1.1=1=0.1 1.5±0.1 3.1±0.3 2.6±0.1 v p n 5.8=1=0.8 3 .9±1.1 25.1=1=3.4 1.7=1=0.5 2.0±0.3 8.8±0.5 12.9=1=1.6 V p l 2 11 .1±2 . 9 4 . 8±2 .2 7 . 3 ± 1 . 5 2.5±0.4 2.6±0.3 9.7±2.6 6 .9±1.8 Table 4.2. VP analysis of superantigen-induced Tregs. Splenocytes from mice treated with TSST-1 , S E A or P B S were cultured in the absence or presence of TSST-1 or S E A for 3 days. Cells were then washed to remove the toxin and cultured in the absence of toxin overnight followed by flow cytometry analysis of VP family of the CD4+ T cells. The numbers shown represented the percent of specific VP positive cells in CD4+ T cells. Data shown were accumulated from three independent experiments, n=3. 96 25-i . j es 2 15-u £ io-09 2 5-to o - L - ^ 1 ' — i — ' 1 P-CD4+T P-CD4+S T-Tregs+T S-Tregs+S Figure 4.10. Assay of V p i 5 expression by R T - P C R . Total R N A was purified from TSST-1 or SEA-induced Tregs and PBS-primed CD4+ T cells stimulated with either TSST-1 or S E A . R T - P C R then was performed to amplify the V p i 5 segment o f the T cell receptor. Constant region of the T cell receptor served as a control. Product of R T - P C R was then measured for it density. The density of each group was then corrected by the density of product of constant region of the T cell receptor. Density o f product o f stimulated CD4+ T cells was then divided by that of the unstimulated CD4+ T cell to calculate the fold of increase of expression of V p i 5 . As shown, stimulation of CD4+ T cells with TSST-1 , but not S E A , led to increased expression of V p l 5 . 97 responses by TSST-l- induced Tregs was not because they contained CD4+ T cells reactive to S E A . 4.4. Discussion To characterize the TSST-l- induced Tregs, a side-by-side comparison of Tregs induced by two different bacterial superantigens, namely TSST-1 and S E A was performed. The results showed that repeated injection of TSST-1 or S E A could induce CD4+ Tregs that were able to down-regulate the cytokine responses of na'ive splenocytes in vitro and protected against lethal shock challenge in vivo. These data demonstrated that the suppressive activities of SEA-induced Tregs were highly antigen specific, as they only suppressed the SEA-triggered responses in vitro and in vivo, but were unable to suppress the TSST-l- induced responses. Similar results were found in a previous study, where repeated mucosal administration of S E A was protective against subsequent SEA-mediated, but not TSST-l-mediated lethal shock challenge, although the authors did not determine i f repeated mucosal administration of S E A was able to induce Tregs that were responsible for these effects (Collins et al., 2002). In contrast to SEA-induced Tregs, the suppressive functions of TSST-l- induced Tregs were more broadly reactive as they suppressed responses triggered by either TSST-1 or S E A . In a previous study, Noel et al. showed that splenocytes of C57BL/6 mice repeatedly injected with S E A or S E B had a similar cytokine profile of high IL-10 and low IL-2 in response to either S E A or S E B stimulation in vitro. This profile was not observed in B A L B / c mice. They hypothesized that this was because S E A and S E B could both stimulate T cells bearing Vp3 , which B A L B / c mice lacked (Noel et al., 2001). 9 8 However, data of V P screening by flow cytometry and R T - P C R did not reveal that TSST-1 or S E A could stimulate a common VP family o f T cells. In addition, i f there is a T cell VP family population reactive to both TSST-1 and S E A , the SEA-induced Tregs should also be activated by TSST-1 and suppressed the TSST-1-triggered responses, which was not observed. Therefore, the ability of TSST-l- induced Tregs to suppress SEA-induced responses was unlikely to be the presence of V p family of T cells reactive to both TSST-1 and S E A . Thus, our data for the first time showed that repeated injection of TSST-1 could generate Tregs with broader suppressive activities as compared to S E A . Interestingly, our data showed that neither TSST-1 nor SEA-induced Tregs suppressed the proliferation of naive splenocytes, which was different from what was previously reported (Feunou et al., 2003; Grundstrom et al., 2003). The discrepancy may be due to the different experimental systems used. In Feunou's studies, Tregs were induced by three intraperitoneal injections of S E B in B A L B / c mice while repeated intravenous injection of S E A or S E B were administered to VP3 or Vp8 transgenic mice to generate Tregs in the other report. The different toxin preparations, dosing regimen, strains of mice and toxin administration methods in these studies may have resulted in these disparate findings. Despite the difference in the ability to control the proliferation of target cells, Tregs generated in our system and others all showed suppressive activities on cytokine responses by naive splenocytes. Generally, as antigen-induced proliferation is an inevitable stage that a naive T cell must undergo when it is activated and develops into an effector T cell, proliferation is often used as a readout for the response of T cells when co-culture experiments are performed to determine the 99 suppressive activities of Tregs. However, it must be borne in mind that proliferation is only an indirect reflection of T cell response, but not a direct index for T cell effector functions. Thus the use of proliferation as the only readout of T cell response in some cases may not accurately reflect the T cell effector functions, as is shown in two recent reports. Ehrenstein et al showed that natural Tregs isolated from autoimmune disease patients prior to anti-TNF-a therapy were able to suppress the proliferation of naive T cells but unable to suppress the cytokine production. However, natural Tregs isolated from patients responding positively to anti-TNF-a therapy were able to suppress both proliferation and cytokine production. These data clearly showed that cytokine response was a more accurate readout for T cell effector functions than the proliferative response in certain circumstances (Ehrenstein et al., 2004). Moreover, L i n et al showed that Tregs generated using non-depleting anti-CD4 antibody were able to maintain transplantation tolerance without suppressing the proliferation of C D 8 T cells mediating rejection in vivo (Lin et al., 2002). It has been also demonstrated that superantigens are able to induce T cell responses that included massive proliferation and proinflammatory cytokines production. However, it is the cytokine responses that mediate the pathological processes triggered by superantigens, such as lethal shock (Arad et a l , 2001; Dinges and Schlievert, 2001; Faulkner et al., 2005; Matthys et a l , 1995). Thus in the current study, cytokine as readout for T cell responses was more accurate to reflect the suppressive activities of TSST-1 or SEA-induced Tregs. Therefore, although TSST-1 and SEA-induced Tregs did not suppress the proliferation of their target cells, they were still able to down-regulate cytokine levels in vitro and inhibit lethal shock in vivo. 100 Previous studies have established that repeated exposure of T cells to S E A or S E B resulted in the increased production of IL-10 and the decreased production of IL-2 and IFN-y. This cytokine profile has also been noted in Tregs induced in different systems, with T r l cells as the most typical example. Our results of cytokine levels in the supernatant of SEA-induced Tregs stimulated with S E A were in line with the previously reported data (Feunou et al., 2003; Grundstrom et al., 2003; Noel et al., 2001). In contrast, stimulation of TSST-l- induced Tregs with TSST-1 not only led to decreased level of IL-2 and increased levels of IL-10, but also significantly increased levels of IFN-y and T N F - a . Recently, several studies showed that T cells with regulatory functions also produced IFN-y (Hong et al., 2005; Kemper et al., 2003; Riemekasten et al., 2004; Sawitzki et al., 2005; Stock et al., 2004). The authors in these studies also demonstrated an essential role of IFN-y either in the regulatory functions, or in the induction of Foxp3+ Tregs. However, the roles of I F N - Y m both the generation and regulatory functions of TSST-l- induced Tregs need to be further investigated. In line with the increased level of IL-10 in the supernatant, flow cytometry analysis showed an increased percent of IL-10 positive CD4+ T cells in TSST-1 or SEA-induced Tregs stimulated with their cognate antigens. However, unexpectedly, flow cytometry analysis demonstrated that these Tregs did express other proinflammatory cytokines, such as I F N - Y , T N F - a and IL-2, regardless of changed levels in the supernatant. These data for the first time showed that the decreased levels of proinflammatory cytokines in the supernatant of stimulated superantigen-induced Tregs might not be due to lack of expression, but might be due to inhibited release or enhanced consumption of them by the Tregs themselves. 101 When TSST-1 or SEA-induced Tregs were not stimulated with their cognate antigens, the cytokine responses in vitro were in some degree similar to their suppressive activities. While SEA-induced Tregs stimulated with TSST-1 produced IL-2, LFN-y and T N F - a comparable to the control, TSST-l- induced Tregs stimulated with S E A produced neglectable level of IL-2 and low level o f LFN-y. Thus, the cytokine responses of SEA-induced Tregs were more antigen-specific while those of T S S T - l -induced Tregs were more broadly suppressive. This might be due to the antigen-non-specific suppressive activities of TSST-l- induced Tregs, so that the responses of S E A -reactive CD4+ T cells in the TSST-l -pr imed splenocytes were suppressed when they were stimulated with S E A . The differences in the cytokine secretion profile further confirmed the findings concerning the differences of the suppressive activities between TSST-1 and SEA-induced Tregs. It has been previously shown that exposure to superantigen led to the activation of superantigen-reactive T cells followed by deletion and anergy o f these T cells (Kawabe and Ochi, 1991; MacDonald et al., 1991; Rellahan et al., 1990; White et al., 1989). However, in our system, TSST-1 or SEA-induced Tregs did not become anergic in response to superantigen restimulation in vitro. Instead, they proliferated more vigorously than the control PBS-primed CD4+ T cells, having characteristics of antigen-experienced T cells. Although the proliferation response of PBS-primed CD4+ T cells to TSST-1 or S E A was similar, TSST-l- induced Tregs proliferated more rigorously than SEA-induced Tregs in response to their cognate antigens. This was demonstrated by higher proliferation of TSST-l- induced Tregs in response to different concentrations of cognate superantigen stimulation at day 2 and day 3. This 102 proliferative state of superantigen-induced Tregs was intriguing. Mult iple previous studies showed that generation of Tregs, including superantigen-induced Tregs, was often associated with the development of an anergic state of the Tregs in response to antigen restimulation (Groux et al., 1997; Jonuleit et al., 2000; Levings et al., 2005), although hyper-proliferative Tregs was reported occasionally (Kemper et al., 2003). Our data for the first time showed that superantigen-induced Tregs could also be hyper-proliferative. The factors that might be responsible for this discrepancy with results from previous studies included the toxin preparation, the injection protocol of superantigen, and the strains of mice used. In terms of the cytokine profile and proliferative response, one remarkable difference between TSST-1 and SEA-induced Tregs was the responses of these cells in the absence of cognate superantigen stimulation. While SEA-induced Tregs showed very modest proliferation and cytokine expression in the absence of stimulation and required SEA-stimulation to fully trigger cytokine expression and proliferation, TSST-l-induced Tregs showed remarkable cytokine expression and proliferation in the absence of TSST-1 stimulation. Thus it seemed that TSST-l - induced Tregs showed a more activated phenotype which was characterized by antigen-independent activities. It could be argued that this might be due to the residual binding of the third dose of superantigen to A P C s of the splenocytes which triggered these activities of T S S T - l -induced Tregs in vitro as these cells were isolated two hours after the last dose of superantigen administration. However, this was unlikely because S E A has been shown to have a stronger binding to A P C s than TSST-1 (Pless et al., 2005), yet SEA-induced Tregs did not show similar antigen-independent activities as TSST-l- induced Tregs. 103 Thus, this feature of TSST-l- induced Tregs was probably due to a unique activation process induced by repeated injection of TSST-1 in vivo, which was also suggested by the analysis of cellular markers of these Tregs. This more activated phenotype of TSST-l- induced Tregs may also be partially responsible for their broader suppressive activities because possessing this phenotype might enable them to respond more easily to other stimuli and exert their suppressive functions. While SEA-induced Tregs did not have this phenotype, they might be required to be activated first by their cognate antigen to exert their suppressive function, thus they were not able to suppress the responses triggered by TSST-1 in the absence of S E A . Further studies are required to test this hypothesis. The cell death of TSST-1 or SEA-induced Tregs was also investigated in this study. In the control group, data showed that PBS-primed CD4+ T cells underwent progressive cell death in the absence of stimulation over a five-day in vitro culture. Addit ion of superantigen did not increase the cell death of CD4+ T cells, but decreased the percentage of CD4+ T cells undergoing cell death. These data demonstrated that in this system lack of stimulation caused B6 CD4+ T cell death while provision of stimulation was a survival signal for CD4+ T cells. However, it should be kept in mind that the in vitro culture was a dynamic system in which cell death caused by lack of stimulation, cell death caused by activation and increased cell number due to proliferation induced by stimulation co-exist. The percent of death cells measured was a net result of these three processes. The results showed that in the absence of stimulation, TSST-l- induced Tregs demonstrated a decreased overall percentage of cell death as compared to SEA-induced Tregs and PBS-primed CD4+ T cells. This was 104 probably because TSST-l- induced Tregs were able to undergo antigen-independent proliferation while the other two were not, as i f TSST-l- induced Tregs were stimulated by their cognate antigen. This factor thus sustained their survival and added proliferated CD4+ T cells into the culture, which might offset the cells undergoing cell death due to lack of stimulation or stimulation-induced cell death. A s the concentration of superantigens increased the difference of cell death between TSST-1 and S E A -induced Tregs began to disappear, suggesting that their survival in vitro in response to higher doses of stimulation was similar. However, their survival in vivo was unknown and further studies are required to investigate this issue. Foxp3 has been shown to be a marker associated with the development of naturally Tregs (Hori et a l , 2003). Although it is a reliable marker for natural Tregs, it can not serve as a reliable marker for antigen-induced Tregs. This is because it has been shown that antigen-induced Tregs did not show enhance expression of Foxp3 (Vieira et al., 2004). In this study, we did not detect a significantly increased expression in TSST-1 or SEA-induced Tregs, which was in line with the previous study where the expression of Foxp3 m R N A in SEB-induced Tregs was assayed (Feunou et al., 2003). These data also further confirm that Foxp3 is not a good marker for antigen-induced Tregs. In contrast, the expression of CD25 and C T L A - 4 on TSST-l-induced Tregs was one fold higher than that on SEA-primed CD4+ T cells. Because increased expression CD25 and C T L A - 4 normally could be seen in stimulated T cells, these data indicated that the generation of TSST-1 or SEA-induced Tregs in vivo by repeated injection of these superantigens was associated with a stimulation and activation process, which has been proposed as a mechanism of antigen-induced Tregs 105 in the periphery (Graca et a l , 2005). However, as the expression of these activation markers was much higher in TSST-l- induced Tregs, this suggested that repeated injection of TSST-1 in vivo has a more potent stimulatory signal than S E A . Moreover, our data also demonstrated that antigen-independent proliferation and cytokine expression was much more remarkable in TSST-l- induced Tregs than in SEA-induced Tregs. A s previous studies showed that stimulated T cells could proliferate without further antigen stimulation (Duthoit et al., 2004; Lee et al., 2002), the proliferation and cytokine profile of TSST-l- induced Tregs also indicated that the generation of TSST-l-induced Tregs in vivo was associated with a distinct stimulation and activation process from that elicited by S E A . Why and how this stimulation and activation process elicited by repeated injection of TSST-1 differ from that elicited by S E A definitely needs more extensive investigation. In summary, our data clearly showed that TSST-l - induced Tregs possess properties distinct from those of SEA-induced Tregs. Generation of Tregs holds great therapeutic potential for various clinical conditions, such as transplant rejection and autoimmune diseases (June and Blazar, 2006). Unique properties o f TSST-l- induced Tregs, including their proliferative potential revealed in this comparative study, may have important implications for selecting TSST-l- induced Tregs as a better candidate to be further studied for their clinical application potential. A s TSST-1 could induce Tregs with broader suppressive activities, they might be applied to the treatment of more diseases regardless of the antigens that cause those diseases. Their more potent proliferative response would be another advantage because recent data suggested that better proliferative ability of Tregs in vivo was correlated with more favorable outcome 106 preventing disease (Trenado et al., 2003). Chapter 5: Possible mechanisms of suppressive properties of TSST-l-induced Tregs 5.1. Introduction Regulatory T cells (Tregs), including naturally occurring CD4+CD25+Foxp3+ Tregs (Sakaguchi, 2004; Sakaguchi, 2005) and antigen-induced Tregs (Bluestone and Abbas, 2003; Vigouroux et al., 2004), have been shown to play an important role in modulating the immune response. While naturally occurring CD4+CD25+ Tregs are generated in the thymus, antigen-induced Tregs are generated in the periphery after antigen encounter. Mechanisms underlying suppression by different types of Tregs are poorly defined. Possible mechanisms included: 1) cytotoxic pathway (Grossman et al., 2004; Janssens et al., 2003; Zhao et a l , 2006); 2) inhibition of activation of target cells (Barthlott et al., 2005; de la Rosa M et al., 2004; George T C et al., 2003; Trenado et al., 2006); 3) infectious tolerance, referring to the ability of Tregs to render their target cells to acquire regulatory functions (Alpan et al., 2004; Dieckmann et al., 2002; Dieckmann et al., 2005; Jonuleit et al., 2002; Unger et al., 2003); 4) soluble factors mediating suppression (Asseman et al., 1999; Fahlen et al., 2005; Grundstrom et al., 2003; McGui rk et al., 2002); and 5) cytokine competition (Barthlott et al., 2005; de la Rosa M et al., 2004; Gunther J et al., 1982; Scheffold A et al., 2005). Staphylococcal superantigens, such as TSST-1 , activate CD4+ T cells by crosslinking M H C class II molecules and the T cell receptor bearing specific VP segments (Dinges et al., 2000). Experimentally, chronic exposure to superantigen has been shown to effectively induce Tregs (Feunou et al., 2003; Grundstrom et al., 2003; Mi l l e r et al., 1999; Noel et al., 2001). In the previous chapter it has been shown that 108 repeated injection of TSST-1 in C57BL/6 mice could induce CD4+ Tregs with distinct characteristics from those induced by S E A . TSST-l- induced Tregs demonstrated suppressive activities on both TSST-1 and S E A in vitro and in vivo. They down-regulated the cytokine responses induced by TSST-1 or S E A in vitro, and suppressed lethal shock induced by either TSST-1 or S E A . In this chapter, the possible mechanisms of TSST-l- induced Tregs are examined. 5.2. Experimental Design The possible mechanisms of TSST-l- induced Tregs were investigated. To determine i f TSST-l- induced Tregs caused cell death or inhibited the activation of target cells, Tregs were co-cultured with CFSE-labelled naive splenocytes in the presence of TSST-1 and then the cell death or expression of activation markers on naive splenocytes were assayed by F C M . To study i f target cells acquired regulatory functions after co-culture with Tregs, the naive splenocytes were co-cultured with CFSE-labelled Tregs and reisolated for assay of their cytokine profile and suppressive functions. To determine the role of soluble factors in the suppression by Tregs, splenocytes containing Tregs were stimulated with TSST-1 and then the supernatant was transferred to co-culture with naive splenocytes. Cytokine responses were assayed by E L I S A . To investigate i f Tregs could consume cytokines produced by target cells, naive splenocytes were stimulated with TSST-1 or S E A , then the supernatant was transferred to co-culture with Tregs. Cytokines in the supernatant were determined by E L I S A . A flow diagram of the experimental design is shown below. 109 P B S , x3 TSST-1 , x3 ^ 2 ^O-Tregs Naive mice ® C F S E staining Naive splenocytes ^^ -j Transfer of supernatant / Co-culture Apoptosis of naive SC by F C M Activation marker ® Naive SC were reisolated by F A C S expression on naive SC by F C M i Splenocytes 1 Assay cytokines of supernatant Transfer of supernatant Assay of cytokine profile and suppressive functions Assay cytokines of supernatant Stimulated with TSST-1 Figure 5.1. Experimental design of studies described in Chapter 5. 110 5.3. Results 5.3.1. TSST-l- induced Tregs did not enhance cell death of the target cells To investigate whether TSST-l- induced Tregs exerted their functions by inducing cell death of their target cells, naive splenocytes were stained with C F S E and co-cultured with TSST-1-indcued Tregs in the presence of TSST-1 . Ce l l death of the target cells was then determined by flow cytometry at different time point post stimulation. A s shown, in Figure 5.2, addition of TSST-l- induced Tregs to naive splenocytes did not lead to enhanced cell death of the target cells at either day 1 or day 2 post stimulation as compared to na'ive splenocytes alone or naive splenocytes co-cultured with PBS-primed control CD4+ T cells. Therefore, these data showed that apoptosis induced by TSST-l- induced Tregs was not responsible for their suppressive activities. 5.3.2. TSST-l- induced Tregs enhanced the activation of target cells Activation is a critical step of the T cell response to the cognate antigens, and is subject to the regulation by Tregs. Thus, efforts were made to determine i f T S S T - l -induced Tregs inhibited the functions of their target cells by suppressing the activation of the target cells in response to TSST-1 stimulation. To this end, naive splenocytes were first labeled with C F S E and then co-cultured with TSST-1-indued Tregs. The activation state of the target cells was then determined by analysis of the expression of CD25 and CD69 on target CD4+ T cells at different time points post stimulation. A s shown in Figure 5.3, surprisingly, co-culture o f naive splenocytes wi th T S S T - l -induced Tregs resulted in enhanced, rather than decreased, expression of CD25 and 111 63 .9% Naive+T 59.4% Naive+TSST-lTre2f5:4)+T NaTve+PBS-T(5:4)+T W t / i u safi v i ^ - . ; - . 5 0 > 8 % i o Q i n ' I D 2 i o 3 7 - A A D : : 51.7% B Naive splenocytes TSST-l-Tregs (5:4) TSST-l-Tregs (5:1) PBS-CD4 T (5:4) PBS-CD4 T (5:1) TSST-1 RPMI + + + + + + + + Figure 5.2. Effect of TSST-l- induced Tregs on the cell death of target cells. Naive splenocytes (2xl0 5 /wel l ) were stained with C F S E and co-cultured with TSST-1 -inducd Tregs or control PBS-primed CD4+ T cells (ratio of na'ive splenocytes to TSST-l- induced Tregs or PBS-primed CD4+ T cells at 5:4 and 5:1) in the presence of TSST-1 . A t 24 and 48 hours, cells were stained with 7 - A A D followed by flow cytometry analysis. A . Representative data of flow cytometry analysis of cells at day L B . Accumulated data from four independent experiments. A s shown, addition of TSST-l- induced Tregs did not result in enhanced cell death of the na'ive splenocytes. Data shown were accumulated results of four independent experiments (n=4). 112 A Naive+RPMI Naive+TSST-1 tf|8 10% • ..".'"".rT; ; i5% o Naive+TSST-1 Treg(5:4HT Naive+TSST-1 Treg(5:l)+T '50%; 35% .Naive+PBS CD4 T(5:4HT k Naive+PBS CD4 T(S:1)+T ta U 31% p i 20% i CD25 F i g u r e 5 . 3 . E f f e c t o f T S S T - l - i n d u c e d T r e g s o n t h e a c t i v a t i o n o f t a r g e t c e l l s . A . R e p r e s e n t a t i v e r a w d a t a o f f l o w c y t o m e t r y a n a l y s i s o f C D 2 5 e x p r e s s i o n o n n a i v e s p l e n o c y t e s c o - c u l t u r e d w i t h T S S T - l - i n d u c e d T r e g s . N a i v e s p l e n o c y t e s ( 4 x l 0 5 / w e l l ) w e r e s t a i n e d w i t h C F S E a n d c o - c u l t u r e d w i t h T S S T - l - i n d u c e d T r e g s o r c o n t r o l P B S - p r i m e d C D 4 + T c e l l s ( r a t i o o f n a i v e s p l e n o c y t e s t o T S S T - l - i n d u c e d T r e g s o r P B S - p r i m e d C D 4 + T c e l l s a t 5 : 4 a n d 5 : 1 ) i n t h e p r e s e n c e o f T S S T - 1 . A t 2 4 a n d 4 8 h o u r s , c e l l s w e r e s t a i n e d w i t h a n t i - C D 2 5 a n d a n t i - C D 4 a n t i b o d i e s f o l l o w e d b y f l o w c y t o m e t r y a n a l y s i s . D a t a s h o w n w e r e r e p r e s e n t a t i v e d a t a o f f l o w c y t o m e t r y a n a l y s i s o f C D 2 5 e x p r e s s e d o n n a i v e c e l l s a t d a y 2 . 1 1 3 B + Q U ^ cu -> w •a JS + Z Q U + Q U ^ Qi 0 s + U O N H Q 50n 40H 3 0 H 20H 10H CD69 Naive Splenocytes + TSST-l-Tregs (5:4) TSST-l-Tregs (5:1) PBS-CD4 T (5:4) PBS-CD4 T (5:1) TSST-1 RPMI + + + + + + *_§*§ J J + + + + + + Day 1 Day 2 i + + + Figure 5.3. B. Data shown were accumulated results of four independent experiments (n=4). *, PO.05, compared with naive splenocytes, paired t-test (Day 1 or Day 2). §, PO.05, compared with nai've splenocytes+PBS-primed CD4+ T cells, paired t-test (Day 1 or Day 2). 114 CD69 on na'ive CD4+ T cells as compared to naive splenocytes alone. In the control groups, adding PBS-primed splenocytes to na'ive splenocytes also increased the expression of CD25 and CD69 on naive CD4+ T cells. Therefore, TSST-l- induced Tregs enhanced, rather inhibited, the activation of their target cells. 5.3.3. TSST-l- induced Tregs did not render their target cells to acquire regulatory functions Since TSST-l- induced Tregs enhanced the activation of their target cells, it was then asked i f TSST-l- induced Tregs could render their target cells to acquire regulatory function. To test this hypothesis, the TSST-l- induced Tregs were first stained with C F S E and then co-cultured with na'ive splenocytes for 48 hours. The naive splenocytes were then reisolated by F A C S and analyzed for their suppressive activities by co-culture experiments. A s shown in Figure 5.4, reisolated naive splenocytes had a proinflammatory cytokine production profile similar to the control groups in response to TSST-1 re-stimulation. Co-culture of the re-isolated splenocytes with fresh na'ive splenocytes did not result in any down-regulation of proinflammatory cytokine levels. Concurrent re-isolated TSST-l- induced Tregs still had regulatory functions, demonstrating that the lack of regulatory activities in the co-cultured naive splenocytes was not due the isolation procedure (data not shown). Thus, infectious tolerance was not involved in the activities of TSST-l- induced Tregs. 115 s = S = o U 1250n 1000-750-500-250-IL-2 0 400i 300-200-10O 0 800-600-400-200-0-IL-12 IFN-y Naive SC(4xl05) + Tregs-cocultured SC (8x10s) Tregs-cocultured SC (4xl05) PBS-T-cocultured SC (8x10s) PBS-T-cocultured SC (4xl05) TSST-1 + + + JL T + + + + + + + + + + + + + + Figure 5.4. TSST-l- induced Tregs did not render the target cells to acquire regulatory functions. Naive splenocytes were first co-cultured with CFSE-stained TSST-l- induced Tregs or PBS-primed CD4+ T cells at the ratio of 5:4 in the presence of TSST-1 for 48 hours. Then dead cells were removed and naive splenocytes were re-isolated by F A C S . The re-isolated splenocytes were co-cultured with freshly prepared naive splenocytes at two ratios or cultured alone in the presence of TSST-1 for 48 hours. Various cytokines in the supernatant were assayed by E L I S A . A s shown, splenocytes co-cultured with TSST-l- induced Tregs still showed a proinflammatory cytokine profile in response to TSST-1 stimulation as compared to freshly prepared na'ive splenocytes and splenocytes co-cultured with PBS-primed CD4+ T cells. They did not show any suppressive activity on the cytokine response of the freshly prepared na'ive splenocytes. Data shown were accumulated results of three independent experiments (n=3). 116 5.3.4. Supernatant of stimulated TSST-l- induced Tregs did not mediate suppressive activities Tregs have been well-known for producing soluble immunosuppressive factors, such as IL-10, to mediate their suppressive activities. To determine the role of soluble factors in mediating their regulatory functions, TSST- l -pr imed splenocytes, which contained TSST-l- induced Tregs and A P C s , were first stimulated with TSST-1 , then the supernatant was harvested and used to culture na'ive splenocytes to determine i f the responses of target cells could be down-regulated in the presence of medium containing soluble factors produced by stimulated TSST-l- induced Tregs. A s shown in Figure 5.5, transfer of TSST-1-conditioned medium from TSST- l -pr imed splenocytes to na'ive splenocytes did not result in decreased levels of IL-2 and IL-12, compared to na'ive splenocytes cultured in control medium. Increasing the number of TSST-l- induced Tregs up to four-fold did not render the supernatant to have suppressive ability. Thus, these data showed that the suppressive activities of TSST-l- induced Tregs were not solely mediated by soluble factors produced by these Tregs. 5.3.5. Suppressive activities of TSST-l- induced Tregs were cell contact-dependent Since the experiments above did not reveal an exclusive role for soluble factors in the suppressive functions of TSST-l- induced Tregs, it was reasoned that T S S T - l -induced Tregs may depend on cell-contact with their target cells to exert their functions. To test this hypothesis, a permeable transwell was added to the co-culture of T S S T - l -induced Tregs and naive splenocytes to determine i f separation o f these two populations would lead to the reversal o f suppression. A s shown in Figure 5.6, 117 150Oi IL-2 -S ~5JD a. e o cs J . c « a o U lain sow 6001 40tH IL-12 20CH 0 Naive Splenocytes Fresh RPMI Tregs supernatant (8><105) Tregs supernatant (2*106) TSST-1 I I I + + + + + + + + + Figure 5.5. The role of soluble factors in the suppressive function of T S S T - l -induced Tregs. TSST-l- induced Tregs ( 8 x l 0 5 / w e l l or 2 x l 0 6 / w e l l ) together with antigen presenting cells were first stimulated with TSST-1 for 48 hours. Supernatant then was collected and used to culture naive splenocytes ( 4 x l 0 5 /well) for another 48 hours in the presence of TSST -1 . Supernatant then was assayed by E L I S A for IL-2 and IL-12 levels. Naive splenocytes cultured in the Tregs supernatant produced similar level of IL-2 and IL-12 as compared to naive splenocyte in fresh medium or control medium, showing that soluble factors contained in the Tregs supernatant were not responsible for the suppressive effects of TSST-l- induced Tregs. Data shown were accumulated results of four independent experiments (n=4). 118 2000-1 fl c 1500-a e 1000-« * c 500-w U 0 X Naive Splenocytes T S S T - l - T r e g s Transwel l membrane TSST-1 PBS-CD4 T • IL-2 1 + + + + + + + + + + + I 1 e c o c o c o c c o c o c o c c o c o c o Figure 5.6. Cel l contact-dependency of TSST-l- induced Tregs. Naive splenocytes (2xl0 6 /wel l ) were co-cultured with TSST-l -pr imed splenocytes in the presence of TSST-1 at the ratio of 1:1 and were separated by a permeable transwell for 48 hours. Supernatant was assayed for IL-2 and IL-12 by E L I S A . A s shown, high levels of IL-2 and IL-12 were detected in supernatant o f naive splenocytes stimulated with TSST-1 . Addition of TSST-l -pr imed splenocytes containing Tregs to naive splenocytes decreased levels of IL-2 and IL-12. Separation of naive splenocytes and TSST-l -pr imed splenocytes with a permeable transwell resulted in restoration of 80% of IL-2 and 100% of IL-12 levels. Addit ion of PBS-primed splenocytes did not led to decreased levels of IL-2 and IL-12. The data showed that suppressive effects of TSST-l- induced Tregs were cell-contact dependent or required the Tregs to be in the vicinity of their target cells. Data shown were accumulated results of four independent experiments (n=4). 119 separation of TSST-l- induced Tregs and naive splenocytes with a permeable transwell membrane did lead to the reversal of suppression, as IL-2 and IL-12 levels in this group were restored to 80% and 100% of control levels, respectively. Therefore, cell contact was required for TSST-l- induced Tregs to exert their functions. 5.3.6. Blockade of IL-10 with an anti-IL-lOR monoclonal antibody did not reverse the suppressive functions of TSST-l- induced Tregs IL-10 was one of the major immunosuppressive cytokines that mediated the suppression by Tregs. In general, soluble IL-10, such as that produced by T r l cells, is responsible for their suppressive activities. However, a recent study did clearly show that IL-10 had to exert its suppressive effects in the context of cell contact (Alpan et al., 2004). Thus, although it was demonstrated above that soluble factors did not exclusively mediate the suppression by TSST-l- induced Tregs and cell contact was actually required for their suppressive activities, it was still possible that IL-10 might play a role in the suppression by TSST-l- induced Tregs. To further determine the role of IL-10, a monoclonal antibody against the IL-10 receptor was added to the co-culture of TSST-l- induced Tregs and naive splenocytes to block the actions of IL-10. The results showed that addition of the anti-IL-10 receptor antibody did not reverse the suppressive activities of TSST-l- induced Tregs as levels of proinflammatory cytokines were still down-regulated (Figure 5.7). This was not due to insufficient concentration of anti-IL-10 receptor antibody which had been raised to 10 times the concentration 120 1 5 0 0 T IOOOI 500-IL-2 & a o v» «s u fl w fl o U o-400" 300" 200-100-o-' 800-600" 400" 200" 0J IL-12 I F N - y TSST-1 + + + + Naive Splenocytes + + + + T S S T - l - T r e g s + + + a - I L - l O R A b + Cont ro l A b + Figure 5.7. Effect of addition of anti-LL-10 receptor antibody on the suppressive function of TSST-l- induced Tregs. Naive splenocytes were co-cultured with Tregs at the ratio of 5:1 (for IL-2 and IL-12) or 5:4 (for IFN-y) in the presence of anti-IL-10 receptor antibody for 48 hours and supernatant was assayed for various cytokines by E L I S A . Addition of anti-IL-10 receptor antibody (lOOpg/mL) did not reverse the levels of IL-2, IL-12 and IFN-y as compared to those in the absence of this antibody or in the presence of control antibody. Data shown were accumulated results of four independent experiments (n=4). 121 previously shown to be sufficient to block IL-10 actions in vitro (O'Farrell A M et al., 1998). Taken together, these data suggest that TSST-l- induced Tregs do not depend on IL-10 to exert their suppressive activities. 5.3.7. Intracellular cytokine expression by naive splenocytes was not suppressed in the presence of TSST-l- induced Tregs Results of the experiments above led us to look for other mechanisms responsible for the suppressive activities of TSST-l- induced Tregs. Several reports have shown that cytokine competition was an important mechanism by which natural Tregs controlled the responses of their target cells (Barthlott et al., 2005; de la Rosa M et al., 2004). Thus, it was hypothesized that cytokine competition also played a role in the suppressive activities of TSST-1-induded Tregs. To this end, it was essential to first determine the cytokine production by naive splenocytes in the co-culture with T S S T - l -induced Tregs. In the co-culture system E L I S A was performed to assay the cytokine levels in the supernatant. However, this method could only determine the net result of cytokine production and consumption. Therefore, a reduction in the level of a specific cytokine might be due to reduced production or enhanced consumption. Intracellular staining of cytokine followed by flow cytometry analysis was performed to address this issue. Naive splenocytes were first stained with C F S E and then co-cultured with TSST-l-induced Tregs for 45 hours, with Golgistop added to the culture for the last 5 hours to stop the cytokine secretion. Cells were then stained for cytokines and analyzed by flow cytometry. A s shown in Figure 5.8, co-culture of TSST-l- induced Tregs with naive splenocytes did not lead to the decreased expression of either IL-2 or IFN-y on 122 Nai've+RPMI Naive+TSST-1 p -1 2.5% 16% MFI:22 u t 17% p f : v ; > v ; ' 'MFI:37 10" 10' 10- 10J IL-2-APC 111 11% |g;v;.>'-;,.MFI:42 10° io1 io* i o 3 io IL-2-APC Naive+TSST-1 Treg(5:l)+T |*U 19% |;>: MFI:24 io° io1 io 2 io 1 io Naive+PBS CD4 T(5:l)+T tvv. 11% ;^f::- -viVlFI:37 io° io1 io- io J io IL-2-APC • • IL-2 Figure 5.8. Expression of IL-2 by naive splenocytes in the presence of TSST-l-induced Tregs. A . Representative raw data of expression of IL-2 by naive splenocytes in the presence of TSST-l- induced Tregs. CFSE-labelled naive splenocytes were co-cultured with TSST-l- induced Tregs at the ratio of 5:1 or 5:4 in the presence of TSST-1 for about 45 hours. Golgistop was added for the last 5 hours. Intracellular staining was performed for IL-2 as described in Methods and Materials followed by flow cytometry analysis. Data shown were IL-2 expression by na'ive splenocytes in the presence of T S S T - l -induced Tregs from one of four independent experiments. Na'ive splenocytes cultured in the absence of TSST-1 stimulation served as the negative control for IL-2 exoression. 123 B 4> > o a. _ | 8 ^ s o U 2 2 ° E e 20-i 15-10-5-0-40-30-20-10-0-Na'ive Splenocytes TSST-l-Tregs (5:4) TSST-l-Tregs (5:1) PBS-CD4 T (5:4) PBS-CD4 T (5:1) TSST-1 X + IL-2 + + + + + + + X " - 1 — + + + X + + + Figure 5.8. B . Intracellular IL-2 expression by naive splenocytes in the presence of TSST-l- induced Tregs. Data shown were accumulated from four independent experiments, n=4. 124 a. 20i > ^ O ^ S i 15H £ s O U 10 X IFN-Y o "S .s + HH W HH B 40i 30H 201 10 0 J Naive Splenocytes TSST-l-Tregs (5:4) TSST-l-Tregs (5:1) PBS-CD4 T (5:4) PBS-CD4T(5:1) TSST-1 X + X + + + + + + + X + + x + + + + Figure 5.8. C. Intracellular IFN-y expression by naive splenocytes in the presence of TSST-l- induced Tregs. Data shown were accumulated from three independent experiments, n=3. 125 stimulated splenocytes. The mean fluorescence intensity (MFI) of the IL-2 positive naive splenocytes co-cultured with TSST-l- induced Tregs was decreased as compared to that of naive splenocytes alone, while the M F I of IFN-y positive naive splenocytes co-cultured with TSST-l- induced Tregs was unaffected. Thus these data suggested that although the levels of proinflammatory cytokines in the supernatant were decreased by TSST-l- induced Tregs, the expression of these cytokines by their target cells might not be suppressed. The results of these experiments led to the hypothesis that cytokines produced by naive splenocytes were either prevented from being released to the supernatant or were consumed by TSST-l- induced Tregs. The latter possibility then was investigated in the following studies. 5.3.8. TSST-l- induced Tregs competed for IL-2 with naive splenocytes To investigate i f TSST-l- induced Tregs could consume cytokines produced by naive splenocytes, naive splenocytes were first stimulated with TSST-1 or S E A for 48 hours and then the supernatant was harvested and cultured with TSST-l- induced Tregs for another 48 hours. Cytokine levels in the supernatant were assayed by E L I S A . A s shown in Figure 5.9, culture o f T S S T - l - p r i m e d splenocytes containing T S S T - l -induced Tregs in TSST-1 or SEA-conditioned supernatant resulted in decreased levels of IL-2, but not IL-12 or IFN-y as compared to the supernatant cultured with no cells or with PBS-primed splenocytes. To further prove that IL-2 in the conditioned medium was consumed, an anti-CD25 antibody was added to the culture to block the interaction of IL-2 and its high affinity receptor CD25. Addition of this antibody restored the level of IL-2 in the TSST-l-conditioned supernatant cultured with TSST-l- induced Tregs 126 s "Si 3 S3 o e u e o U 2000' 1500' 1000' 500' TSST-l-conditioned supernatant IL-2 o 1000-800-600-400-200-0-1000' 800' 600' 400' 200' 0-IL-12 IFN-y TSST-l-conditioned supernatant TSST-l-Tregs (0.8x10s) TSST-l-Tregs (3.2*105) PBS-CD4 T (0.8x10s) PBS-CD4 T (3.2x10s) + + + + - r -+ + + + + Figure 5.9. Competition of IL-2 by TSST-l- induced Tregs. Naive splenocytes (2x l0 6 /we l l ) were stimulated with TSST-1 or S E A for 48 hours. Then the supernatant was collected and used to cultured TSST-l- induced Tregs (0 .8x l0 5 /we l l or 3 .2x l0 5 /wel l ) in the presence of antigen presenting cells for another 48 hours. Supernatant was then assayed for various cytokines by E L I S A . A . Culture of TSST-1 conditioned naive splenocyte supernatant with T S S T - l -induced Tregs resulted in the significantly decreased level of IL-2 as compared to supernatant cultured with no cells or with PBS-primed C D 4 + T cells. IL-12 and IFN-y levels were not affected by the co-cultured of TSST-1 or S E A -conditioned naive splenocyte supernatant with TSST-l- induced Tregs. The data indicated that TSST-l- induced Tregs could consume IL-2 produced by the naive splenocytes. *, P<0.05, compared with TSST-l-conditioned supernatant alone, paired t-test. Data shown were accumulated results from four independent experiments (n=4). 127 B ta s "bio C-C o S3 i-s <j B © U 2000' 1500' 1000-500-0-1000 800 600 400 200 0 1000-800-600-400-200-0-SEA-conditioned supernatant IL-2 SEA-conditioned supernatant TSST-l-Tregs (0.8x10s) TSST-l-Tregs (3.2x 10s) PBS-CD4 T (0.8x10s) PBS-CD4 T (3.2xl05) IL-12 IFN-y + + + + + + + + + Figure 5.9. B . Culture of SEA-conditioned naive splenocyte supernatant with TSST-l- induced Tregs resulted in the significantly decreased level of IL-2 as compared to supernatant cultured with no cells or with PBS-primed CD4+ T cells. IL-12 and IFN-y levels were not affected by the co-cultured of TSST-1 or SEA-conditioned naive splenocyte supernatant with TSST-l- induced Tregs. The data indicated that TSST-l- induced Tregs could consume IL-2 produced by the naive splenocytes. *, P O . 0 5 , compared with SEA-conditioned supernatant alone, paired t-test. Data shown were accumulated results from four independent experiments (n=4). 128 —1 s a o i -C w u C o 2000' 1500' 1000' 500' TSST-l-conditioned supernatant 0-»-r 2000' 1500' 1000' 500-0' SEA-conditioned supernatant TSST-l/SEA-conditioned supernatant + + + + + + + TSST-l-Tregs (0.8xl05) + ' + + TSST-l-Tregs (3.2xl05) + + + a-CD25Ab + + Control Ab + + Figure 5.10. Effect of anti-CD25 antibody on the uptake of IL-2 by T S S T - l -induced Tregs. In experiments described in F ig 5.9, groups were set up where monoclonal antibody against CD25, the high affinity receptor subunit of IL-2 receptor, was added to the co-culture of TSST-1 or SEA-conditioned naive splenocyte supernatant and TSST-l- induced Tregs. A s shown, TSST-1 or S E A -conditioned naive splenocyte supernatant contained high level of IL-2 when it was cultured with no cells for 48 hours. In the presence of TSST-l- induced Tregs, IL-2 level decreased to undetectable level while addition anti-CD25 antibody (10|j.g/mL) restored the level of IL-2 to that in the absence of any cells. Control antibody was not able to restore the IL-2 level. The data further confirmed that IL-2 was uptaken via IL-2 receptor. Data shown were accumulated results of four independent experiments (n=4). *, P<0.05, paired t-test, n=4. 129 TSST-l-Tregs PBS-CD4 T cells Figure 5.11. Expression of CD25 on TSST-l- induced Tregs in the co-culture with naive splenocytes. In experiments described in Figure 5.3, the expression of CD25 on TSST-l- induced Tregs was also assayed simultaneously. TSST-l- induced Tregs could be identified as C F S E negative and C D 4 positive population. A s shown, TSST-l- induced Tregs had higher expression of CD25 than PBS-primed CD4+ T cells. Data were accumulated from four independent experiments. *, P<0.05, compared with P B S - C D 4 T cells, t-test, n=4. 130 (Figure 5.10). In addition, the expression of CD25 on TSST-l- induced Tregs in the co-culture was also assayed. A s shown in Figure 5.11, CD25 was highly expressed on TSST-l- induced Tregs. It could be as high as 70% of the TSST-l- induced Tregs at days of the co-culture. Thus collectively, these data showed that TSST-l- induced Tregs down-regulated the IL-2 level by active consumption of this cytokine. 5.3.9. Addition of exogenous IL-2 did not reverse the suppression of TSST-l- induced Tregs Since IL-2 plays an important role in the T cell responses and TSST-l- induced Tregs were able to deprive their target cells of this important cytokine, it was reasonable to ask i f TSST-l- induced Tregs suppressed their target cells by preventing their target cells from accessing IL-2. To address this issue, exogenous IL-2 was added to the co-culture of naive splenocytes and TSST-l- induced Tregs. A s shown in Figure 5.12, the addition of exogenous IL-2 did not reverse the suppressive activities of TSST-l-induced Tregs, suggesting that the presence of additional mechanisms to control cytokine responses of naive splenocytes by TSST-l- induced Tregs. 131 800n IL-12 -J s "ex 3 a .© « ts U c o V 600-400-200-0-800n 600-400' 200' I F N - y Naive Splenocytes + T S S T - l - T r e g s TSST-1 + IL-2 (10 ng/ml) + + + + + + + + + + Figure 5.12. Effect of addition of exogenous IL-2 on the suppression mediated by TSST-l- induced Tregs. Naive splenocytes were co-cultured with TSST-l- induced Tregs at the ratio of 5:4 in the presence of TSST-1 and exogenous IL-2 (lOng/mL) for 48 hours and supernatant was assayed for IL-12 and IFN-y by E L I S A . A s shown, IL-12 and LFN-y were produced by naive splenocytes in response to TSST-1 . Addit ion of exogenous IL-2 (lOng/mL) to naive splenocytes did not alter the IL-12 and LFN-y- Co-culture of naive splenocytes with TSST-l- induced Tregs resulted in decreased levels of IL-12 and LFN-y. Addition of IL-2 to the co-culture did not restore the levels of IL-12 and IFN-y. The data suggested that regulation of IL-12 and LFN-y by TSST-l- induced Tregs differed from that of IL-2. Data shown were accumulated results of four independent experiments (n=4). 132 5.4. Discussion Previous studies on the mechanisms of Tregs induced by superantigens, such as S E A or S E B , showed that these cells mainly rely on soluble factors to mediate their effect (Miller et al., 1999; Noel et a l , 2001). In the last chapter, our studies showed that co-culture of TSST-l- induced Tregs with naive splenocytes in vitro led to decreased levels of proinflammatory cytokines. Thus in this chapter, the possible mechanisms of TSST-l- induded Tregs were investigated. We first determined i f TSST-l-induced Tregs induced apoptosis of their target cells. This mechanism has been noted in Tregs in several published studies with different antigens (Grossman et al., 2004; Janssens et al., 2003; Zhao et a l , 2006). However, in our system, co-culture of TSST-l-induced Tregs did not result in enhanced cell death of the target cells, showing that induction of cell death of target cells was not the major mechanism. It has been demonstrated in vivo and in vitro that activation of target cells could be inhibited by naturally occurring Tregs as up-regulation of activation markers, such as CD25, was suppressed. Interestingly, co-culture of TSST-l - induced Tregs did not lead to down-regulation of CD25 and CD69 expression; instead, up-regulation of CD25 and CD69 was noted. These data showed not only that the activation of naive CD4+ T cells was not inhibited, but also that TSST-l- induced Tregs might cause activation of their target cells. We then further determined i f co-culture of these Tregs with their target cells could render the target cells to acquire regulatory functions. However, re-isolated naive splenocytes co-cultured with TSST-l- induced Tregs did not show any suppressive activities in secondary co-culture experiments, indicating that 133 activation of target cells in the presence of TSST-l- induced Tregs did not render them to acquiring regulatory functions. Soluble factor-mediated suppression was the most well-documented mechanism of Tregs, where IL-10 was one of the major immunosuppressive cytokines. Our previous data did show that TSST-l -pr imed splenocytes produced high levels of IL-10 and a high percentage of TSST-l- induced Tregs expressed IL-10 by flow cytometry analysis. Yet, our data in this study did not reveal any role for soluble factors or IL-10 in the suppression mediated by TSST-l- induced Tregs. Thus, in contrast to previous reports with S E A or SEB-induced Tregs (Noel et a l , 2001), TSST-l- induced Tregs in C57BL/6 mice did not depend exclusively on IL-10 or other soluble factors to mediate their suppressive effects, as least in vitro. These data are in line with the observation that the suppressive activities of TSST-l- induced Tregs are mainly cell-contact dependent, as separation of TSST-l- induced Tregs from their target cells using a permeable membrane almost completely reversed their suppressive effects. For some types of Tregs, their cytokine profile is highly associated with their suppressive activities. For instance, T r l cells depend on the production of high levels of soluble IL-10 to exert their functions, as neutralization of IL-10 can totally block their suppression (Groux et al., 1997; McGui rk et al., 2002). However, some groups have reported that soluble IL-10 or other immunosuppressive factors were not exclusively responsible for the suppressive effects mediated by Tregs in different systems (Chen et al., 2003b; Steinbrink et al., 2002; Tang et a l , 2004). Thus, there are non-redundant mechanisms for Treg-mediated suppression, depending on the local environment and immune response. 134 Cytokine competition has long been recognized as one of the important arms in the control mechanisms for immune responses. Several recent papers have suggested that cytokine competition may be one of the major mechanisms for the regulation mediated by CD4+CD25+ natural Tregs (Barthlott et al., 2005; de la Rosa M et al., 2004). These naturally occurring Tregs constitutively express IL-2 receptor CD25 but fail to produce IL-2 when activated. However, they can compete with their target cells for IL-2, leading to suppression of activation or effector function of their target cells. Our data clearly showed that TSST-l- induced Tregs could consume IL-2, but not other cytokines produced by target cells stimulated with TSST-1 or S E A . This can explain why TSST-l- induced Tregs could down-regulate IL-2 level when they were co-cultured with naive splenocytes stimulated with either TSST-1 or S E A . In contrast to inhibition by naturally occurring Tregs, where the addition of IL-2 to the co-culture can reverse the inhibition, addition of exogenous IL-2 did not reverse the suppression mediated by TSST-l- induced Tregs. This could be explained by the role of IL-2 in different suppression assay systems. In the co-culture system used to assay the suppressive functions of natural Tregs, proliferation of target cells, which is highly IL-2 dependent, is used as the readout of response (de la Rosa M et al., 2004). However, for superantigen-induced proliferation and cytokine production, IL-2 might not play a central role as T cells from IL-2 or IL-2 receptor deficient mice still responded normally to S E B stimulation as compared to wi ld type mice (Jin et al., 2006). Thus, although exogenous IL-2 was added to offset the decreased level of IL-2 available to the target cells due to consumption by TSST-l- induced Tregs, suppression of IL-12 and IFN-y by TSST-l- induced Tregs still existed. 135 These findings do not necessarily mean that IL-2 is not important in the superantigen-induced responses. IL-2 is one of the main cytokines induced by superantigens (Rink L et al., 1996). The role of IL-2 in superantigen-induced responses could be shown by the suppressive effects o f cyclosporine A on lethal shock, which targeted the activation o f T cells by blocking IL-2 production (Miethke T et al., 1993). Additionally one study also demonstrated that blockade of IL-2 by antibody in vivo could partially inhibit lethal shock induced by superantigens (Mountz et al., 1995). Thus, it is possible that consumption of IL-2 by TSST-l- induced Tregs was one of the major mechanisms for these cells to exert their suppressive functions. Further studies are required to confirm its role in vivo. In contrast to the regulation of IL-2 by TSST-l- induced Tregs, mechanisms of regulation of IL-12 and IFN-y by these cells are less clear. Our data excluded cytokine competition as the responsible mechanism. Although flow cytometry analysis showed that target cells did express these cytokines induced by TSST-1 , in the presence of TSST-l- induced Tregs, the levels of these cytokines in the supernatant were decreased. Taken together, these data suggest that TSST-l- induced Tregs may inhibit the release of these cytokines by the target cells. The identification of specific molecules that mediate this effect w i l l be one of the focuses in future studies. In summary, we sought to elucidate the mechanisms of suppression mediated by TSST-l- induced Tregs. We found that TSST-l- induced Tregs regulated the actions of IL-2 by competition for and consumption of IL-2 from their target cells. They suppressed cytokine responses other than IL-2 via a cell-contact dependent manner. 136 The molecules responsible for the latter mechanism are unknown and require further investigation. 137 Chapter 6: Potential application of TSST-l-induced Tregs in the control of acute graft-versus-host disease 6.1. Introduction Although allogeneic hematopoietic stem cells transplantation ( A H S C T ) is a curative therapy for certain blood malignancies, a G V H D remains a major complication and main barrier to A H S C T . A s mentioned earlier, superantigen administration has been shown to regulate certain diseases, including transplantation rejection (Pan et al., 2003). One previous study investigated the effects of S E B administration on acute graft-vs-host response using a non-lethal murine model where parent splenocytes were infused to unconditioned F l mice (Takenaka et al., 2001). They showed that S E B administration could down-regulate the allogeneic responses due to the ability of S E B to induce deletion of SEB-reactive T cells which happened to be alloreactive. However, this model is of limited clinical relevance and it was not known i f superantigen-induced Tregs could mediate dominant suppression on a G V H D or not. In the previous chapter, it was shown that TSST-l- induced Tregs were able to suppress the T h l cytokine response by naive splenocytes. Because T h l cytokine response has been shown to play a critical role in the pathogenesis of a G V H D , it can be hypothesized that T S S T - l -induced Tregs may also be able to suppress a G V H D . In this chapter, a chemotherapy-conditioned parent-to-Fl lethal a G V H D model is used to test this hypothesis. 6.2. Experimental Design To determine i f TSST-l- induced Tregs are able to suppress a G V H D , splenocytes containing Tregs from B6 (H-2 b) mice injected with 3 doses of TSST-1 were 138 adoptively transferred to B6D2F1 recipient mice (H-2 b / d ) that were conditioned by cyclophosphamide (300 mg/kg) 24 hours before. Recipient mice were then given either no TSST-1 , one dose, or two doses of TSST-1 (4 pg/mouse/injection at 4-day interval). Survival was monitored. If suppression of a G V H D was observed, the effects of Tregs on the donor T cells, host antigen presenting cells, cytokine production and serum endotoxin levels were further studied. The role of IL-10 and G V T effects were also investigated. A flow diagram of the experimental design is shown below. B6 (H-2 b), TSST-1 , x3 » r^. Splenocytes containing ' , Tregs ^ D 9 2 J d ! Cyclophosphamide I m " z (300 mg/kg) f 24 h later No stimulation One dose of Two doses of TSST-1 TSST-1 V v J Survival was monitored Effects of Tregs on I donor T cells and host I f a G V H D Jas suppressed A P C s M Effects of Tregs on cytokine . G V T effects production and serum ~ ~ - _ . -endotoxin level Figure 6.1. Experimental design of studies described in Chapter 6. 139 6.3. Results 6.3.1. Development and optimization of an a G V H D animal model Multiple murine models exist for the study of a G V H D (Korngold R et al., 2005). Most of the models use total body irradiation as the conditioning approach because irradiation is the most commonly used conditioning method clinically in human A H S C T . Irradiation is used because it can effectively ablate the immune system and hematopoietic cells of the immune-competent host, allowing the engraftment of donor T cells and hematopoietic cells. A s the donor T cells are not rejected, they become activated and thus can mediate lethal a G V H D . M i c e undergoing a G V H D in these models have clinical symptoms, such as hunched back, diarrhoea, body weight loss and death. Survival and body weight were reliable gross indices for the severity of a G V H D . However, when the proposed studies were planned, unfortunately no irradiation facility could be accessible. Thus, a non-irradiation conditioning approach was utilized for this study. Several such models exist. One model of a G V H D is established by infusing parent mice splenocytes to their hybrid F l recipient mice. According to the literature, when this donor-recipient combination is used, lethal a G V H D can happen whether conditioning is used or not used (Ellison et al., 1998; H i l l et al., 1998). In reports where no conditioning is used, a large number of parent lymphoid cells are infused to unconditioned F l hosts. a G V H D develops and is followed by lethality 4 weeks after cell transfer (Ellison et a l , 1998). For non-irradiation conditioning approaches, cyclophosphamide, is commonly used, and infusion of a much smaller number of parent lymphoid cells resulted in lethal a G V H D (Owens, Jr. and Santos, 1968; Rybka W B , 1994). Thus, based on these reported studies, 140 initial studies were performed first to establish a cyclophosphamide conditioned model for this study. Out of the many parent and F l strain combinations, the B6 parent and B6D2F1 recipient combination is one of the most well established ones. Lethal a G V H D has been reported in this donor and recipient combination whether conditioning is used or not. Thus, this combination was chosen for the initial study. In one experiment, a large number of B6 splenocytes (1 .2x l0 8 /mouse) were infused into the unconditioned B6D2F1 mice, and clinical symptoms of a G V H D , lethality and body weight were monitored. A s shown, in Figure 6.2, infusion of this high number of splenocytes did lead to transient a G V H D symptoms, such as hunched back, body weight loss, while recipients of F l splenocytes did not show any of these symptoms. Body weight was observed to drop from day 7 to day 20, and then recovered to that prior to cell transfer. Lethality only occurred in one of 6 mice. In another experiment, recipient mice were first given cyclophosphamide at the dose of 300 mg/kg intraperitoneally for conditioning 24 hours prior to cell transfer. Then, B6 splenocytes were transferred to these conditioned recipients at the dose of 4><107 /mouse. After cell transfer, clinical symptoms of a G V H D , such as hunched back, were observed as early as day 4 post cell transfer. Body weight showed persistent loss in recipients of B6 splenocytes, and lethality occurred in 5 out of 5 recipients. The body weight did not recover and this was well correlated with lethality (Figure 6.3). Moreover, histological studies of the target organs demonstrated typical pathological changes of a G V H D (Figure 6.4). Recipients of F l splenocytes did not show any clinical symptoms of a G V H D and no lethality was observed. Therefore, based on the 141 100' 03 3 W5 c u 75H 50H 25H 0' B6 SC Fl SC 0 — i — 10 20 30 —r— 40 Time pos t - t ransp lan t (days) Figure 6.2. a G V H D in B6^uncondit ioned B6D2F1 mouse model. 1.2xl0 8 B6 splenocytes were intravenously transferred to unconditioned B6D2F1 mice ( A , n=6/group). In the control group, same number of F l splenocytes was transferred (•, n=3/group). Survival (A) and body weight (B) were monitored. A s shown in A , one out of six mice died. Body weight of recipients of B6 splenocytes decreased from day 7 to day 20, indicating a G V H D . However, after three weeks post cell transfer, body weight began to increase to that prior to cell transfer. N o lethality and body weight loss were observed in the control group. 03 > a Vi 100 ~ 25H c u s-u P H 75H 50H B6 SC Fl SC — i — 30 40 — i — 50 B6 SC Fl SC 20 60 0 10 Time pos t - t ransp lan t (days) — i — 30 — i — 40 — i — 50 - i 60 Figure 6.3. a G V H D in B6^cyclophosphamide-conditioned B6D2F1 mouse model. 4 x l 0 7 B6 splenocytes were intravenously transferred to B6D2F1 mice receiving 300mg/kg cyclophosphamide 24 hours ago ( A , n=6/group). In the control group, same number of F l splenocytes was transferred (•, n=3/group) to cyclophosphamide conditioned F l mice. Survival (A) and body weight (B) were monitored. A s shown in A , five out of five mice died. Body weight of recipients of B6 splenocytes decreased persistently after cell transfer, indicating progressive severe a G V H D . In control group, no lethality was observed. Body weight of mice in the control group dropped slightly at day 3 post cell transfer and then rose to level prior to cell transfer promptly. 142 Figure 6.4. Histology of target organs of mice undergoing a G V H D . Recipient B6D2F1 mice were first injected with cyclophosphamide (300 mg/kg) and then injected with B6 splenocytes (4><107/mouse). M i c e in the control group received conditioning and F l splenocytes. Survival was monitored. a G V H D target organs were removed at the time when mice died of a G V H D . Tissues were fixed, sectioned and stained with hematoxylin and eosin. A and C. Liver and intestine of control mice respectively. N o pathological changes were observed. B and D . Liver and intestine of mice undergoing a G V H D respectively. B . Marked lymphocyte infiltration o f the portal space was observed, which was typical pathology o f the liver related to a G V H D (arrow). D. Remarkable atrophic v i l l i and hyperplastic crypts with lymphocyte infiltration were observed in the intestine mucosa ( x 125). 143 preliminary results, the model using cyclophosphamide as conditioning was chosen for the subsequent studies because of the following advantages as compared to other models: 1) based on lethality, clinical symptoms, and histological studies, a G V H D in this model was more similar to that in models where irradiation is used for conditioning; 2) lethal a G V H D occurred more consistently in this model, suggesting that survival may be used as a reliable index for the severity of a G V H D following treatment with Tregs; 3) persistent body weight loss was well correlated to a G V H D progression, making it also a reliable index for the severity of a G V H D ; 4) since cyclophosphamide-conditioned mice were much more sensitive to lethal shock induction, fewer numbers of donor cells were needed and the observation period could also be shortened; 5) cyclophosphamide is commonly used in human A H S C T clinically and thus this conditioning method is also clinically relevant. 6.3.2. Activation of TSST-l- induced Tregs led to suppression of a G V H D Since the studies in Chapter 4 showed that TSST-l- induced Tregs have potent ability to control proinflammatory cytokine responses, we asked i f these Tregs could also be used to inhibit a G V H D where proinflammatory cytokines play a critical role in its pathogenesis. To test this hypothesis, we used a well-characterized parent-to-Fl lethal murine a G V H D model, where splenocytes from B6 mice receiving three injections of TSST-1 were infused into cyclophosphamide-conditioned B6D2F1 mice. A s shown in Figure 6.5-A, transfer of 4 x l 0 7 splenocytes from F l mice, either TSST-1 or P B S primed, did not cause any clinical signs of a G V H D and mice survived the 60-144 A - A - T S S T - 1 B6 SC -A- PBS B6 SC - 0 - TSST-1 F l SC - • • - P B S F l SC - 1 — 40 —1— 50 70 10 20 30 60 B 100-I--50H 25H - « - TSST-1 B6 SC + T(xl) - D - TSST-1 B6 SC + P(xl) - • - P B S B6 SC + T(xl) - O PBS B6 SC + P(xl) i 11 ! ^ — i - r -10 i •k 20 30 40 —t— 50 60 - • - T S S T - 1 B6 SC+T(x2) * -O-TSST-1 B6 SC+P(x2) * - « - P B S B6 SC+T(x2) - O P B S B6 SC+P(x2) i i i i — 10 20 30 40 50 60 Time post-transplant (days) 70 Fig 6.5. 145 21n Time post-transplant (days) Figure 6.5. Activation of TSST-l- induced Tregs attenuated a G V H D . B6D2F1 (H-2 b d ) recipient mice were conditioned by cyclophosphamide at the dose of 300mg/mouse 24 hour before transfer of B6 (H-2 b ) donor splenoytes. 4 x l 0 7 T S S T - l -primed or PBS-primed B6 or F l splenocytes, were transferred to the B6D2F1 recipient mice intravenously 24 hours later. A . TSST-1 or PBS-primed B6 or F l splenocytes were transferred to conditioned F l recipient mice and survival was monitored. Transfer of TSST-1 (0) or PBS-primed (•) F l splenocytes did not result in a G V H D (n=5/group). A l l recipients of TSST-l -pr imed B6 splenocytes ( A ) , which contained TSST-l- induced Tregs, died from a G V H D as recipients of P B S -primed B6 spleocytes (A). Survival of both group was not significantly different (15.6±0.3 vs 16.2±0.25 days, P>0.05, n=10/group). B . Recipient mice of TSST-1 or PBS-primed B6 splenocytes were given one injection of TSST-1 (4u.g/mouse) or P B S at day 0. Recipients of TSST-l -pr imed B6 splenocytes with one dose of TSST-1 (• and dotted line) had significantly longer survival as compared to recipients of TSST-l -pr imed B6 splenocytes with P B S injection ( • and dotted line) (26.3±1.9 vs 16.6±0.6 days, P O . 0 5 , n=10/group). Recipient mice of PBS-primed B6 splenocytes and given TSST-1 (• and dotted line) or P B S (o and dotted line) all died from a G V H D (16.4±2.2 or 15.3±0.25 days, n=8/group). C. Recipient mice were given two doses of TSST-1 or P B S after transfer of TSST-1 or PBS-primed B6 splenocytes. Survival time of recipients of TSST-l -pr imed B6 splenocytes with two doses of TSST-1 at day 0 and day 4 (•) was significantly prolonged as compared to recipients of TSST-l -pr imed B6 splenocytes with injection of two doses of P B S (•) (52.1±5.3 vs 15.1±0.26, P O . 0 5 , n=10/group). Survival rate was also increased (80% vs 0%). Survival of recipient mice of PBS-primed B6 splenocytes and two doses of TSST-1 (•, n=9/group) or P B S (o, n=8/group) was also significantly shorter than that of recipient of TSST-l -pr imed B6 splenocytes and two doses of TSST-1 (21.6±4.9 or 16.5±0.7 days, P O . 0 5 ) . *, P O . 0 5 , compared with TSST- l -pr imed B6 splenocytes+one/two dose/doses of P B S , or PBS-primed B6 splenocytes+/one/two dose/doses of P B S , or PBS-primed B6 splenocytes+one/two dose/doses of TSST-1 . D . Body weight of mice receiving B6 splenocytes. M i c e receiving TSST-l -pr imed B6 splenocytes showed less severe body weight loss. Groups were the same as those i n C . 146 day observation period (n=5/group). In contrast, adoptive transfer o f same numbers of PBS-primed B6 splenocytes resulted in clinical signs of a G V H D , such as body weight loss and hunched- back, and all recipient mice died by 21 days post transplant (mean survival 16.2±0.25 days, n=10). Recipient mice o f TSST- l -pr imed splenocytes, which contained TSST-1-indued Tregs, showed the similar morbidity and mortality as compared to recipient mice of PBS-primed splenocytes (15.6±0.3 days, n=T0, P>0.05). Therefore, inclusion of TSST-l- induced Tregs in the graft alone did not confer protection" against a G V H D . The lack of protection by the inclusion of TSST-l- induced Tregs in the graft led us to hypothesize that this might be due to the lack of activation TSST-l- induced Tregs in vivo. We then tested this hypothesis by giving one dose or two doses of TSST-1 to recipient mice post cell transfer. A s shown in Figure 6.5-B, recipient mice of TSST-l -pr imed B6 splenocytes receiving one dose of TSST-1 at the time of cell transfer had prolonged survival (26.3±1.9 days, n=10, P O . 0 5 ) as compared to mice receiving TSST-1 (16.6±0.6 days, n=10) or PBS-primed (15.3±0.25, n=8) B6 splenocytes and one dose of P B S , although all mice still died from a G V H D . This beneficial effect required the presence of TSST-l- induced Tregs in the graft, as recipients of PBS-primed splenocytes receiving one dose of TSST-1 all died from a G V H D (16.4±2.2, n=8). When two doses of TSST-1 were given to mice receiving TSST-l -pr imed splenocytes, the survival time was further significantly prolonged (52.1±5.3 days, n=10, P O . 0 5 ) as compared to that of mice receiving TSST-l -pr imed B6 splenocytes and two doses of P B S (15.1±0.26 days, n=9) or mice receiving P B S -147 primed B6 splenocytes and two doses of P B S (16.5±0.7 days). Again , this effect was not due to the two doses of TSST-1 alone, as most of the recipient mice of PBS-primed B6 splenocytes receiving two doses of TSST-1 still died from a G V H D by day 25 (21.6±4.9, n=9). Moreover, in contrast to one dose of TSST-1 , mice receiving T S S T - l -primed B6 splenocytes and two doses of TSST-1 had a much higher survival rate than those receiving TSST-l -pr imed B6 splenocytes and two doses of P B S (80% vs 0%) (Figure 6.5-C). In addition, recipients of TSST-l -pr imed B6 splenocytes and two doses of TSST-1 also demonstrated less severe body weight loss as compared to other control groups (Figure 6.5-D). Therefore, these data showed that activation of T S S T - l -induced Tregs in the graft could attenuate a G V H D . 6.3.3. TSST-l- induced CD4+ Tregs mediated suppression of a G V H D To further confirm that the TSST-l- induced CD4+ Tregs were responsible for this protection, CD4+ T cells ( lx l0 7 /mouse) were isolated from splenocytes of mice treated with three doses of TSST-1 and co-transferred with naive B6 splenocytes (4xl0 7/mouse) to B6D2F1 recipient mice. In the control groups, CD4+ T cell-depleted TSST-l -pr imed B6 splenocytes ( 3 x l 0 7 /mouse) or PBS-primed B6 CD4+ T cells ( lx l0 7 /mouse) were co- transferred with naive splenocytes to recipient mice. Co-transfer groups all received two doses of TSST-1 at day 0 and day 4 since our earlier experiments have shown that activation of TSST-l- induced Tregs led to control of a G V H D . A s shown in Figure 6.6, co-transfer of l x l O 7 TSST-l- induced CD4+ Tregs with two doses of TSST-1 post cell transfer prolonged the survival of recipient mice (46.2 ± 5.8 days, n=10, P O . 0 5 ) and increased the survival rate o f the recipient mice 148 100-75H 50H 254 0-L naive naive+Treg+T(2x) naive+Treg-depleted SC+T(2x) naive+PBS-CD4+T(2x) JL 1 1 i 1 0 10 20 30 40 50 60 70 Time post-transplant (days) Figure 6.6. Activation of TSST-l- induced CD4+ Tregs mediated.suppression of a G V H D . B6D2F1 recipient mice were conditioned with 300 mg/kg cyclophosphamide 24 hour before transfer of B6 (H-2 b ) donor splenoytes. Recipient mice then were injected intravenously with 4 x l 0 7 naive B6 splenocytes alone (•) or with l x l O 7 TSST-l- induced Tregs (•), l x l O 7 P B S -primed B6 CD4+ T cells (0) or 3 x l 0 7 CD4+ T cell-depleted TSST- l -pr imed B6 splenocytes (•). Mice in the later three groups all received two injections of TSST-1 (4 u.g/mouse) at day 0 and day 4. Survival time of recipients of T S S T - l -primed CD4+ T cells with two doses of TSST-1 at day 0 and day 4 was significantly increased as compared to that of recipients of naive B6 splenocytes alone (46.2±5.8 vs 16.4±1.8 days, P O . 0 5 , n=10/group). Co-transfer of CD4+ T cell-depleted TSST-l -pr imed B6 splenocytes (17.2±1 days) or PBS-primed CD4+ T cells (17±0.61 days) did not confer protection, showing that T S S T - l -induced CD4+ Tregs were responsible for suppression of a G V H D . *, P O . 0 5 , compared with naive alone, naive+Tregs-depleted TSST- l -pr imed B 6 splenocytes + two doses of TSST-1 , or naive+PBS-primed CD4+ T cells+two doses of TSST-1 . 149 (60%) as compared to mice receiving naive splenocytes alone (16.4±1.8 days, 0% survival, n=10). In contrast, co-transfer of PBS-primed CD4+ T cells with two doses of TSST-1 (17±0.61 days, n=10) or CD4+ T cell depleted TSST- l -pr imed B 6 splenocytes with 2 doses of TSST-1 (17.2±1 days, n=10) did not confer protection. Thus, these data further confirmed that donor derived TSST-l- induced CD4+ Tregs when activated in vivo with two doses of TSST-1 . 6.3.4. Activation of TSST-l- induced Tregs did not affect donor T cell expansion or enhance host antigen presenting cell elimination in vivo Ample studies have shown that donor T cells and host A P C s are critical for a G V H D as host A P C s initiate and donor T cells maintain a G V H D . Thus it was logical to determine the effects of activated TSST-induced Tregs on these cellular components. To this end, the splenocytes of recipient mice were analysed at day 3, 7 and 14 post-transplant to track the donor T cells and host A P C s . Four groups were included in this study. The experimental group consisted of recipient mice receiving TSST-l -pr imed splenocytes with two doses of TSST-1 injection post transplant, and was previously shown to confer protection against a G V H D . Recipient mice of TSST-l -pr imed splenocytes with P B S injection post transplant served as a control group. The other two control groups were cyclophosphamide-conditioned mice receiving no cell transfer but TSST-1 injection or receiving TSST-l -pr imed F l splenocytes with two injections of TSST-1 . Donor and host cells could be distinguished by their expression of H - 2 d antigens, which was only expressed on host cells. Results in Figure 6.7 showed that there was no significant difference between the absolute numbers o f donor C D 4 and 150 e u o i-« s 3 C "33 fl o s s e "3 U A. Donor CD4+ T cells T-B6 SC+T T-B6 SC+P fl 8 IO.O a ve O 7.5' i-v X) S 3 C 5.0 2.5' r j 0.0' Day 3 Day 7 Day 14 T-B6 SC+T: Recipients of TSST-l-primed B6 SC + two doses of TSST-1 T-B6 SC+P: Recipients of TSST-l-primed B6 SC + two doses of PBS T: No cell transfer + two doses of TSST-1 T-F l SC+T: Recipients ofTSST-1-primed F l SC + two doses of TSST-1 B. Donor CD8+ T cells 20-CZ3T-B6 SC+T 15- T • l-llii SC+P 10-5-1 n i 0-Day 3 Day 7 Day 14 C. Host MHC 11+ APCs -r C H T - B 6 SC+T • T-B6 SC+P C U T • 1 T - F 1 SC+T D Day 3 Day 7 Time post-transplant (days) Figure 6.7. Effect o f activation of TSST-l- induced Tregs on donor T cells and host A P C s . B6D2F1 mice were conditioned with 300mg/mouse cyclophosphamide and transferred with TSST- l -pr imed B6 or F l splenocytes. Two doses o f TSST-1 or P B S were given to the recipient mice. A t day 3, 7 and 14, spleens of the recipient mice were removed and the percent of donor CD4 and C D 8 T cells and host A P C s were determined by flow cytometry analysis. The absolute number of these cells was then calculated. Donor cells were identified as H - 2 d negative cells while host cells were H -2 d positive. A P C s were identified as M H C Class II positive cells. A donor C D 4 T cell. B . donor C D 8 T cell. C. host A P C s . Four mice were included in each group for each time. N o significant difference was found between the T-B6 SC+T and T-B6 SC+P groups in terms of the number of donor C D 4 and C D 8 T cells and host A P C s . 151 C D 8 T cells recovered in the spleens of recipient mice at all three time points, except for C D 8 T cells at day 7 when there was significantly higher number of C D 8 T cells in recipients of TSST-l -pr imed B6 splenocytes and two doses of TSST-1 . These data suggested that activation of TSST-l- induced Tregs did not affect the engraftment and expansion of donor T cells in vivo. Analysis of host A P C s by their expression of M H C class II molecules also revealed no significant difference between the experimental group and the a G V H D control group (Figure 6.7-C). Therefore, the activation of TSST-l- induced Tregs did not influence the donor T cell engraftment and expansion, and did not enhance the elimination of host A P C s . 6.3.5. Activation of TSST-l- induced Tregs modulated cytokine production and serum endotoxin level Proinflammatory T h l cytokines, such as T N F - a and IFN-y, have been shown to play critical roles in the pathogenesis of a G V H D . Thus, it would be important to determine how the levels of these cytokines were changed in the presence of activated TSST-l- induced Tregs. In the same experiment to study the effects o f activated Tregs on the donor T cells and host A P C s , sera of recipients were obtained when the mice were sacrificed. Serum levels of IL-2, IL-12, IL-10, T N F - a and IFN-y were then assayed by E L I S A . A s shown in Figure 6.8-A, serum level of T N F - a in both experimental and a G V H D control groups increased from day 3 to day 14, with no difference detected at each time point between these two groups. A t day 7 and 14, levels of T N F - a in the experimental group appeared to be higher than that of the a G V H D group, although the difference was not statistically significant. In contrast, 152 levels of serum IFN-y in the experimental group (61.5±28.6 pg/mL, n=4, P<0.05) were significantly lower than in the a G V H D group (280±44 pg/mL, n=4) at day 7 post transplant. These data suggest that activation of TSST-l- induced Tregs might control a G V H D by down-regulating levels of IFN-y. Amounts of IL-10 were not significantly different between the experimental and a G V H D groups at all time points. In the control groups, no cell transfer or transfer of TSST-l -pr imed F l splenocytes with two doses of TSST-1 did not lead to detectable levels of T N F - a and IFN-y, confirming that the increased levels of T N F - a and IFN-y in the other two groups were due to alloresponses mediated by donor cells. Serum levels of IL-2 and IL-12 were below detection levels (data now shown). In vitro mixed lymphocyte reactions ( M L R ) using T cells recovered from the recipient mice at different time points post transplant as responder cells further confirmed the effect of activated TSST-l- induced Tregs on the production of cytokines induced by alloresponses. A s shown in Figure 6.8-B, when T cells from recipient mice of TSST-l -pr imed B6 splenocytes and two doses of TSST-1 were stimulated by F l A P C s , production of IL-2, IL-12 and LFN-y was significantly lower than those of T cells from the a G V H D group at day 7 and day 14 post transplant. T N F - a production was not affected. IL-10 level was higher in the experimental group than in the a G V H D group only at day 3 post transplant. Endotoxin translocation has been shown to be a critical step in the pathogenesis of a G V H D . Efforts were made to study how the activated TSST-l- induced Tregs affected endotoxin translocation. A s shown in Figure 6.8-C, serum levels of endotoxin in recipient mice of the experimental group were significantly lower than those of the 153 — I 1 " l Day 3 Day 7 Day 14 Time post-transplant (days) Figure 6.8. Effect of activated TSST-l- induced Tregs on cytokine production in vivo and in vitro and serum endotoxin levels. A . In the same experiment described in F ig 6.7, sera were collected at each time point when mice were sacrificed to remove the spleens of recipient mice. Serum levels o f cytokines were determined by E L I S A . A . A s shown, no significant difference of serum T N F - a between recipients of TSST-l -pr imed B6 splenocytes and two doses of TSST-1 (•, experimental group) and of TSST-l -pr imed B 6 splenocytes and two doses of P B S (•, a G V H D group) at three time points post transplant. In contrast, significant lower level of serum LFN-y was detected in the experimental group as compared to the a G V H D group (61.5±28.6 vs 280±44 pg/mL, P O . 0 5 , n=4). IL-10 levels of the experimental group and a G V H D group were not significantly different at all time points. *, P O . 0 5 , compared with T-B6 SC+P (2*). T ( T ) : N o cell transfer + two doses of TSST-1 ; T - F l SC + T (•): Recipients of T S S T - l -primed F l SC + two doses of TSST-1 . 154 350 300 250 200 150 IOOH 50 0 TIL-2 1  III nlll I Day 3 Day 7 Day 14 CUB 600 500 400-300-200-100-0 I F N - y ft c = i A I IB Mi' Day 3 Day 7 Day 14 200 150-1 100 50 0 X til Day 3 Day 7 Day 14 I C 300-1 IL-12 200- T JL I -111 ill i • A CD B Day 3 Day 7 Day 14 -i * 750' 500 250' IL-10 Q A C U B Day 3 Day 7 Day 14 Time post-transplant (days) G r o u p s R e s p o n d e r c e l l s S t i m u l a t o r c e l l s A T c e l l s f r o m r e c i p i e n t s o f T S S T - l - p r i m e d B 6 s p l e n o c y t e s + t w o d o s e s o f T S S T - 1 T c e l l - d e p l e t e d F l s p l e n o c y t e s B T c e l l s f r o m r e c i p i e n t s o f T S S T - l - p r i m e d B 6 s p l e n o c y t e s + t w o d o s e s o f P B S T c e l l - d e p l e t e d F l s p l e n o c y t e s C N a ' i v e B 6 T c e l l s T c e l l - d e p l e t e d F l s p l e n o c y t e s F i g u r e 6 . 8 . B . M i x e d l y m p h o c y t e r e a c t i o n s . C y c l o p h o s p h a m i d e - c o n d i t i o n e d F l m i c e w e r e t r a n s f e r r e d w i t h T S S T - l - p r i m e d B 6 s p l e n o c y t e s a n d r e c e i v e d t w o d o s e s o f T S S T - 1 o r P B S . A t d i f f e r e n t t i m e p o i n t s p o s t t r a n s p l a n t , T c e l l s f r o m s p l e e n s o f r e c i p i e n t m i c e w e r e s t i m u l a t e d w i t h T c e l l - d e p l e t e d m i t o m y c i n C -t r e a t e d F l o r B 6 s p l e n o c y t e s f o r 5 d a y s . C y t o k i n e l e v e l s i n t h e s u p e r n a t a n t s w e r e a s s a y e d b y E L I S A . S p l e n i c T c e l l s c o n t a i n i n g a c t i v a t e d T S S T - l - i n d u c e d T r e g s ( g r o u p A ) p r o d u c e d d e c r e a s e d l e v e l s o f I L - 2 , I L - 1 2 a n d I F N - y a t d a y 7 a n d d a y 1 4 w h e n s t i m u l a t e d w i t h a l l o g e n e i c s t i m u l a t o r c e l l s a s c o m p a r e d t o s p l e n i c T c e l l s o f m i c e f r o m a G V H D g r o u p ( g r o u p B ) . 4 m i c e w e r e i n c l u d e d f o r e a c h g r o u p a t e a c h t i m e . * , P O . 0 5 , g r o u p A c o m p a r e d w i t h g r o u p B , n = 4 . 1 5 5 Figure 6.8. C. Serum levels of endotoxin in recipients of activated T S S T - l -induced Tregs. In separate experiments as described in F ig 6.7, sera were collected at each time point and serum endotoxin level was determined. A s shown, serum endotoxin level in recipient mice of TSST- l -pr imed B6 splenocytes plus two doses of TSST-1 (•) was significantly lower than that of the a G V H D mice (•).*, P O . 0 5 , compared with • : T-B6 SC+P (2x), n=4. T ( T ) : N o cell transfer + two doses of TSST-1 ; T - F l S C + T (•): Recipients ofTSST-l-primed F l SC + two doses of TSST-1 . 156 a G V H D group at day 14 post transplant, indicating that at this point in time, the presence of activated TSST-l- induced Tregs resulted in suppression of a G V H D leading to attenuated tissue injury of the target organs and diminished translocation of endotoxin from the gut. 6.3.6. IL-10 blockade with IL-10 receptor monoclonal antibody did not reverse the suppression mediated by TSST-l- induced Tregs It has been shown that suppression mediated by T r l cells in vivo, which is dependent on the production of IL-10, was not associated with increased serum IL-10 levels (Cottrezet al., 2000). In addition, elevated IL-10 levels were detected in the mixed lymphocyte reaction described in Figure 6.7-B, suggesting that IL-10 might play a role in the suppression mediated by activated TSST-l- induced Tregs on a G V H D . Thus, the ro le .of IL-10 was further studied using a monoclonal antibody, I B 1.2, against the IL-10 receptor which has been previously shown to block the actions of IL-10. A s shown in Figure 6.9, recipients of TSST-l -pr imed B6 splenocytes with two doses of P B S all died from a G V H D by day 20 (14±0.45 days, n=8) while recipients receiving the same cells with two doses of TSST-1 showed prolonged survival (55±5 days, n=8) and increased survival rate (87.5%). Administration of anti-IL-10 receptor antibody did not shorten the survival (55±4.4 days, n=8) time or decrease the survival rate (87.5%) of the recipients as compared to recipients without the antibody or recipients of control antibody. Therefore, these data suggest that activated T S S T - l -induced Tregs did not depend on IL-10 exclusively to exert their suppression on a G V H D in vivo. 157 o 25H • • i i i 0 i q A, El -•-T-B6 SC+P(2x) - Q -«-T-B6SC+T(2x) - A - T-B6 SC+T(2x)+anti-rL-10R Ab -O- T-B6 SC+T(2x)+control Ab — i — 1 1 1 1 1 0 10 20 30 40 50 60 70 Time post-transplant (days) Figure 6.9. Role of IL-10 in the suppression mediated by TSST-l- induced Tregs on a G V H D . Conditioned B6D2F1 recipients mice were transferred with 4*10 7 TSST-l-pr imed B6 splenocytes and received two doses of TSST-1 . They were further injected with anti-IL-10 receptor antibody ( A , lmg/mouse at day 0 and 0.5mg/mouse/week thereafter). M i c e receiving TSST-l -pr imed B6 splenocytes and two doses of P B S served as a G V H D positive group (•). Other control groups included mice receiving TSST-l -pr imed B6 splenocytes and two doses of TSST-1 (•) and mice receiving TSST-l -pr imed B6 splenocytes, two doses o f TSST-1 and control antibody ( o ) . A s shown, mice in a G V H D group (•) died by 20 days post-transplant while mice receiving TSST-l -pr imed B6 splenocytes and two doses of TSST-1 had prolong survival (•). Neutralization of IL-10 did not reverse the suppression by activated TSST-l- induced Tregs ( A ) as compared to group without neutralization (•) or mice receiving control antibody ( o ) . 158 6.3.7. Control of a G V H D by activation of TSST-l- induced Tregs did not block graft-vs-tumor ( G V T ) response a G V H D and G V T effect are two closely related processes that are triggered by alloresponses. Data above demonstrated that activation of TSST-l- induced Tregs was able to suppress a G V H D mediated by donor T cells. Thus, it was important to further investigate i f G V T effects were preserved or not because G V T effects have been shown to be associated with a favourable outcome in clinical A H S C T . To address this issue, l x l O 4 P815 tumor cells, which are derived from D B A 2 mice, were added to the inoculums for each recipient mouse. Results demonstrated that recipients of T S S T - l -primed F l splenocytes with two doses of TSST-1 did not confer protection against the tumor attack. Lethality began from 11 days post transplant and all recipient mice died by day 14 (11.8=1=0.3 days, n=8). Due to syngeneic transplantation mice did not suffer from a G V H D as reflected by the lack.of clinical signs and severe body weight loss typical of a G V H D (Figure 6.10-A and B) . Histological study and autopsy demonstrated the typical infiltration of tumor in the liver (Figure 6.11-B). Recipients of T S S T - l -primed B6 splenocytes with P B S survived a little longer, demonstrating the presence of G V T effects, however, they all died from a G V H D (15±0.38 days, n=8) as revealed by clinical signs and severe body weight loss (Figure 6.10-B). Autopsy and histological study did not reveal the typical tumor nodules in the liver (Figure 6.11-C). In contrast, recipients of TSST-l -pr imed B6 splenocytes had significantly prolonged survival as compared to the two control groups described above (45.6±5.3 days, n=10, P O . 0 5 ) . Three out o f ten mice died from a G V H D , as determined by cl inical signs and the absence of infiltration of tumor cells to the liver. Three mice ultimately died from 159 100 cs > c ii u s-75H 50H 25H OH * - T-B6 SC+T(2x)+P815 O - T-B6 SC+P(2x)+P815 • - T - F 1 SC+T(2x)+P815 T-Fl SC+T(2x) 10 4 i -LX. 20 30 40 —r-50 — l -60 70 22.5-B 20.0-.SP17.S-V, 15.0-tt 12.5-- • - T - B 6 SC+T(2x)+P815 -B- T-B6 SC+P(2x)+P815 - • - T - F l SC+T(2x)+P815 -0- T - F l SC+T(2x) 0 3 7 14 21 28 35 42 49 56 63 Time post-transplant (days) Figure 6.10. Effect o f activated TSST-l- induced Tregs on G V T response. After conditioning with 300mg/kg cyclophosphamide, recipient F l mice were injected with TSST-l -pr imed B6 splenocytes and given two doses of TSST-1 or P B S , or with TSST-l -pr imed F l splenocytes and given two doses of TSST-1 . 10,000 P815 cells were added to each mouse. A . Survival of mice. Recipient mice of T S S T - l -primed F l splenocytes, two doses of TSST-1 and P815 cells (•) all died from tumor progression by day 14 (11.8±0.3 days, n=8). Survival o f recipients o f T S S T - l -primed B6 splenocytes, two doses of P B S and P815 cells (•) was slightly prolonged (15±0.38 days, n=8), but they all died from a G V H D . In contrast, recipients of TSST-l-primed B6 splenocytes, two doses of TSST-1 and P815 cells (•) were significantly prolonged (45.6±5.3 days, n=10, P O . 0 5 ) as compared to the other groups. Three mice died from a G V H D and three mice died from tumor progression at day 50. Thus, activation o f TSST-l- induced Tregs could control a G V H D while sparing G V T response. *, P O . 0 5 , compared with T - F l SC+T(2x)+P8 15(D). Control mice receiving TSST-l -pr imed F l splenocytes and two doses of T S S T - l without tumor cells (0) were healthy and survived the whole observation period. B . Body weight of the mice as a reflection of a G V H D severity. Recipient mice of TSST-l -pr imed F l splenocytes, two doses of T S S T - l with (•) or without (0) P815 cells did not showed persistently severe body weight loss, indicating absence o f a G V H D . Recipients o f TSST-l -pr imed B6 splenocytes, two doses of TTST-1 and P815 cells (•) showed less severe body weight loss as compared to a G V H D recipients of TSST-l -pr imed B6 splenocytes, two doses of P B S and P815 cells (•), indicating attenuated a G V H D . 160 Figure 6.11. Histological study of tumor infliltration of the liver undergoing tumor challenge. Liver tissues were taken from experiments described in Fig 6.10. Typical tumor nodules were formed in the livers of recipients o f TSST-l -pr imed F l splenocytes, two doses of TSST-1 and P815 tumor cells (B, arrow pointed to the tumor nodule). Tumor nodules were not found in recipient mice of T S S T - l -primed B6 splenocytes, two doses of either TSST-1 or P B S and P815 tumor cells (C, D) , or in recipient mice of TSST-l-pr imed F l splenocytes and two doses of TSST-1 without tumor cells (A). H . E stain, x l 2 5 . 161 tumor progression at day 50 as they had tumor infdtration of the liver but lacked clinical signs of a G V H D . Surviving mice did not show any signs of a G V H D or tumor infiltration of the liver at day 60 post-transplant. The overall less severe body weight loss of this group further demonstrated the attenuated a G V H D (Figure 6.10-B) and prolonged survival against tumor infiltration. Histological studies of the liver taken from this group also did not reveal tumor nodule formation (Figure 6.11-D). Recipients o f TSST-l -pr imed F l splenocytes without tumor cells did not show any signs o f illness through the whole observation period except a slight body weight loss induced by the conditioning protocol, which recovered promptly by day 7 (Figure 6.10-B). Histological study did not reveal any pathological changes in the liver (Figure 6.11-A). The cause of death of each group was summarized in Table 6.1. Thus, activation of TSST-l- induced Tregs could attenuate a G V H D while sparing G V T effects mediated by the allogeneic donor T cells. Table 6.1. Summary of cause of death in each group of mice. G V H D mortality Tumor mortality Overall Survival T - F l SC+T(2x) 0/4 (0%) 0/4 (0%) 4/4(100%) T - F l SC+T(2x)+P815 0 (0%) 8/8 (100%) 0 (0%) T-B6 SC+T(2x)+P815 3/10(30%) 3/10 (30%) 4/10 (40%) T-B6 SC+P(2x)+P815 8/8 0 (0%) 0 (0%) 162 6.4. Discussion Our data showed that transplantation of allogeneic graft containing T S S T - l -induced Tregs with two doses of TSST-1 administration post-transplant remarkably attenuated the mortality and morbidity of a G V H D using a parent-to-Fl murine model. This was due to the active suppression mediated by the TSST-l- induced CD4+ Tregs rather than the effects of superantigen administration alone. In a previous study, Takenaka et al showed that administration of S E B could attenuate the graft-vs-host response due to the deletion of SEB-reactive T cells bearing Vp8 which were alloreactive in unconditioned B6D2F1 recipients transplanted with B6 donor cells (Takenaka et al., 2001). However, in our model, CD4+ T cells bearing V p i 5 were TSST-1 reactive but not alloreactive (Patterson and Korngold, 2001). Deletion or anergy induced by TSST-1 on donor T cells was not responsible for the attenuation of a G V H D . This was further supported by the finding that recipients o f nai've allogeneic donor T cells plus two doses of TSST-1 were not protected against a G V H D . Moreover, the data that co-transfer of TSST-l- induced CD4+ Tregs but not the CD4+ T cell depleted TSST-l -pr imed splenocytes protected the recipient mice further demonstrated the presence of active suppression mediated by TSST-l- induced Tregs on a G V H D . The lack of alloresponses mediated by CD4+ TSST-l- induced Tregs might also account for the requirement of cognate antigen stimulation for their suppressive effects, as these cells were not stimulated by alloantigens at an early stage after transplantation. Therefore, our results demonstrated that TSST-l- induced CD4+ Tregs could mediate active bystander suppression on a G V H D . 163 Bystander suppression has been noted in multiple animal disease models. For example, the typical T r l cells could mediate bystander suppression of colitis (Groux et al., 1997), Th2 response (Cottrez et al., 2000) or immune-mediated cardiovascular pathologies (Mallat et al., 2003) only when the mice were fed or immunized with O V A to activate the T r l cells. IL-10 was required for T r l cells to exert the bystander suppression effects as neutralization of IL-10 blocked the bystander suppression (Cottrez et al., 2000; Groux et al., 1997). Oral tolerance has also been shown to elicit potent bystander suppression on autoimmune diseases, such as experimental allergic encephalitis (Chen et al., 1996). One of the mechanisms of inhibition of autoimmune diseases by oral tolerance has also been shown to be the induction of Tregs, such as Th3 cells which exerted their functions by TGF-p (Chen et al., 1994). In' the transplantation context, it has recently been shown that induction of CD4+CD25+ Tregs specific for a model antigen in recipients could prevent rejection of the M H C mismatched donor skin graft (Karim et al., 2005). Hashimoto et al demonstrated that activation of N K T cells by their cognate ligand could also attenuate a G V H D (Hashimoto et al., 2005). What these Tregs had in common was that the Tregs must be activated by their cognate antigens to suppress a response elicited by antigens different from their cognate antigens. Despite these studies, it is not known i f a G V H D could be suppressed via bystander inhibition by conventional ap C D 4 or C D 8 T cells. Thus our study was the first to demonstrate that a G V H D could be controlled via bystander inhibition mediated by conventional ap C D 4 Tregs induced by a bacterial superantigen. Engraftment, activation and expansion of allogeneic donor T cells by host A P C s are the critical steps in the initiation of a G V H D . Measures to block this 164 interaction have been shown to effectively inhibit a G V H D . For example, Lan et al showed that using a conditioning protocol that enriched the host N K T cells were able to prevent donor T cell engraftment and expansion (Lan et al., 2001). In addition, co-transfer o f ex vivo expanded allospecific natural CD4+CD25+ Tregs was able to suppress the expansion of donor T cells, resulting in the suppression of a G V H D (Trenado et al., 2006). The central role of host A P C s in initiating a G V H D was best demonstrated by the study showing that recipient mice, whose A P C s had been replaced by donor type A P C s , were resistant to lethal a G V H D induction (Shlomchik et al., 1999). However, our data did not reveal a role of TSST-l- induced Tregs in blocking these initiating steps of lethal a G V H D , as no differences have been found in terms of expansion of donor T cells and the host A P C s . This is in line with our in vitro studies in Chapter 5 on the mechanisms o f TSST-l- induced Tregs, where it was found that TSST-l- induced Tregs did not inhibit the proliferation o f their target cells in vitro. Interestingly, a more vigorous expansion of CD8+ donor T cells was detected at day 7 in the recipients of TSST-l- induced Tregs receiving two doses o f TSST-1 . A possible explanation is that TSST-l- induced Tregs were able to enhance the proliferation of CD8+ T cells because data in vitro in Chapter 5 showed that naive splenocytes cocultured with TSST-l- induced Tregs showed enhanced proliferation! Further work is needed to elucidate the exact cause of this observation. Despite the enhanced expansion of donor CD8+ T cells, a G V H D in this group was not deteriorated, suggesting a minor role of CD8+ T cells in mediating a G V H D in this model. Therefore, it was further examined i f TSST-l- induced Tregs could affect the production of cytokines and endotoxin translocation that are important in the 165 pathogenesis of a G V H D (Reddy and Ferrara, 2003). Serum LFN-y was found to be down-regulated at day 7 post-transplant while serum levels of T N F - a were not affected at any time points assayed. In vitro mixed lymphocyte reactions confirmed that the presence of activated TSST-l- induced Tregs could down-regulate the proinflammatory cytokines induced by allo-responses, namely IL-2, IL-12 and LFN-y. The role of these proinflammatory cytokines in a G V H D is critical. These T h l proinflammatory cytokines have been found to potentiate a G V H D . For instance, IFN-y can prime monocytes to produce T N F - a , increased adhesion molecule expression by endothelial cells and promote C T L generation, thus leading to progression of a G V H D . In addition, pro-inflammatory cytokines could also directly cause tissue injury of the target organs (Reddy and Ferrara, 2003). Multiple methods which suppress a G V H D have been shown to be associated with the inhibition of proinflammatory cytokine production (Edinger et al., 2003; H i l l et al., 1998). Our data also suggest that TSST-l- induced Tregs can suppress a G V H D by down-regulation of T h l proinflammatory cytokines. Moreover, previous work by Ferrara's group demonstrated that in a parent-to-Fl murine a G V H D model using B6 and B6D2F1 donor and recipient combination, a G V H D was mediated by CD4+ T cells (Teshima et al., 1999). Thus taken together, our data suggest that the suppressive effects of TSST-l - induced Tregs were exerted on donor CD4+ T cells. Endotoxin translocation is another important factor in the progression of a G V H D . Host conditioning or alloresponses can lead to gastrointestinal tract damage, which results in leakage of endotoxin into the systemic circulation. The translocated endotoxin can greatly potentiate the alloresponses. This positive feedback eventually 166 results in the progression of a G V H D (Hi l l et al., 1997; H i l l et al., 1999; H i l l and Ferrara, 2000). Ample evidence has been accumulated that interruption of this vicious circle can attenuate a G V H D (Cooke et a l , 2001; H i l l et al., 1998; Krijanovski et al., 1999). Our data showing that recipient mice of activated TSST-l- induced Tregs had both suppressed production of proinflammatory cytokines and lower serum level of endotoxin suggested that TSST-l- induced Tregs attenuated a G V H D by preventing the potentiative effects of both proinflammatory cytokines and translocation of endotoxin. One interesting observation in this study is that the production of TNF-a was not affected by the activated TSST-l- induced Tregs. TNF-a has been shown to be an important proinflammatory cytokine in the pathogenesis of a G V H D , as blockade with ant i-TNF-a could prevent lethality of a G V H D (Teshima et al., 2002). TNF-a derived from donor T cells (Schmaltz et al., 2007), CD4+ T cells in particular (Ewing et al., 2007), plays an important role in enhancing a G V H D . One possibility for our observation is that in addition to TNF-a produced by alloreactive donor T cells, it was also produced by the non-alloreactive TSST-l- induced Tregs as data in Chapter 4 showed that these Tregs expressed and secreted TNF-a when they were reactivated in vitro. It was also demonstrated that CD8+ donor T cells could also be a source of TNF-a in a G V H D (Ewing et al., 2007). Since G V T effects, which is l ikely mediated by CD8+ donor T cells (Hi l l et al., 1999), were not affected in our system, it is highly probable that TSST-l- induced Tregs did not inhibit CD8+ T cell function, and thus production of TNF-a by CD8+ T cells might not be suppressed. Even though the production of TNF-a was not suppressed by TSST-1 induced Tregs, a G V H D was definitely attenuated, suggesting that other proinflammatory cytokines (such as IFN-y) 167 might be more important, either alone or in combination with T N F - a , to mediate the pathology of a G V H D in target organs (Reddy and Ferrara, 2003). Although T N F - a is a critical mediator of a G V H D , it also plays a pivotal role in G V T effects (Hi l l et al., 1999; Schmaltz et al., 2007). Thus it w i l l be worthwhile to further determine the source of T N F - a and its role in a G V H D and G V T effects in our system in further studies. It has been well documented that IL-10-producing Tregs can regulate transplantation rejection. H i l l et al recently described IL-10-producing Tregs induced by dendritic cells harvested in donor administered with G - C S F . These Tregs were able to control a G V H D while sparing G V T effects (Morris et al.,2004). In our study, it was shown that TSST-l- induced Tregs produced high levels of IL-10. Although serum IL-10 was not elevated, it could still function locally. Administration of anti-IL-10 receptor antibody, however, did not reverse the suppression mediated by activated TSST-l- induced Tregs on a G V H D , suggesting that these Tregs did not depend on IL-10 to exert their functions in vivo. Alternately, the systemic administration of anti-IL-10R antibody may not have been locally effective. These data were also in line with results described in chapter 5, where it was shown that blockade o f IL-10 did not reverse the suppressive activities of TSST-l- induced Tregs in vitro. Although TSST-l- induced Tregs suppressed a G V H D , our data showed that the G V T response was preserved. One major reason might be the production of T N F - a was not affected. H i l l et al has shown using the same combination of donor and recipient mice that T N F - a played an essential role in mediating G V T effects against P815 cells since neutralizing T N F - a abrogated G V T effects (H i l l et al., 1999). Moreover, the data showing that the engraftment and expansion of both CD4+ and CD8+ donor T cells 168 were not affected by TSST-l- induced Tregs suggested that they might still mediate alloresponses that resulted in G V T effects, although the alloresponses were attenuated leading to suppression of lethal a G V H D . This is another critical contributing factor to the G V T effects in this study. It was also shown by Ferrara's group in a G V H D using the same donor and recipient combination that G V T effects against P815 cells were dependent on both CD4+ and CD8+ T cells, with CD8+ T cells mediating more potent G V T effects and CD4+ T cells providing help to achieve the optimal G V T effects (Teshima et al., 1999). The preserved G V T effect in our study was probably due to the unaffected C D 8+ T cell response. Although the CD4+ T cell response was attenuated by TSST-l- induced Tregs, these cells were still able to provide sufficient help to aid the G V T response while they were prevented from mediating lethal a G V H D . One drawback of the use of powerful immunosuppressants such as cyclosporin is their non-specific immunosuppressive activities. Although they are able to suppress G V H D , they may also blunt the G V T responses. In some clinical settings, intentional withdrawal of immunosuppressive drug was actually used to allow G V T effects. However, this w i l l also cause the occurrence of uncontrolled a G V H D . In our study, since TSST-l- induced Tregs were not alloreactive, it is possible that activation of this population may lead to non-specific immunosuppression similar to that caused by immunosuppressive drugs. However, the presence o f G V T effects, despite the inhibition of a G V H D , suggested that unlike these immunosuppressants, activation of this population suppressed the pathogenic responses leading to a G V H D specifically while leaving favourable G V T responses intact. It remains to be determined whether 169 activation of TSST-l- induced Tregs affects the ability o f immune systems to fight infections. This should clearly be further investigated in the future. In summary, we demonstrated that CD4+ Tregs induced by TSST-1 were able to control lethality of a G V H D via bystander inhibition while sparing G V T responses. This may have important clinical implications. Although human natural CD4+CD25+ Tregs are able to suppress allo-responses and murine studies have confirmed their potential use in the clinical setting, the scarcity of these cells in vivo mandates extremely laborious ex vivo expansion. In contrast, as superantigens are potent stimulators for T cells and can activate large numbers of T cells, they may have the potential to generate large number of Tregs in vitro with relative ease. Indeed, we did show previously that human Tregs can be generated in vitro by repeated stimulation of peripheral blood mononuclear cells during a 5-day period (Kum W W et al., 2006). Our study w i l l further raise the possibility of using donor-derived TSST-l- induced Tregs to prevent a G V H D . However, the requirement to administer cognate superantigen for the suppressive functions of these cells w i l l make this therapy impossible in the clinical settings, as superantigens themselves may cause potentially lethal effects. Nevertheless, it has been shown that mutants of superantigens with T cell stimulation ability but without causing lethality can be generated (Dinges et a l , 2000). This may be a potential solution for the clinical use of superantigen-induced Tregs in a G V H D , and clearly warrants further study. 170 Chapter 7: General discussion, conclusions and future directions 7.1. General discussion Tregs have been one of the major developments in immunology in recent years. Research in this field has generated insightful knowledge of the mechanisms of immune tolerance, leading to a better understanding of physiological processes and the pathogenesis of many diseases as well as novel therapeutic strategies for these diseases. Thus, experimental systems to induce Tregs are important to advancing the field of research in Tregs. One problem with inducing Tregs with nominal antigens is that they can only activate a very low number of antigen-specific T cells, making it difficult to track and study these antigen-induced Tregs. In this case, use of repeated injection of superantigens to induce Tregs may provide a solution for this problem, as superantigens have potent stimulatory effects on T cells and are able to activate a large number of superantigen-reactive T cells, making it easier to generate antigen-reactive Tregs. Moreover, due to the VP specific effects, the VP segments can also serve as useful markers to detect T cell responses. However, due to the differences of recognition of superantigens by T cells from that of nominal antigens, more studies are needed to elucidate i f the biologic properties of superantigen-induced Tregs are different from those induced by nominal antigens. Furthermore, as this study has indicated, Tregs induced by different superantigens may also display different biological properties. Results of this study showed that repeated injection of TSST-1 induced Tregs which share properties similar to T r l cells, such as the production of high levels IL-10. However, many of the characteristics were different from those of T r l , for instance, the hyperproliferative profile and their independence on IL-10 to 171 exert the suppressive functions. This implied that the generation of TSST-l- induced Tregs was associated with unique signals different from those in the induction of T r l cells. Thus the ability of TSST-1 to induce Tregs with distinctive properties may offer an excellent opportunity to further investigate the unique signals that drive the development of these Tregs. Although quite a lot of research has been done in the field of Tregs, progress in elucidating the mechanisms of Tregs is still slow. It has been wel l established that immunosuppressive cytokines, such as IL-10 and TGF-P, are main players in mediating suppression by Tregs. However, these mediators could not completely explain the mechanisms of suppression in many cases, especially contact-dependent suppression. Although cytokine competition was responsible for the regulation of IL-2, other molecules may be involved in modulating other cytokines, which were not identified in this study. These data demonstrate that unlike T r l cells, the cytokine profile of TSST-l-induced Tregs might not be causally related to their suppressive function. Identifying molecules mediating the suppressive function of TSST-l- induced Tregs w i l l not be an easy task. Advances in molecular techniques may be helpful in this regard. A s unique properties have been found in the mechanisms of TSST-l- induced Tregs, use of these cells for proteomic analysis may be helpful in identifying more novel molecules responsible for their suppressive functions. The promise of potential therapeutic applications of Tregs is one of the main reasons that fuel research in this area. A s mentioned in chapter 1, a lot of work has previously focused on natural Tregs. However, many problems have been identified, such as the difficulty to obtain sufficient number of natural Tregs for study and the 172 existence of functional defects in some situations. Antigen-induced Tregs can be a very useful alternative for study. In this thesis research, it was clearly shown that T S S T - l -induced Tregs were able to exert bystander inhibition of a G V H D in vivo while sparing G V T effects. These data are in line with other reports showing that bystander suppression mediated by antigen-induced Tregs has great potential for the treatment of multiple diseases. Several important issues have to be addressed regarding the use of superantigen-induced Tregs in the clinical setting. First, more work needs to be done to determine i f TSST-l- induced Tregs can be applied to treat other diseases, such as autoimmune diseases. Second, it remains to be determined i f other TSST-1 mutants are also able to generate Tregs with the same ability to suppress a G V H D or other diseases. Third, efforts should be made to generate non-toxic TSST-1 mutants for clinical application. A s TSST-1 has been implicated in the cause o f TSS , it may not be possible to use TSST-l- induced Tregs in humans as these Tregs need to be reactivated by their cognate antigens. However, it is possible to generate superantigen mutants that have T cell stimulatory effects but have lost the ability to cause lethal shock. Generation of such mutant superantigens w i l l be a necessary step in promoting the clinical application of TSST-l- induced Tregs. Taken together, knowledge gained in this study of TSST-l- induced Tregs not only extends our understanding of superantigen-induced Tregs, but also sheds new light on potential therapeutic applications of TSST-l- induced Tregs. 173 7.2. Conclusions Based on the results of this study, the following conclusions can be drawn: • Repeated subcutaneous injection of TSST-1 to C 5 7 B L / 6 mice induced CD4+ Tregs with potent suppressive activities. These Tregs could downregulate the TSST-1-triggered proinflammatory cytokine responses of na'ive splenocytes in vitro and inhibit TSST-l- induced lethal shock in vivo. • Repeated injection of TSST-1 induced CD4+ Tregs with distinctive properties as compared to those induced by S E A . TSST-l- induced Tregs had broader suppressive activities in vitro and in vivo. They were hyperproliferative in vitro in the absence or presence of TSST-1 restimulation in vitro and had more potent proliferative ability. They also produced T N F - a and IFN-y in response to TSST-1 stimulation and had enhanced expression of CD25 and C T L A - 4 . Moreover, TSST-l- induced Tregs demonstrated antigen-independent proliferation and cytokine expression in vitro. • TSST-l- induced Tregs depend on cell-contact to mediate their suppressive activities and cytokine competition for IL-2 appears to be a major mechanism for their suppressive function on target cells. Induction of cell death, infectious tolerance and secretion of soluble factors are not directly involved in the suppression mediated by TSST-1 ^ induced Tregs. • Transfer of TSST-l- induced Tregs to allogeneic recipients followed by TSST-1 injection to activate them attenuated a G V H D . This suppression was not due to inhibition of donor T cell engraftment and expansion, or enhanced elimination of host A P C s , but associated with decreased proinflammatory cytokine 174 production. Furthermore, control of a G V H D by TSST-l- induced Tregs did not jeopardise G V T effects. 7.3. Future directions This study revealed distinctive properties of TSST-l - induced Tregs. Further studies should be devoted to aspects concerning the development of TSST-l- induced Tregs. First, the origin of TSST-l- induced Tregs remains unclear. Data in this study showed that TSST-l- induced Tregs did not demonstrate enhanced expression of Foxp3, a cellular marker for natural Tregs. However, CD25 and C T L A - 4 expression were enhanced in TSST-l- induced Tregs. Theoretically, during in vivo chronic exposure to TSST-1 , any TSST-1-reactive CD4+ T cells could develop into TSST-l- induced Tregs. Since CD4+ T cells could be divided into natural Tregs and conventional T cells, both of which contain TSST-l-reactive CD4+ T cells, it is possible that TSST-l- induced Tregs consist of TSST-l-reactive CD4+ T cells derived from both compartments. To address the origin of TSST-l- induced Tregs, CD25+ natural Tregs-depleted CD4+CD25- conventional T cells should be isolated, mixed with CD25+ natural Tregs isolated from congenic mice bearing markers for their identification and then adoptively transferred to SCID mice. These recipient mice then can be given repeated injection of TSST-1 . The transferred T cells w i l l be re-isolated and separated according to the congenic marker and their suppressive functions reassayed. These experiments w i l l lead to better understanding of the origin of TSST-l- induced CD4+ Tregs. Second, the role of natural Tregs in the generation of TSST-l- induced Tregs is another interesting issue for further investigation. Studies have shown that the presence 175 of natural Tregs are required for the generation of antigen-induced Tregs (Taylor et al., 2001) , possibly due to the ability of natural Tregs to limit the proinflammatory responses and thus to ensure a non-inflammatory environment that is more conducive for the generation of Tregs. The role of natural Tregs in the generation of T S S T - l -induced Tregs may be studied by transferring natural Tregs-depleted conventional T cells to SCLD mice followed by repeated administration of TSST-1 to these recipient mice. The transferred T cells can then be assayed for their regulatory functions. Another approach is to deplete the natural Tregs in vivo by the administration of a depleting antibody against CD25 combined with removal of the thymus. Then these mice may be repeatedly injected with TSST-1 and the functions of the CD4+ T cells can be assayed to determine i f conventional T cells can develop into Tregs in the absence o f natural Tregs. Third, the role of certain cytokines should also be further clarified. In a previous study, it was demonstrated that repeated mucosal administration o f S E A resulted in tolerance to this superantigen which was IL-10 dependent (Collins et al., 2002). IL-10 was also required for the induction of Tregs via mucosal tolerance (Massey et al., 2002) . Thus it is important to determine the role of IL-10 in the generation of T S S T - l -induced Tregs as these cells appear different from SEA-induced Tregs in many aspects. LFN-y has been shown to play a role in the induction of Foxp3+ Tregs (Hong et al., 2005) and data in this study showed that TSST-l- induced Tregs secreted LFN-y, suggesting that it may play a role in the generation or suppressive function of these Tregs. 176 Studies on the mechanisms of TSST-l- induced Tregs revealed two main findings. TSST-l- induced Tregs depend upon cell contact to mediate their suppression and cytokine competition may be one important mechanism. The role of IL-10 in the suppression mediated by TSST-l- induced Tregs should be further studied. Results in this study did not show a role of IL-10 for suppressive activities of TSST-l- induced Tregs both in vitro and in vivo. It should be kept in mind that using antibody to block actions of IL-10 may have intrinsic drawbacks. For example, the in vivo administered antibody may not reach sufficient concentrations locally to totally block the actions of IL-10. Thus results of this study certainly have not ruled out the possibility that TSST-l-induced Tregs may depend on IL-10 to exert their functions. It is also worthwhile to use more stringent methods to investigate the role of IL-10. The IL-10 knockout mouse is an excellent candidate for such studies. Thus identifying the molecules responsible for their action should be a major priority in future studies. One of the potential targets is C T L A - 4 as this molecule has been shown to mediate suppression by Tregs and its expression has been found to be up-regulated in TSST-l- induced Tregs, suggesting a potential causative role for this molecule. However, searching for the relevant molecules amongst the hundreds of proteins expressed by suppressive T cells w i l l not be an easy task. Powerful molecular biology methods have been developed to screen for gene expression or down-regulation, and using a genomics and proteomics approach may greatly facilitate this process. Multiple molecules involved in Tregs function were identified using this technique. For example, C T L A - 4 , GITR, Foxp3, L A G - 3 were among the most typical ones (Hori et a l , 2003; Huang et al., 2004; Shimizu et a l , 2002; Takahashi et al., 2000). It must be kept in mind that the change of 177 gene expression of certain proteins must be confirmed by functional assays to ascertain their causative role in the suppression mediated by Tregs. The data showing that TSST-l- induced Tregs suppressed a G V H D while sparing G V T effects clearly suggests the potential therapeutic application of these cells in the clinical setting. Further studies should be directed to some aspects that are of clinical importance; for example, the feasibility to obtain sufficient numbers o f cells for study of their efficacy and effectiveness. In this setting, ex vivo repeated stimulation of human P B M C to generate human TSST-l- induced Tregs is desirable and this has already been shown to be feasible in our laboratory. The next step is to further develop an ex vivo method for the generation of TSST-l- induced Tregs using murine cells and then determine i f these ex vivo generated TSST-l- induced Tregs could be expanded to obtain sufficient numbers for transplantation and i f the expanded Tregs could suppress a G V H D in the murine model. Another important issue in A H S C T is immune reconstitution (Porter and June, 2005). Due to the conditioning before A H S C T , the recipient's immune system is severely suppressed and may result in an immune deficiency that can last 1 to 2 years after A H S C T . Life-threatening infections can develop prior to completion of the immune reconstitution. a G V H D can further cause delayed immune reconstitution leading to more severe immune deficiency. 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