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Innate regulation of TSST-1 induced immune response by peripheral blood γδ T cells Kalyan, Shirin 2006

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INNATE R E G U L A T I O N OF TSST-1 INDUCED I M M U N E RESPONSE B Y PERIPHERAL B L O O D y5 T CELLS by SHIRIN K A L Y A N B.Sc , The University of British Columbia, 1999 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH C O L U M B I A April 2006 © Shirin Kalyan, 2006 Abstract Toxic shock syndrome toxin-1 (TSST-1) is the staphylococcal superantigen (sAg) identified as being the primary causative agent of menstrual toxic shock syndrome (TSS), and it is among the main sAg's responsible for non-menstrual TSS. Superantigens are the most potent T cell mitogens known. They induce the massive secretion of inflammatory cytokines by bypassing the processing and presentation requirement of conventional peptide antigens and by binding as intact proteins directly to the M H C class II on antigen presenting cells and to specific V(3-chains of the oc(3 T cell receptor. This interaction results in an inflammatory cascade that has the potential to lead to hypotension, shock, and death. TSST-1 is expressed by at least 20% of S. aureus clinical isolates; however, not everyone exposed to this sAg succumbs to TSS. Apart from protective antibodies, the factors in the immediate microenvironment which dictate the course of the immune response leading to a deleterious inflammatory reaction are largely unknown. y8 T cells are a unique subset of innate T lymphocytes that are credited with linking the innate and adaptive immune system as they serve as the first line of defense against both infectious and non-infectious stress. The objective of this study was to determine if and how human peripheral blood y5 T cells may influence the course of TSST-1 induced inflammation. High mobility group box-1 protein (HMGB-1) was investigated as an indicator of the downstream consequence of y8 T cell activity in TSST-1 pathogenesis. HMGB-1 is a nuclear protein that was previously found to be secreted by LPS-stimulated macrophages, and was determined to be the late mediator of endotoxin shock since neutralizing its effects as late as 24 hours following sepsis was sufficient to rescue mice from lethality. This study established that y8 T cells potently exacerbate the early inflammatory response to TSST-1 and enhance the subsequent secretion of HMGB-1 . y8 T cells mediate this effect by inducing early monocyte maturation and upregulating the expression of CD40 which plays a pivotal role in TSST-1 pathogenesis. The results of this study lend support to the notion that y8 T cells play an important role in linking the innate and adaptive immune responses to TSST-1. i i Table of Contents Abstract ii Table of Contents i i i List of Tables vi List of Figures vii List of Abbreviations and Symbols x Acknowledgements xi Dedications xii Manuscripts, abstracts, and presentations that resulted from this thesis xii Chapter 1: Introduction 1 1.1. y8 T cells: A sorority of lymphocytes in a class of their own 1 1.1.1 Ontogeny and distribution 5 1.1.2 Vy9V52 T cells: human peripheral blood y8 T cells - a primate inheritance 10 1.1.3 Stressed out: Function in health and disease 14 1.2. Toxic Shock Syndrome Toxin-1: A superantigen in a class of its own 18 1.2.1 Phylogeny, distribution, and cause of disease 20 1.2.2 Stressing out the host - not just a primate privilege 25 1.2.3 y8 T cells run into superantigens: stressing the implications. 27 1.3.1 High Mobility Group Box-1 Protein: An inflammatory cytokine in a class of its own 29 1.4.1 Getting a handle on stress: Aims and objective of study 33 Chapter 2: Study approach, material and methods 35 2.1 Study approach 35 2.2 Material and methods 37 2.2.1 Cell preparation and culture conditions 37 2.2.2 Cytokine analysis 38 2.2.3 Apoptosis studies 39 2.2.4 HMGB-1 expressions studies 41 2.2.5 Cells surface analysis of costimulatory molecules 43 i i i Chapter 3: Determining the influence of y5 T cells in TSST-1 induced inflammation and apoptosis 46 3.1 Introduction 46 3.2 Results Part 1 49 3.21 Discussion 57 3.3 Results Part H 59 3.31 Discussion 69 Chapter 4: Regulation of HMGB-1 expression by staphylococcal Toxic Shock Syndrome Toxin-1 73 4.1 Introduction 73 4.2 Results 76 4.2.1 Translocation of nuclear HMGB-1 in human P B M C 10 h post-TSST-1 stimulation 76 4.2.2 Secretion of translocated nuclear HMGB-1 from the surface of differentiated and adherent cells 24 h post-TSST-1 stimulation 80 4.2.3 Requirement of activated T cells in TSST-1-induced nuclear translocation and secretion of HMGB-1 in human P B M C 85 4.3 Discussion 89 Chapter 5: A means to an end - Human Vy9V82 T cells enhance the inflammatory response to TSST-1 by up-regulating HMGB-1 and CD40 expression 91 5.1 Introduction 91 5.2 Results 93 5.2.1 Activated y8 T cells significantly potentiate the translocation and secretion of nuclear HMGB-1 following TSST-1 stimulation...93 5.2.2 y5 T cells regulate the maturation of antigen presenting cells and the cell surface expression of HMGB-1 (amphoterin) 96 5.2.3 JPP-primed y8 T cells induced the up-regulation of CD40 on antigen presenting cells 106 5.2.4 IPP activated y8 T cells do not appear to directly affect a,p T cells 109 5.3 Discussion I l l Chapter 6: Summary, Discussion and Conclusions 115 6.1. Summary of findings 115 6.2. Discussion 116 6.3. Considerations, potential significance, and future endeavours... 127 iv Bibliography List of Tables Chapter 1 Table 1-1. Major differences between human a P and y8 T cells 1 Table 1-2. Comparative diversity of the a , P , 8, and y TCR chains in mice and men 5 Table 1-3. Comparative diversity and percent composition of y8 T cells between species 9 Table 1-4. Diagnostic criteria for Staphylococcal Toxic Shock Syndrome...25 Chapter 2 Table 2-1. Outline of the parameters investigated and the methodology used 45 vi List of Figures Chapter 1 Figure 1-1. Comparison of the common features shared by y8 T cells with N K cells, a(3 T cells and professional APCs 4 Figure 1-2. Developmentally guided distribution of y8 T cell subsets in the mouse 7 Figure 1-3. Developmental change in y8 TCR in humans 8 Figure 1-4. The classical mevalonate and alternative metabolic pathways leading to the production of JPP which stimulates human Vy9V82T cells 11 Figure 1-5. Structural properties of Vy9VS2 T cells 13 Figure 1-6. Schematic diagram of the time and response of y8 T cells during infection 16 Figure 1-7. Superantigen mediated immune hijacking 19 Figure 1-8. Phylogenetic tree of staphylococcal and streptococcal superantigens 21 Figure 1-9. Ribbon structure of TSST-1 and identified binding sites for TCR and M H C II 24 Figure 1-10. Regulation of HMGB-1 , and its downstream effects in systemic inflammatory disorders 31 Chapter 2 Figure 2-1. Experimental approach for the study of the immune regulatory function of human peripheral blood Vy9V82 T cells in TSST-1 induced inflammatory response 36 Chapter 3 Figure 3-1. The potential role of human y8 T cells in modulating the inflammatory response to TSST-1 48 Figure 3-2. Quantification of cytokines in culture supernatants from IPP primed or non-primed human P B M C after stimulation with TSST-1, RPMI medium, or IPP alone at different time intervals 51 vii Figure 3-3. Proportion and MFI of y8 and aP T cells expressing intracellular IFNy post-TSST-1 stimulation 55 Figure 3-4. Representative data from a single donor, showing the effect of IPP-activated y8 T cells on apoptosis of P B M C following stimulation with 1 nM of TSST-1 for4h 62 Figure 3-5. Effect of IPP-activated y8 T cells on apoptosis of TSST-1 stimulated PBMCs 63 Figure 3-6. IPP-activated y8 T cells did not induce apoptosis of TSST-1 reactive ap T cells or upregulated CD95 (fas) expression in P B M C following TSST-1 stimulation 65 Figure 3-7. Direct cytolysis of TSST-1 stimulated autologuous monocytes by expanded y8 T cells 68 Figure 3-8. Summary of the modulatory effect of IPP-activated y8 T cells on TSST-1 induced proinflammatory cytokines and apoptosis in human P B M C 71 Chapter 4 Figure 4-1. The potential role of HMGB-1 in TSST-1 induced pathology. ..75 Figure 4-2. Nuclear translocation of HMGB-1 in human P M B C following TSST-1 stimulation 78 Figure 4-3. Nuclear translocation in T cells following TSST-1 stimulation 79 Figure 4-4. Secretion of membrane associated HMGB-1 in the extracellular milieu following TSST-1 treatment 82 Figure 4-5. Surface expression of HMGB-1 on PBMCs cultured in RPMI medium in the absence of TSST-1 84 Figure 4-6. Requirement of T cells for HMGB-1 secretion following TSST-1 stimulation 87 Figure 4-7 T cells are required for the detection of HMGB-1 in culture supernatants following TSST-1 stimulation but not LPS 88 viii Chapter 5 Figure 5-1. Translocation and secretion of nuclear HMGB-1 from IPP pretreated or untreated P B M C following TSST-1 stimulation 94 Figure 5-2. Investigation by fluorescence microscopyof the change in cell surface expressed HMGB-1 (amphoterin) on cultured adherent and differentiated monocytes in P B M C following IPP and TSST-1 treatment....98 Figure 5-3. Flow cytometery analysis of surface expressed HMGB-1 in the presence or absence of peripheral blood y8 T cells 102 Figure 5-4. Assessment of the loss of surface HMGB-1 and Western blot analysis of secreted HMGB-1 104 Figure 5-5. Flow cytometery analysis of the increase of cell surface expressed HMGB-1 in culture overtime 105 Figure 5-6. Modulation of costimulatory molecules on APCs by activated y5T cells 107 Figure 5-7. Assessment of T cell activation by studying the change in CD25 and CD28 expression after both IPP and TSST-1 treatment 110 Chapter 6 Figure 6-1. y8 T cells in TSST-1 induced inflammation 119 Figure 6-2. Hypothetical model illustrating how y5 T cells and HMGB-1 intersect in the regulation of TSST-1 pathogenesis 124 Figure 6-3. Model illustrating how human peripheral blood y5 T cells exacerbate the acute and systemic inflammatory response to TSST-1 126 ix List of Abbreviations Abbreviation Definition 7 - A A D 7-aminoactinomycin D AICD Activation-induced cell death APC Antigen presenting cell CD95 Fas receptor CFSE 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester C T L Cytotoxic T lymphocyte D region diversity region of lg or T cell receptor for Ag ELISA Enzyme-linked immunosorbent assay FBS Fetal bovine serum FGF Fibroblast growth factor FETC Fluorescein isothiocyanate HMGB-1 High mobility group box-1 protein LENy Interferon gamma lg Immunoglobulin IL- Interleukin-TPP Iso-pentyl pyrophosphate K G F Keritinocyte growth factor KIR Killer Ig-like receptor LPS Lipopolysaccharide M H C Major histocompatibility complex N K cells Natural killer cells P B M C Peripheral blood mononuclear cell PBS Phosphate buffered saline PE Phycoerythrin R A G E Receptor for advanced glycation end products SCID Severe combined immunodeficiency SDS-PAGE SDS-polyacrylamide gel electrophoresis SEA Staphylococcal enterotoxin A SEB Staphylococcal enterotoxin B SECi,2,3 Staphylococcal enterotoxin C 1,2,3 SED Staphylococcal enterotoxin D SEE Staphylococcal enterotoxin E SPEA Streptococcus pyogenes exotoxin A SPEC Streptococcus pyogenes exotoxin C sAg Superantigen TBS Tris-buffered saline TCR T cell receptor TNFa Tumour necrosis factor a TSS Toxic shock syndrome TSST-1 Toxic shock syndrome toxin-1 vp Variable region of the T cell receptor P chain X Acknowledgments "I not only use all the brains I have, but all I can borrow." — Woodrow Wilson With that apt sentiment, I express my deep gratitude to Dr. Anthony W. Chow. One can only hope for a mentor with a sincere desire to nurture the development of an independent investigator who balances introspection, intuition, and focus. Dr. Chow travels a step further in his guidance by acknowledging and appreciating the strengths and idiosyncrasies of each of his trainees and cultivates those strengths to their full potential. His trust in my abilities served as a strong buttress in my own developing confidence as a scientist. I don't fathom I can thank you enough, Dr. Chow - other than by cutting down on my infamous verbiage in the expression of my gratitude. I lucked out, and I have to say it was a good match. I consider myself very fortunate to have been among the many students to have been honed under your experienced tutelage, and I have little doubt that I may be among the more memorable trainees many years down the road. I can only hope the affect of time will draw those memories out with an aura of fondness. In continuation with the mentoring and guidance aspect on my journey towards a doctorate degree, I give many thanks to Dr. Vince Duronio, Dr. Jan Dutz, Dr. Fumio Takei, and Dr. Sara Townsend for taking the helm of my PhD committee. Your time, patience, and essential feedback during this time were both useful and much appreciated. Of course, no list of acknowledgment would be near complete without due accolades to Dr. Winnie Kum - who serves impeccably as a friend, sounding board, discussion partner, patient mentor, and support system all in one. Through the last few years, life experiences have put things in perspective, and I am grateful for Dr. Kum's company through these years of maturation. With much pleasure and gratitude for their company, support, and brainstorming - 1 recall the many members of the Chow lab who have made the journey through this right of passage appreciated by the few gluttons for punishment aiming for a graduate degree in the life sciences commencing with the unforgettable Dr. Ryan Hung. He is certainly in a class of his own in every way imaginable. Thanks, Ryan, for our many theological meanderings. Dr. Sabine Ivison, the ever stimulated scientist, thank you for our many exchanges in introspections and bouts of caffeine; and, finally, the current and past posse of trainees - I know you will undoubtedly succeed at whatever you set your sites on. I would also like to take this opportunity to thank the supportive institute enshrined within this country to support academic research and hope that Canada continues in its investment in higher training of Canadian scholars. I truly am in deep gratitude for the support I've been given throughout my entire academic career as an undergraduate and then doctoral candidate from UBC, the Canadian Institute of Health Research, and the Michael Smith Foundation of B.C. xi Dedications I dedicate this body of work (both mine and that which is a product of mine) to my family. Last but never least (cliches are oft repeated because they are poignantly true), I give everything - the good, the bad, and the downright unscrupulous to my right hand -the appendage that can be held accountable for my waywardness. Without your support, none of this would have been possible. I am currently investigating how to keep all of you cryopreserved until the end of at least my time (and you know I don't dabble in idle threats). Mom and Dad - 1 can only say that the heterogeneous advantage worked in this case. Thank you for first making me and then loving me with no strings attached. A simple, yet powerful, formula. Jamil, you should be thankful you were spared the neurotic gene of the idealistic academic and were given the psychotic gene of the pragmatic. Together, we near perfection (some may say, close, but no cigar). And Amin, only Rumi knew the value of a life line - and I have some time now to write my version of his Diwan-i Shams-i Tabriz in your honour. xii Manuscripts, abstracts, and presentations that have resulted from the work done towards the completion of this thesis. Manuscripts in final preparation of submission: Kalyan S, Chow A W . Differential regulation of apoptosis by Vy9V82 T cells following stimulation with staphylococcal superantigen, Toxic Shock Syndrome Toxin-1. Kalyan S, Chow A W . Staphylococcal Toxic Shock Syndrome Toxin-1 induces the translocation and secretion of High Mobility Group-1 Protein which is dependent on both activated T cells and monocytes Kalyan S, Chow A W . Enhancement of CD40 and HMGB-1 expression on APC by peripheral human Vy9V52 T cells: Linking the adaptive and innate immune response to Toxic Shock Syndrome Toxin-1. Kalyan S, Chow A W . The immunoregulatory role of y8 T cells: tipping the balance. (review) Refereed Journal Articles: Kalyan S, Chow A W (2004) Human peripheral y8 T cells potentiate the early proinflammatory cytokine response to staphylococcal toxic shock syndrome toxin-1. J Infect Dis 189: 1892-1896 Kum WW, Cameron SB, Hung RW, Kalyan S, Chow A W (2001) Temporal sequence and kinetics of proinflammatory and anti-inflammatory cytokine secretion induced by toxic shock syndrome toxin 1 in human peripheral blood mononuclear cells. Infect Immun 69: 7544-7549 National and International Meetings: Kalyan S, Chow A W . Both activated T cells and monocytes are required for the translocation and secretion of nuclear High Mobility Group-1 Protein in Toxic Shock Syndrome Toxin-1 induced inflammation. 18 t h Annual Spring Meeting, Canadian Society of Immunology. Whistler, B C , Canada. April 7-10, 2005. Kalyan S, Chow A W . Toxic Shock Syndrome Toxin-1 Regulates the Intracellular Expression of High Mobility Group-1 Protein in T Cells as Well as Monocytes. Presented at the 12th International Congress of immunology. Montreal, Canada, July 18-23, 2004. Abstract number 4106. Clin Invest Med 27 (4): T32.18. Kalyan S, Chow A W . Activated yS T cells heighten IFNy expression in ap T cells in response to toxic shock syndrome toxin-1. Presented in part at the Western American Federation for Clinical Research annual meeting, Carmel, C A . February 1, 2004. J Invest Med 52(1): S151-S151 J A N 2004. xiii Kalyan S, Chow A W . Peripheral y8 T cells potentiate the early inflammatory response to toxic shock syndrome toxin-1. Presented in part at the Western American Federation for Clinical Research annual meeting, Carmel, C A . February 1, 2003. J Invest Med, 51: 389 Suppl. 1 FEB 2003 Kalyan S, Chow A W . Immunoregulation by y5 T cells in TSST-1 Pathogenesis. Poster presented at the National CIHR Student Symposium and Poster Competition, Winnipeg, M A , May 13-14, 2003. Local Presentations: Kalyan, S. Toxic Shock Syndrome-1 Toxin Regulates the Intracellular Expression of High Mobility Group-1 Protein - the Late Mediator of Sepsis. Poster presentation for Student Experimental Research Day, Vancouver, BC, Nov. 11, 2004. Kalyan, S. Enhanced secretion of IFNy in human P B M C by Toxic Shock Syndrome Toxin-1 following priming of y8 T cells is attributed to both an increased proportion of IFNy-producing y8 T cells, and their capacity to increase aP T cell responsiveness. Oral presentation for Student Experimental Research Day, Vancouver, BC, Oct. 31, 2003. Kalyan, S. Peripheral y5 T cells potentiate the inflammatory response to toxic shock syndrome toxin-1. Oral presentation for Student Experimental Research Day, Vancouver, B C , Nov. 2002. Kalyan, S. Activation of y5 T cells by staphylococcal toxic shock syndrome toxin-1: role in dysregulation of the innate immune response? Poster presentaion for Student Experimental Research Day, Vancouver, BC, Nov. 2001. Kalyan, S. Role of y8 T cells in toxic shock syndrome toxin-1 induced immune activation. Oral presentation for Student Experimental Research Day, Vancouver. xiv Chapter 1: Introduction 1.178 T cells: A sorority of lymphocytes in a class of their own. y8 T cells entered the consciousness of immunologists over 20 years ago when the y gene was serendipitously discovered during the molecular characterization of the a and P T cell receptor (TCR) genes (1, 2, 3, 4, 5). Within this span of time, much has been discerned about the antigen recognition mechanism and raison d'etre of ap T cells. However, y8 T cells have proven to be more challenging to solve and are clearly different in both form and function from their fraternal counterparts. Table 1-1 lists some of the major disparities between the two subsets of T cells in both mice and men. Table 1-1. Major differences between human ap and y5 T cells. Feature Frequency in blood MHC restriction CD4/CD8 expression ap T cells • 65-75% of P B M C • CD4+: M H C class II • CD8+: M H C class I • -60% CD4+; -30% CD8+;<1% double positive; • <1% double negative Antigen recognition • Processed peptide/MHC • Large TCR V gene germ line repertoire TCR diversity Function Very diverse Adaptive immunity y5 T cells • 1-5% of P B M C (25-60% gut) • No M H C restriction • Possible roles of CD 1 & MICA/B • Majority (> 70) double negative; <1% CD4+; -30% CD8+aa (as TELs in gut) • Unprocessed, not peptides • Small • Very restricted; expression variance dictated by tissue • Immuno-regulation and immuno-surveillance 1 Part of the reason why characterization of y8 T cell has lagged behind that of aP T cells is due to the early assumption by immunologists that their antigen recognition and function would be similar to aP T cells. It has come to light that, not only is the role of y8 T cells different, but the type and nature of the antigens that stimulate this unique subset of lymphocytes are inherently distinct from that of ap T cells. Recognition by y8 T cells is not typically in the context of classical M H C class I or II molecules and neither is antigen processing required (6). These observations corroborate the fact that the majority of y8 T cells are CD8 and CD4 negative. Crystal structure analysis of the y8 T cell receptor (TCR) determined that the length and conformation of y8 T cells resemble immunoglobulins more than the aP TCR, suggesting that antigen recognition by y8 T cells may be more similar to the binding of antibody to antigen rather than the MHC/peptide complex recognized by ap T cells (7). The very basis of foreign antigen recognition by lymphocytes of the adaptive immune system is a point of contradiction for y8 T cells. Lymphocytes that bear similar features to y8 T cells are the natural killer (NK) cells. N K cells are considered constituents of innate immunity, recognize stressed or transformed cells, play a major role in antiviral protection, and are cytolytic lymphocytes. These are all characteristics shared by y8 T cells. Furthermore, y8 T cells also express killer Ig-like receptors (KIRs), such as the C-type lectin CD94 paired with N K G 2 A which binds certain M H C class I aleles to negatively regulate N K cytotoxicity (7). The versatile functionality of y8 T cells is further extended by their ability to assume the role and appearance of professional antigen presenting cells (APCs) (8). In addition to this list of diverse functions, y8 T cells are also vital for maintaining epithelial integrity and homeostasis by secreting growth factors such as KGF, FGF-7 as 2 well as insulin growth factor-1 (6, 9, 10, 11, 12). Figure 1-1 gives an amalgamated representation of the emerging portrait of these multifaceted lymphocytes. The most unambiguous aspect that can be said about these unconventional T cells is that they work well under stress. 3 Figure 1-1. Comparison of the common features shared by y5 T cells with NK cells, ap T cells and professional APCs. Like N K cells, y8 T cells express stimulatory and inhibitory N K receptors (eg NKG2D and other KIRs) which either induce or inhibit cytotoxicity when triggered by their ligands on neighbouring cells. y8 T cells also have the ability to present and activate ap T cells, like professional APC, during infection. Lastly, like aP T cells, y8 T cells express their TCR complex consisting of CD3^, y, 5, and e as well as many of the co-stimulatory molecules usually found on T cell subsets, such as CD25, CD28, and CD45 - though the expression of these co-receptors are more variable than they normally are on aP T cells. 4 1.1.1 Ontogeny and distribution. As is befitting cells of innate immunity, the appearance of y8 T cells during development comes before the expression of a(3 T cells. y8 TCR gene rearrangement can be detected at embryonic day 13 in the murine thymus (13) and week 8 in humans (14). Unlike ap T cells, there are limited numbers of Vy and V8 gene segments. Table 1-2 lists the relative number of vp, Va , Vy, and V8 gene segments in mice and men. The 8 genes are embedded within the TCRa locus and some segments can be used as both V a and V8 (referred to as ADV) ; thus, it is difficult to ascertain diversity completely within this gene segment (15, 16). The development and tissue distribution of y8 T cells in mice and men is illustrated in Figures 1-2 and 1-3 respectively. Table 1-2. Comparative diversity (based on the relative number of gene segments) of the a, p, 6, and y TCR chains in mice and men. Human Mice Vy ~ 6 (5 from same family, 1 distantly ~ 6 (2 from same family, 4 broadly related) diverged) V 8 ~ 8 functional genes (only 3 ~ 16 (6 homologous, 10 distinct) commonly used) Va ~ 42 -75 Vp ~ 47 ~ 23 As Figure 1-2 illustrates, some subsets of y8 T cells occur entirely prenatally and make their way to certain anatomical locations where all the y8 TCRs within that tissue microenvironment express invariant germ-line encoded receptors. For obvious reasons, the development of yS T cells during fetal maturation in humans has not been elucidated with as much detail, but the changes in both the receptor repertoire and junctional 5 diversity (DJ) following birth has been well documented from sampling of cord blood in which y8 T cells make up -2% of the CD3+ cells (17). 6 Development waves & distribution of 78 TCR chains in the fetal mouse a p T c e U s p r e d o m i n a t e a f t e r d a y 1 6 ; and juactional diversity appears LYMPH Vy2V55 (V5 6 or 7) 14 16 DAYS BEFORE BIRTH SKIN V73/V8I y8 T cells at mucosal sites: no junctional diversity •INTESTINE - p o s s i b l e extrathymic o r i g i n Vy2\ 85 REPRODUCTIVE TRACT/TONGUE Vy4/V81 PROINFLAMMATORY Figure 1-2. Developmentally guided tissue distribution of 78 T cell subsets in the mouse. Schematic diagram of the developmental waves of selective y5 T cell chains observed in the fetal mouse is depicted. The y8 T cell receptor repertoire seems to be pre-programmed as specific pairs of the y and 5 chains occur prior to any foreign antigen exposure and are targeted to specific anatomical locations where they are driven to expand by local endogenous stimuli. Certain subsets of 78 T cells, such as those present in the circulation and the lungs, have been characterized as being pro-inflammatory in nature and having a definite Th l bias. 7 cord blood V81 + 50%DJ1»DJ2 <10% in periphery DJ1 minor V52+ 25VJDJ3>>DJ2^gjT~ 1 ,70-75% in periphery DJI major) DJ1 major V53+ DJ1 minor 1st 2nd i 3rd B I R T H 1.5 yrs 6.5 yrs 20 yrs 60 - 80 yrs Trimester of gestation Postnatal Adult Figure 1-3. Developmental change in y 8 T C R expression in humans. A schematic representation of the developmental changes observed in the human y8 T cell expression over time is shown. The most striking feature is the change from having fairly diverse pairs of y8 T cells (of which V81+ serve as the majority at birth) to increasingly restricted pairings, with Vy9V52 T cells becoming the major subset with very limited receptor diversity by adulthood. Postnatal expansion of the Vy9V82 T cell subset in the periphery goes from a mean of 2% to 75% of circulating y8 T cells (18). This preferential increase appears to be antigenically driven and continues through puberty, after which the overall number of y8 T cells declines with age (19). 8 In both mice and primates, yd T cells comprise a small population of peripheral T cells (averaging 5% of the total peripheral T cell pool) (6), and they are poorly represented within conventional T cell zones found in lymphoid tissue (6). In contrast, sheep, cattle, rabbits, and chickens have much higher levels of circulating y5 T cells (20). Although the reason behind the particular constitution of y8 T cells between and within species is currently unknown, the discrepency seen between "low" and "high" y5 T cell species was correlated with the level of TCR variable (TCR-V) genes that is mirrored in the diversity present in the immunoglobulin (IGV) genes (20). Many of the species having higher numbers of circulating y8 T cells have apparently lost entire TCR-V subgroups. Table 1-3 gives the proportional representation of y8 T cells in the periphery of various animal species. Table 1-3. Comparative diversity and percent composition of y 5 T cells between species. Percent y 5 T cells Diversity of TCR-V genes Diversity of IGV genes Human ~ 5% (low) high high Mouse ~ 5% (low) high high Rabbit ~ 20% (high) low low Sheep ~ 30% (high) low low Cattle -30% (high) low low Chicken -20% (high) low low In comparison to the relatively low numbers in the murine and primate circulation, y5 T cells are much more prevalent in epithelial tissue such as the skin, 9 intestine, lungs, uterus, vagina, and tongue (15, 17, 21, 22) where they serve to fortify the first line of defense against both infectious and non-infectious stress as part of the local intraepithelial lymphocytes (IEL) (17, 23). 1.1.2 Vy9V52 T cells: predominant human peripheral blood yd T cells - a primate inheritance. The predominant subset of systemic y§ T cells in the peripheral blood of healthy adult Homo sapiens, and some other primates such as the rhesus monkey (24), bear the canonical Vy9V82 T C R (25, 26). This unique posse of innate T cells and the class of antigens they recognize have no equivalent in either rodents or ruminants. The antigens that stimulate human peripheral blood y5 T cells are non-peptidic, low molecular weight molecules that are ubiquitous in nature. The first Vy9V82 T cell antigen identified, isopentyl pyrophosphate (IPP), was isolated from lysates of mycobacteria (27). It was later determined that IPP is a metabolite in both the mevalonate pathway for cholesterol metabolism and the deoxyxylulose pathway commonly used by organisms that lack the critical H M G - C o A reductase enzyme of the mevalonate pathway (28). Virtually all l iving organisms produce isoprenoids as essential metabolites of their cellular biology (Figure 1-4). Thus, the antigens that stimulate the main subset of human peripheral yS T cells can be anticipated to be elevated during both infectious and non-infectious stress. 10 • Most eukaryotes • Archaebacteria Mevalonate Pathway (Classical) f»sd<je1a*a if i Anti-cancer drugs that stimulate Tfl T cells block pathways downstream of IPP 4 M«v»kxT4te-SP 4 4 Cyanobacteria Uebacteria Algae & plastids Non-Mevalonate Pathway (Alternative) i 0*oxyxyMc*«>S>P ' 4 PfcyM Vi twnn K V temk i E G**»«¥l PP Zoledronate I P a mi dronate F«mesy» PP — 4 G*T«r*(*0tMrHnyt PP 4 OanctnyWM PP 4 tswmenyMftNA De*chots Ma-am A ^ Ub«quinows Uttr+tymus fwoleln* (ras. I*min cxhertl (rap, n*b, I M , «t>o<»j hotmoti— 0<« Mile Vwrwn • L l p o p t O t t m * Figure 1-4. The classical mevalonate and alternative metabolic pathways leading to the production of IPP which stimulates human Vy9V82 T cells (29,30). IPP and its isomer, dimethylallyl pyrophosphate ( D M A P P ) , are molecules at the vital metabolic bottleneck shared across the board. Many tumour cell lines that induce Vy9V52 T cel l-mediated cytolytic activity is a result of the build up of endogenous IPP (31). Some anti-cancer drugs, such as palmidronate and zoledronate, actually block the mevalonate pathway downstream of IPP which results in a build up of this substrate and subsequent stimulation of peripheral y8 T cells (32, 33). 11 More recently, a second class of low molecular weight antigens have been found to be stimulatory for this same pool of human peripheral blood y5 T cells. Like the phospho-antigens described above, this second class of molecules, the alykylamines, are naturally occurring antigens that are ubiquitous in bacterial secretions, certain foods, and even body fluids (34). Interestingly, L-theanine, an amino acid found in tea, is converted into ethylamine (an alkylamine antigen) in the body by the liver and stimulates Vy9V52 T cells both in vivo and in vitro (35). Some of the structural properties of Vy9V82 T cell antigens and their relative potencies are illustrated in Figure 1-5. It is still unknown how these antigens are presented, although it is well established that recognition is independent of presentation or processing involving classical M H C class I or II molecules; however, cell to cell contact for cytokine secretion and effector functions appears to be a prerequisite (36). 12 Phospho-antigens Alkyl amines N'srac Potency Molten t* Pettncy isojx-nlenyl pyrophosphate (IPH}(C5) rthylamine +++ — ^ m«hybmli>c -germylpyrophospfate (GPP) CIO) t i i i isobutylamine faracslypFophosphttic +++ wx-feytyJarnine Gcrcwyl g m n y l pyrophosphate (GEPPKC20) ise-amylaniine Uridine I I I H dhanolamine -thymidine (TUBag 3) 11 i M Figure 1-5. Structural properties of nonpeptidic antigens recognized by Vy9V52 T cells. [Modified from (37) with permission]. Structural properties of the two families of molecules, the phospho-antigens and alkylamines, found to be stimulatory for human peripheral blood Vy9V82 T cells. Some recent research suggests that the potency of the antigens is determined by the bond strength conferred by the active group (i.e. the phosphate or amine group, respectively). 13 In contrast to the Vy9V82 T cells in circulation, over 70% of tissue localized human y8 T cells express V81 (38) and recongize stress inducible M H C class-I-chain-related (MIC) A and MICB expressed on epithelial cells (39). These stress-induced molecules also serve as ligands for NKG2D on N K cells and CD8+ a0 T cells (40, 41, 42). It was recently determined that this same subset of V81 T cells also recognize C D l c directly in the absence of any bound antigen, and this interaction plays a vital role in inducing dendritic cell maturation (43). 1.1.3 Stressed out: Function in health and disease. y8 T cells produce EFNy and TNFa very early upon ligand recognition, and this event is a very important stimulus for macrophages early in infection. Response to alkylamine and organophosphate antigens occurs within two hours of stimulation (30). The early pivotal role of human Vy9V52 for resistance against both gram-positive (S. aureus) and gram-negative bacteria (E. coli and Morganella morganii) was demonstrated using a chimeric severe combined immunodeficiency (SCID) mouse (hu-SCID) model in which resistance was evident at day 1 after infection, and bacteria were cleared well before y8 T cell expansion (44, 45). Furthermore, treatment with Vy9V82 T cell antigens markedly increased clearance (45). Expansion of y8 T cells was not detected until about day six post-infection and after bacterial clearance - indicating that proliferation of y8 T cells is not a requirement for their antibacterial response. It is evident that y8 T cell-mediated early immuno-protective functions are independent of proliferation or clonal expansion. In reality, y8 T cells are poor producers of IL-2 and are dependent on CD4+ T cells for the production of this T cell growth factor (46, 47). Figure 1-6 depicts the biphasic activity of y8 T cells during the course of 14 infection. The bi-phasal response of y5 T cells serve opposite functions. The first phase involves initiation of a pro-inflammatory cascade and the second phase usually involves a down-regulatory function in which the recruited y5 T cells kill off activated, transformed, and infected cells and initiate wound healing (11, 16,48, 49). There is mounting evidence that the "anti-inflammatory" y8 T cells that take over the "clean-up" task post-infection are a different subset than those that set off the early alarm bells (50, 51). In both human and murine experimental systems, the tissue localized y8 T cells have proven to serve a potent anti-inflammatory role; whereas, the circulating group of yS T cells have a very strong T h l , pro-inflammatory bias (52, 53). 15 Time since infection Figure 1-6. Schematic diagram of the biphasal response of yd T cells during infection. Illustration of the activity of y8 T cells throughout the course of infection. During the early phase of the immune response, y8 T cells are found to exhibit a pro-inflammatory effect prior to the activation of the adaptive immune response; thus, they act in concert as cells of innate immunity. After pathogen clearance, y8 T cells have proven to be important players in down-regulating the immune response by killing off activated/infected cells and secreting growth factors important in initiating wound healing. 16 An additional impediment that has made solving the y8 T cell conundrum difficult has been the lack of a suitable model to study the role of these ambivalent cells in human health and disease. There recently has been progress in this area by the finding that our relative, the maque monkey, shares a very similar y8 T cell repetoire which includes the phospho-antigen reactive peripheral Vy9VS2 T cell subset (24). The inherent properties of self-recognition and association with inflammation have had y5 T cells prematurely linked to inflammatory and autoimmune diseases. In retrospect, one may surmise that evolution would hardly favour conservation of such a development to be encoded into our germ-line blueprint of immune regulation. Nevertheless, some fundamental concepts have been revealed through the use of murine models selectively lacking either y8 or a P T cells, hi most cases, immune protection and the development of adaptive immunity is largely unimpaired in y8 T cell deficient mice except in two instances: 1) when infection is of the very young (54, 55); and 2) when the bacterial load is high. In these cases, y8 T cell deficient mice suffer from extensive bacteremia indicating that they contribute in early immunity - both in terms of the developmental maturation of the immune system and the kinetic course of the immune response. However, the most profound defects in y5 T cell deficient mice have been in 1) wound healing (9); 2) terminating the immune response by killing off infected or activated cells (56), and 3) preventing carcinogenesis (57). Mice lacking y8 T cells have exacerbated inflammation due to tissue necrosis (16, 58), as well a greater susceptibility to certain types of cancer (59). These findings have taken y5 T cells out of the "immuno-protective" category and positioned them as homeostatic cells having an "immune regulation and surveillance" role (37, 60). 17 1 . 2 . Toxic Shock Syndrome Toxin-1: A superantigen in a class of its own. The term "superantigen" was coined by Marrack et al. in 1989 when they demonstrated that staphylococcal enterotoxins (SE) induced massive expansion of T cells that all shared the same T cell receptor variable (V) P chain domains (61). Over the last 4 years, much to the credit of microbial genome sequencing projects, 41 superantigens have been identified (62) and this list is likely to grow. Superantigens, as the term signifies, are the most potent T cell mitogens that have been discovered to date (63). Less than 0.1 pg/ml is enough to lead to uncontrolled T cell proliferation, possibly culminating in fever, shock, and death (64). Conventional peptide antigens are processed and presented by classical M H C molecules to aP T cells. This interaction has the potentional to stimulate between 0.001 - 0.0001% of the total T cell pool (63, 65). In contrast, superantigens bind to invariant regions of M H C class II molecules outside the peptide binding groove as intact proteins that are presented to T lymphocytes expressing certain VP chains (see Figure 1-7). This results in polyclonal activation of upto 30% of the T cell repetoire (66) and the induction of massive secretion of pro-inflammatory cytokines such as TNFa, LENy, and IL-lp from both T cells and APCs. 18 Staphylococcal e.g.. TSST-1/ exotoxins ( a p T C e l l I Cell Proliferation 1 m V fcupenintiguu T S S T - 1 IFNy T X F u , IL 2 Antigen Presenting Cell f..;. gfefi.;.f,. Figure 1-7. Superantigen mediated immune hijacking. Superantigens, such as TSST-1, by-pass the regular antigen presenting requirements of Ag-specific T cells recognizing processed peptides in the context of M H C molecules on professional APCs. In contrast, sAg bind to certain V-P elements (VP2 for TSST-1) outside the peptide-binding groove (as shown), thereby bridging together and activating a large number of T cells and A P C s leading to massive T cell proliferation and the release of pro-inflammatory cytokines which can result in a systemic response culminating in death as a consequence of multi-organ failure. 19 1.2.1 Phylogeny, distribution, and cause of disease. Most superantigens are toxins ranging 20-30 kDa in size and are produced by a number of bacteria including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus equi, Streptococcus dysgalactiae, Clostridium perfringens, Yesinia pseudotuberculosis, Mycoplasma arthritidis, as well as some viruses such as mouse mammary tumor virus (62, 65, 67). Staphylococcal and streptococcal pyrogenic superantigens are the best studied and most often cited virulence factors as cause of disease (68). Together these superantigens build a large protein family (Figure 1-8) suggesting phylogenetically that they may have all evolved from a single progenitor. The genes for these toxins are located on mobile elements allowing for horizontal transfer between and within bacterial species (62). It is evident by disease pathogenesis as well as structural groupings, that TSST-1 forms a class of its own. This outlier toxin shares little homology at the amino acid level to the other staphylococcal sAgs but possesses a similar overall structure (shown in Figure 1-9). 20 -TSST-1 - SMEZ-1 - SMEZ-2 — S P E - C • SPE-J — S P E - G - S E G S S A S E B SEC1 SEC2 S E C 3 — S P E - A — SPE-A7 _ S E H - S E D _ S E J - S E P — S E A - S E E — S E N — S E O r SPE-H L SePE-H — S E L — SEI — S E K — S E M — S E Q r S P E - l T S e P E - l - SPE-Gdys r SePE-L H _ SPE-L r S P E - M * ~ i - S D M r - SePE-M " t -SPE-M Figure 1-8. Phylogenetic tree of staphylococcal and streptococcal superantigens (62). An alignment of streptococcal and staphylococcal superantigens based on amino acid sequence is depicted. As shown, TSST-1 is a clear outlier, and, in fact, shares more similarity with some of the streptococcal sAg's than the other staphylococcal sAg's. Despite varying degree of similarity at the amino acid level, sAg as a family of exotoxins share a very similar overall structure shown in Figure 1-9. 21 Staphylococcal superantigens have been implicated in many diseases and immunological disorders such as multiple sclerosis (69), diabetes mellitus (70), rheumatoid arthritis (71), atopic dermatitis (72), Kawasaki disease (73), and psoriasis (72). TSST-1 has even been linked to sudden infant death syndrome (SEDS) by virtue of the isolation of toxin producing S. aureus from the kidneys of 18% of victims (74). Unlike the well characterized staphylococcal enterotoxins (SE), SEA and SEB, TSST-1 is not a cause of staphylococcal food poisoning and lacks the ability to induce emesis when ingested as is the case with SEs (75). This difference between sAgs was made evident when TSST-1 was administered orally to monkeys and, instead of symptoms of food poisoning, it induced systemic symptoms of toxic shock syndrome (TSS) which is the most serious and life-threatening illness caused by staphylococcal superantigens (75). Like endotoxin-mediated shock, TSS is a cytokine-mediated disease. It is considered a capillary leak syndrome characterized by high fever, hypotension, hypoalbuminemia, generalized non-pitting edema (76), and involves three or more organ systems potentially culminating in death (77). It is currently thought that pyrogenic superantigens sensitize individuals to endotoxin shock by priming the innate immune cells to respond to LPS (76). Table 1-4 lists the C D C s diagnostic criteria for staphylococcal TSS. TSS was initially brought to the attention of the medical community in 1978 when it was identified as a major systemic illness associated with staphylococcal infections in children (78). It is thought that TSST-l's unique ability to cross vaginal mucosal epithelial tissue is the basis for its being the major causative agent of menstrual-associated toxic shock syndrome (75). TSST-1 is also responsible for at least 50% of non-menstrual cases of TSS (64) including post-surgical TSS (79), influenza-associated TSS (80), recalcitrant 22 erythematous desquamating syndrome (81), and TSS associated with the use of contraceptive diaphragm (79). Burn patients are also susceptible to TSS due to the frequent colonization by 5. aureus on burn wounds; and children, in particular, are more likely to develop TSS following burn incidents (82). At present, the main treatment of TSS is the appropriate use of toxin neutralizing antibodies by administration of intravenous immunoglobulin (IYIG) (83). Supportive care is critical to prevent associated problems, such as renal failure and respiratory distress syndrome. 23 N-terminjs SEA SEC2 SEB T S S T -1 Figure 1-9. Ribbon structure of TSST-1 and related superantigens with TCR and MHC II binding sites identified (84, 85). Ribbon diagrams of TSST-1 and related staphylococcal and streptococcal exotoxins are depicted, showing the characteristic structure of bacterial superantigens. TSST-1 is a single polypeptide chain having a molecular weight of 22 kDa. As illustrated, the molecule can be divided into two domains: Domain A, which contains a long central a-helix near the C-terminus (shown in red), and Domain B characterized with the P-barrel, or claw (shown in green on the right). Regions of the molecule serving as the binding sites to M H C II and the TCR were elucidated using selective mutants having specific amino acid substitutions. 24 Table 1-4. Diagnostic criteria for staphylococcal toxic shock syndrome 1. Fever 2. Hypotension 3. Diffuse macular rash with subsequent desquamation 4. Three of the following organ systems involved: i. Liver ii. Blood iii. rRnal iv. Mucous membranes v. Gastrointestinal vi. Muscular vii. Central nervous system 5. Negative serologies for measles, leptospirosis, and Rocky Mountain spotted fever as well as negative blood or cerebral spinal fluid cultures for organisms other than S. aureus. Additional diagnostic criteria for staphylococcal TSS includes: (a) isolation of S. aureus from a mucosal or normally sterile site, (b) production of TSS-associated sAg by isolate, (c) lack of antibody to the implicated toxin at the time of acute illness, and (d) development of antibody to the toxin during convalescence. (CDC diagnostic criteria) 1.2.2 Stressing out the host - not just a primate privilege. S. aureus frequently inhabits the epithelial and mucosal membranes of people; but clinical infection occurs in a relatively small percentage of cases (86). The gene for 25 TSST-1 is found in roughly 20% of clinical isolates taken from both blood and nasal specimens (87). The epidemiological data on staphylococcal TSS show that 93% of cases reported from 1979 to 1996 were women with a median age of 22 (79). This finding is not surprising given that the majority of cases being reported were due to menstrual associated TSS. What is more intriguing is that the reported non-menstrual cases of TSS were also predominantly in women (73%). Host immunity plays a large role in determining the response rendered upon exposure to sAg-secreting bacteria. The main risk factor is the lack of neutralizing anti-sAg antibody production. Very low or negative titers of TSST-1 specific antibodies were found in acute-phase serum samples of TSS patients (88), and less than half of these patients developed sero-positive titers to TSST-1 within two months of their illness (88). It is not fully known what factors in the immunological microenvironment upon exposure to TSST-1 leads to a failure to develop protective antibodies, but it has been long suspected that a strong initial Thl bias characterized by secretion of IFNy, TNFa, and EL-2 blunts the Th2 response required for B cell support and subsequent antibody production (89). Superantigen genes have also been found among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry (90). A significant source of staphylococcal superantigen-induced angst comes from the bovine and ovine world where clinical staphylococcal mastitis is a constant threat and liability for the dairy industry (91, 92). In many ways the situation seen in clinical bovine inflammatory disorders originating from staphylococcal infections resembles the colonization of mucosal membranes with potential recurrent clinical infections in humans (86). Rabbits have been extensively used to investigate the etiology of menstrual associated TSS as the disease progression is very 26 similar to that seen in women using tampons (93). Mice, however, are notoriously resistant to the development of toxic shock syndrome and need to be pre-sensitized with D-galactosamine (D-gal) for murine models of toxic shock syndrome (94). D-gal is a hepatotoxic agent which depletes hepatocytes of UTP by accumulation of UDP-galactosamine (95). Nevertheless, studies done in both mice and rabbit models have clearly identified TNFa as one of the key critical initiator of both TSS and sepsis (94); but attempts to target this inflammatory cytokine for treatment of systemic inflammation have proven unsuccessful partly due to the very early and transient secretion of TNFa into the blood stream following superantigen stimulation (96). 1.2.3 yd T cells run into superantigens: stressing the implications. Given the body of evidence that suggests that y8 T cells could have some immuno-regulatory role in bacterial superantigen inflammatory disorders, it is surprising that this area of research has not been further investigated. Initial studies in the late 1980's and early 1990's had, in fact, attempted to study the response of human y5 T cell lines to staphylococcal superantigens by assessing their cytolytic activity upon sAg presentation (97, 98). SEA seemed to be the most promising candidate for direct recognition by the majority of peripheral y8 T cells. However, given what is now known about the role and antigen recognition of Vy9V82 T cells, this speculation warrants further clarification - especially given the fact that despite the ubiquitous nature of y8 T cell antigens, toxic or septic shock has never been directly attributed to their antigen reactivity to phospho-antigens or alkylamines. One of the most intriguing clinical cases of finding the possible involvement of human y8 T cells with stapylococcal superantigens comes from multiple sclerosis (MS) 27 patients. Both activated y8 T cells, which have been speculated to contribute to the demyelination process in MS (99, 100, 101), and staphylococcal sAgs were isolated from the cerebral spinal fluid and blood of MS patients (100). These studies established a union of two prominent suspects in deleterious autoreactive inflammatory disorders discovered together at the scene of disease pathology. This finding inevitably renewed interest of their potential role in MS and other autoimmune disorders (101, 102). Another compelling case for the involvement of yo T cells in sAg-induced inflammation once again comes from bovine inspiration. Blood samples from cows with confirmed staphylococcal and streptococcal mastitis had increased numbers of y8 T cells. However, the most dramatic changes in leukocyte distributions occurred in the milk samples from these infected cows, with a 75% increase in a P T cells levels and a 100% increase in y8 T cell levels relative to the amount in milk samples from healthy animals (103). As mentioned previously, sAg's have an extensive history in contributing to this costly dairy problem, and y8 T cells are now seen as prominent participants in this pathology. Thus, yS T cells and sAg's were once again united in immune corruption. It was subsequently shown that bovine y8 T cells induced the production of IL-12, TNFa, and EFNy in the presence of superantigens, and this effect was partly dependent on the presence of exogenous IL-2 and dendritic cells (104, 105). Nonetheless, like the dual roles y8 T cells play in human immune regulation, the same is observed in bovine studies in which sAg's serve as virulence factors in staphylococcal mastitis. It became evident that there are two different populations of y8 T cells that are recruited to the site of the inflammatory response that bear different cell surface markers and apparently mediate different 28 functions in inflamed cows (106). It has even been speculated that y8 T cells may have a suppressive role in staphylococcal mastitis (106). 1.3. High Mobility Group Box-1 Protein: An inflammatory cytokine in a class of its own. High mobility group box-1 protein (HMGB-1) was not initially a cognizant part of the project which had a primary focus on y8 T cells in the context of TSST-1 induced inflammation. HMGB-1 was discovered over 30 years ago as a fast migrating chromatin-associated protein on an electrophoresis gel (107). It was subsequently characterized as a 30 kD protein that is abundantly expressed in virtually all nucleated cells and that has been highly conserved through evolution (99% identity in mammals). Since this initial discovery, HMGB- l ' s versatility has been appreciated in many different roles - from leading axons in neural development (where it was first identified as a 30 kD protein called amphoterin) (108) to erythroleukemia cell differentiation (109, 110) to mediating sickness behaviour and anorexia (111). It was not until 1999 that HMGB-1 inflamed the septic field when it was revealed this non-histone DNA-binding protein was secreted by LPS activated macrophages (112, 113). More impressively, HMGB-1 was determined to be the late mediator of endotoxin lethality (112, 113). The excitement over this finding stemmed from the fact that this pivotal molecule broadened the therapeutic window for sepsis and endotoxemia (114). As the late mediator of septic shock, it is secreted during the later phase of the inflammatory response; in contrast to TNFa which is secreted within the first couple of hours during the onset of sepsis (96). Neutralizing the effect of HMGB-1 as late as 24 hr post-sepsis induction, either by inhibitors or neutralizing antibodies against HMGB-1 , rescued mice from lethality (112). Not surprisingly, 29 HMGB-1 is currently being evaluated as a therapeutic agent for sepsis and other inflammatory disorders such as arthritis (115, 116, 117). One of HMGB- l ' s key receptors is R A G E (receptor for advanced glycation end products) which is a member of the lg superfamily that is ubiquitously expressed on a wide number of cell types including monocytes, dendritic cells, endothelial cells, vascular smooth muscle cells, and activated (but not resting) T cells (118, 119, 120, 121, 121, 122, 122). The translocation and subsequent release of nuclear HMGB-1 from monocytes is dependent on stimulation by TNFa, IL- lp , and EFNy that follows early upon LPS signaling (123). Figure 1-10 illustrates and summarizes the role of HMGB-1 in endotoxin shock. 30 TISSUE D A M A G E or NECROSIS F«vtr Anorexia / Weight loss Taste sverewc Pmn Neuftte oud^puwth H^, yJ-,,1. TllllHl III g ivtwrapM iiwiBaon Endotnella activation InfVammfltion BflHMI t Nrtnc oxide aynthsae Loss of epftheJia barrier function Refostsa of Irvef enzyrne I 1 Macrophage actuation Monocyte activation Nautnjpti activation Endothallal acBvation lTt-PA?PAf Figure 1-10. Regulation of H M G B - 1 , and its downstream effects in systemic inflammatory disorders. HMGB-1 i s d e t e c t e d i n t h e l a t t e r p h a s e o f t h e i m m u n e r e s p o n s e t o L P S o r t i s s u e d a m a g e a f ter t h e e a r l y r e l e a s e o f T N F a , ILlf3, a n d B F N y . T h i s m o l e c u l e w a s s h o w n t o m e d i a t e m a n y o f t h e p a t h o l o g i c a l s y m p t o m s c h a r a c t e r i s t i c o f s e p s i s , s u c h as s i c k n e s s b e h a v i o u r , i n f l a m m a t i o n , e d e m a , a n d v a s c u l a r p e r m e a b i l i t y , a n d i t w a s d e m o n s t r a t e d t h a t n e u t r a l i z i n g i t s e f f e c t s p r e v e n t e d s e p s i s - i n d u c e d l e t h a l i t y . O n e o f t h e k n o w n r e c e p t o r s o f HMGB-1 i s RAGE ( r e c e p t o r f o r a d v a n c e d g l y c a t i o n e n d p r o d u c t s ) e x p r e s s e d o n m a n y c e l l t y p e s s u c h as m o n o c y t e s , e n d o t h e l i a l c e l l s , a n d a c t i v a t e d T c e l l s . S t i m u l a t i o n w i t h e x t r a c e l l u l a r HMGB-1 a c t s as a n a c t i v a t i o n f e e d b a c k 31 loop leading to the release of another dose of pro-inflammatory cytokines which is often observed during sepsis. 32 Taken together, HMGB -1 poses as an attractive tool to evaluate the downstream influence of y8 T cells in TSST-1 induced inflammation. Immune pathology induced by TSST-1 essentially overlaps with that of HMGB- l ' s role (as outlined in Figure 1-10) in endotoxin shock. The difference between the two is the cooperative requirement of both T cells and monocytes for superantigen-induced inflammation as opposed to the singular role of monocytes in endotoxin shock. The main impediment in using HMGB-1 as a downstream effector molecule for the evaluation of TSST-1-mediated inflammation is the fact that the secretion and modulation of HMGB -1 expression upon sAg exposure has not been formally assessed. Given the potential importance of HMGB-1 in TSST-1 pathogenesis, this possibility was examined using human peripheral blood mononuclear cells (PBMCs) with a focus on the role of y8 T cells in TSST-1 induced inflammation, as further elaborated upon below. 1.4. Getting a handle on stress: Aims and objective of study. The overall objective of the study was to determine the role and influence of human peripheral blood y8 T cells in TSST-1 induced inflammation. The specific aims that were addressed in this study are as follows. 1. To characterize the potential immuno-regulatory activity of human peripheral blood y8 T cells in the course of the inflammatory response to TSST-1 by assessing a) early and late cytokine secretion; and 2) analysis of early and late apoptosis of immune cells. 2. To determine if HMGB -1 plays a downstream role in TSST-1 induced inflammation. 33 3. To determine the potential consequence of activated y8 T cells in TSST-1 induced inflammation by assessing the regulation of HMGB-1 expression. 4. And lastly, to gain further insight as to the means and mechanisms by which y8 T cells may mediate their influence during the inflammatory response to TSST-1 by assessing the regulation of co-stimulatory molecules on antigen presenting cells (APC) and T cells in the presence and absence of activated peripheral blood y5 T cells. 34 Chapter 2: Study Approach, Material and Methods 2.1. Study Approach. The hypotheses being examined in this thesis are: 1) whether yb T cells augment or dampen the proinflammatory or apoptotic responses of human peripheral mononuclear cells (PBMCs) following TSST-1 stimulation in vitro! 2) Whether the expression and secretion of High Mobility Group Box-1 (HMGB-1) protein in human P B M C is altered following TSST-1 stimulation in vitro! 3) Whether gd T cells potentiate the proinflammatory cytokine response of human P B M C to TSST-1 stimulation by altering the regulation of HMGB-1 expression or activation of other co-stimulatory molecules? The overall experimental paradigm adopted is depicted in Figure 2-1. In brief, human peripheral blood mononuclear cells (PBMCs) were purified from normal donors. For each experimental question being investigated, parallel cultures of P B M C either with or without pretreatment with y5 T cell antigen, IPP, were subsequently incubated with TSST-1. To further ensure that any observed difference in the experimental results between groups was attributable to the population of y8 T cells in peripheral blood, yo T cell depleted cultures were assayed as controls. The methods utilized to examine these hypothesis, and the chapters in which the findings are reported, are summarized in Table 2-1. 35 RPMI PBMCs (with or without y8 T cells) IPP RPMI TSST-1 RPMI TSST-1 immune response Figure 2-1. Experimental approach for the study of the immuno-regulatory function of human peripheral blood Vy9V82 T cells in TSST-1 induced inflammatory response. Human peripheral blood mononuclear cells (PBMCs) were purified from normal donors by Ficoll-Paque Plus density centrifugation. One experimental group was treated with the y8 T cell antigen, IPP, and the other group was left untreated in growth medium (RPMI). The cells were then stimulated with TSST-1 or left untreated (RPMI control) and certain parameters of the immune response were subsequently assayed. To ensure that any observed variance in the response was due to y8 T cell activity, y8 T cell depleted PBMCs were analyzed in parallel experiments as a control. 36 2.2 Material and Methods 2.2.1 Cell preparation and culture conditions. Toxin Purification. Recombinant TSST-1 was purified from culture supernatants of S. aureus strain RN4220 previously transformed to carry the tst gene, using both preparative isoelectric focusing and chromatofocusing (124). Toxin purity was assessed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 14% acrylamide gels, and LPS activity was undetectable by the Limulus amoebocyte lysate test (sensitivity limit, 10 pg/ml). Preparation of human PBMC and culture conditions. Fresh human P B M C from healthy donors were obtained by Ficoll-Paque PLUS (Amersham Biosciences Corp., Piscataway, NJ) density centrifugation, and cultured in 24-well flat-bottom plates at 1.5xl06 cells/ml in complete culture medium consisting of RPMI 1640 (Stem Cell Technologies Inc., Vancouver, BC, Canada), 10% heat-inactivated fetal bovine serum (HyClone Laboratories Inc., Logan, UT), 2 m M L-glutamine (Stem Cell), 25 mM Hepes buffer (Stem Cell), and 2 pg/ml of polymyxin B sulphate (Sigma-Aldrich Corp., St. Louis, MO). Depletion of y8 T cells was by immunomagnetic negative cell selection using the StemSep™ protocol (Stem Cell). Briefly, human P B M C were incubated for 15 min at room temperature with an anti-pan-y8 TCR M A b (clone Immu510, BD Biosciences Pharmingen Inc., San Diego, CA) conjugated in a tetrameric array with an anti-dextran M A b (Stem Cell). This was followed by incubation for 15 min at room temperature with a magnetic-colloid consisting of dextran iron particles (Stem Cell), and y8 T bound to the tetrameric-antibody-magnetic-colloid complexes were passed through a magnetic column for depletion. Purity of the yS T cell-depleted P B M C was analyzed in a 37 flow cytometer (model EPICS X L - M C L , Beckman-Coulter Inc., Miami, FL) using phycoerythrin (PE)-conjugated anti-V52 and anti-aP TCR antibodies (BD Biosciences Pharmingen). Data analysis was by the EXP032 A D C software (Beckman Coulter) with a minimum of 10,000 events collected for each sample. Consistent of published literature, V82 + T cells constituted 1-5 percent of total P B M C among different healthy donors studied. Following immunomagnetic separation, greater than 99.8% efficiency in y8 T cell depletion was achieved in all experiments. Treatment of PBMC with IPP and TSST-1. P B M C with or without y8 T cells were treated with either 45 u M isopentyl pyrophosphate (IPP) (Sigma-Aldrich) or RPMI culture medium for 16 h prior to incubation with 1 nM TSST-1 at 37°C. 2.2.2 Cytokine analysis (Chapter 3) by ELISA and intracellular staining: Cytokine assays. Culture supernatants from cells stimulated with TSST-1 or RPMI medium were assayed for IFN-y, TNF-a, IL-2 and IL-10 by enzyme-linked immunosorbent assay (ELISA), using commercial sandwich ELISA kits containing recombinant human cytokine standards, murine monoclonal capture antibodies, and biotinylated goat anti-human cytokine detecting antibodies (125). The sources of various ELISA kits for the different cytokine assays (and their sensitivity limits) are as follows: IFN-y (62 pg/ml), TNF-a (62 pg/ml) and IL-2 (62 pg/ml) from R & D Systems, Minneapolis, M N ; IL-10 (31 pg/ml) from BD Biosciences Pharmingen, San Diego, C A . Intracellular staining for IFNy. Detection of intracellular IFNy after 4 hours of TSST-1 stimulation was determined using fluorescein isothiocyanate (FLTC) conjugated anti-IFNy antibody (clone 25723.11) as directed by manufacturer. Briefly, P B M C or y8 T cell-depleted P B M C , either pretreated with IPP or IPP-untreated, were stimulated with 1 38 nM of TSST-1 in the presence of GolgiPlug (containing a protein transport inhibitor) to prevent the release of intracellular cytokines. After 4 hours of TSST-1 stimulation, cells were washed; stained for cell surface molecules using PE conjugated mouse anti-human aP T cell antibody (clone T10B9.1A-31) and mouse anti-human V82 T cell antibody (clone B6); fixed with Cytofix Buffer; and kept overnight in the dark at 4° G. Cells were permeabilized with PermWash buffer and stained with FITC-anti-IFNy and analyzed by flowcytometry (all reagents and antibodies from Pharmingen). Data analysis. The GraphPad PRISM software (version 3.01; GraphPad Software, Inc., San Diego, CA) was used for data analysis. Cytokine assays by ELISA were determined in duplicate, and data were obtained from a minimum of 3 different donors. Differences in cytokine levels between different treatment groups at different time intervals, or between different time intervals in the same treatment group were assessed by the paired Student t-test (P < 0.05). 2.2.3 Apoptosis studies (Chapter 3): Analysis of apoptosis and cell surface markers by flow cytometery. Cell surface expression of PBMCs was performed using the following fluorescently labelled antibodies: phycoerythrin (PE)-conjugated anti-human V82, PE anti-human CD14, PE anti-human CD3, and PE anti-human CD95 antibodies (all from B D Biosciences Pharmingen). Apoptosis was determined using a combination of APC-conjugated Annexin V and 7-Amino-actinomycin D (7-AAD) to differentiate between early and late apoptosis as described by the manufacturer (BD Biosciences). Data was analyzed by flow cytometry (FACSCalibur™ Flow Cytometry System, BD Biosciences Pharmingen) with a minimum of 10,000 events collected for each sample. 39 Expansion of yd T cell lines and monocyte cytolysis. Expansion of y8 T cells was established by treating freshly purified PBMCs plated at 1 x 106 cells/ml in 24 well culture plates with 4 m M of isobutylamine (IBA) with 20 U/ml of rIL-2 (R&D Systems) supplementation as previously described (126). Expansion of y5 T cells from P B M C took between 10 to 12 days at which time cells were harvested and a(3 T cells were removed by magnetic cell separation using the same protocol described above (Stem Cells). Autologous monocytes were purified from cryopreserved PBMCs from the same donor using Monocyte RosetteSep (Stem Cells). Purified monocytes were cryopreserved (cryopreservation solution: 40% RPMI, 40% FBS, and 20% DMSO) and thawed at the time of cell lysis experiments when y5 T cell lines had been generated. y8 T cell mediated specific lysis of TSST-1 activated monocytes was performed using a modified version of the fluorometric technique described by Sheehy et al. (127) using PKH-26 (Sigma-Aldrich) stained effector cells and carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Invitrogen) labelled target cells. CFSE passively diffuses into live cells where it reacts with intracellular amines, and lysis of target cells results in loss of CFSE fluorescence. The protocol for CFSE and PKH-26 is described below. y8 T cells and monocytes were plated at a 1:1 ratio for 24 h in the presence or absence of 1 nM TSST-1, and direct lysis was assessed by gating the CFSE labeled monocytes in both TSST-1 treated and untreated cultures. CFSE stained monocytes plated in the absence of yS T cells (with and without TSST-1) served as a control to confirm that direct killing of activated monocytes was indeed mediated by y8 T cells. CFSE and PKH-26 cell staining. For CFSE staining, cells were resuspended at a concentration of Ixl0e7/ml cells in PBS (total of 1 ml). An equal volume of the prepared 40 10 u M CFSE/PBS solution was added to the cells for a final CFSE concentration of 5 uM. The target cells were subsequently incubated at room temp for 10 min. The reaction for CFSE staining was stopped by adding an equal volume (2 ml) of FBS, and the CFSE stained cells were then washed cells 3X in warm RPMI medium. For PKH-26 staining of effector y8 T cell effector cells, a cell concentration of Ixl0e7/ml cells were incubated with a 2 u M of PKH-26. Cells were mixed by inversion and gentle rotation at room temperature (for 5 minutes), and the reaction was stopped by adding an equal volume of FBS. Cells were then washed 3X prior to resuspending in complete growth medium. 2.2.4 H M G B - 1 expression studies (Chapters 4 and 5). Detection of HMGB-1 by fluorescent microscopy. P B M C were first surface-stained for expression of CD3 with mouse anti-human CD3 IgG (Pharmingen) followed by anti-mouse IgG antibody conjugated to Alexa594 (Molecular Probes); cells were subsequently fixed using Cytofix buffer (Pharmingen). For the intracellular detection of HMGB-1 , cells were permeabilized with Cytoperm (Pharmingen) and incubated for 30 min (4°C) with rabbit polyclonal anti-HMGB-1 antibodies (Orbigen BioCarta, San Diego) diluted in blocking buffer (phosphate buffered saline, 3% FBS) followed by a secondary antibody with Alexa488-conjugated goat anti-rabbit IgG. The anti-HMGB-1 antibodies were raised against the peptide sequence corresponding to amino acids 166-181 that were previously shown to be specific for HMGB-1 and not HMGB-2 (128). Surface expression of HMGB-1 on P B M C was established by culturing cells (37°C, 5% C O 2 ) directly on coverslips to allow attachment of adherent cells overnight before stimulation with 1 nM of TSST-1. Cells were then fixed and surface-stained as above (but without the permeabilization step). Fluorescent-labeled P B M C were subsequently 41 stained with Hoechst 3342 nuclear dye (Molecular Probes) according to manufacturer's directions, and mounted on slides using Prolong Antifade reagent (Molecular Probes). Cells were visualized with an AxioPlan II fluorescence microscope equipped with a CCD camera using Northern Eclipse software (Epix) for acquisition of images. Images were taken with the 63 x oil immersion objective lens, and Adobe Photoshop 6.0 software was used for image layout. Flow cytometric analysis of surface-expressed HMGB-1 on differentiated cells. Surface expressed HMGB-1 was analyzed by flow cytometery (FACSCalibur™ Flow Cytometry System, B D Biosciences Pharmingen) using the anti-HMGB-1 antibody and secondary Alexa488-conjugated antibody described above in conjunction with phycoerythrin (PE)-conjugated anti-CD3 and anti-CD14 antibodies (BD Biosciences Pharmingen), with a minimum of 10,000 events collected for each sample. Western blot analysis of secreted HMGB-1 from culture supernatants after treatment with either TSST-1 or LPS. For analysis of secreted HMGB-1 in cell culture supernatants, P B M C were plated in 24-well flat-bottom plates in Opti-MEM I reduced serum medium (Gibco). THP-1 cells (human monocytic cell line) were obtained from A T C C and kept in recommended growth media until use, at which time the cells were plated at a concentration of 1.5xl06 cells in Opti-Mem I medium. T cell depletion of human P B M C was performed using a column-free method of magnetic bead separation (EasyCep from StemCell Technologies) by positive selection of T cells using an anti-CD3 conjugated antibody according to manufacturer's directions. Cells were treated with either 500 ng/ml LPS or 1 nM TSST-1, and culture supernatants were collected at 24 h post-treatment, microcentrifuged at 800 x g for 5 min, and frozen at -70°C until protein 42 analysis. At time for Western blot analysis performed, thawed supernatants were concentrated 10-fold from original volume using Amicon Ultra centrifugal filters with a molecular weight cut-off of 10 kDa (Millipore). In some cases, samples from the P B M C culture supernatants had been further prepared using the SDS-PAGE Clean-up kit (Amersham) according to the manufacturer's directions prior to running on a 12% polyacrylamide gel. Western blotting was performed by semi-dry transfer of proteins (Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell, BioRad) onto a Immobilon-P PVDF membrane (Millipore) which had been blocked for 1 h at room temperature with 1% BSA, 0.5% Tween in Tris buffered saline (TBS) prior to overnight incubation (at 4°C) with rabbit polyclonal anti-HMGB-1 antibody. The membrane was subsequently incubated with anti-rabbit IgG horse-radish-peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature on a shaker. Detection of HMGB-1 was performed using Super Signal substrate (Pierce) and developed as well as analyzed using the Alpha Innotech 3400 Gel Documentation system (Alpha Innotech). Statistical analysis. Statistical analysis was performed using Prism 3.0 software package (GraphPad). Categorical parameters in HMGB-1 nuclear translocation were analyzed using 2 by 3 Chi-square test. The proportion of P B M C expressing HMGB-1 following treatment with TSST-1 or RPMI in different donors were compared by paired student t test. Differences were considered significant if p < 0.05. 2.2.5 Cell surface analysis of costimulatory molecules by flow cytometery (Chapter 5). Flow cytometric analysis of the expression of costimulatory molecules on APC and T cells. Cell surface expression of costimulatory molecules was analyzed by flow 43 cytometry (FACSCalibur™ Flow Cytometry System, BD Biosciences Pharmingen) using the following fluorescently-conjugated antibodies: APC anti-human H L A - D R (LN3), PE-Cy5 anti-human CD86 (B7-2) (both from eBioscience, Inc), FITC anti-CD40 (5C3), APC anti-CD25 (M-A251), and PE-Cy5 anti-CD80 (B7-1) (all from BD Biosciences, PharMingen). Cells were subsequently washed, resuspended in buffer containing 2 m M EDTA (to facilitate adherent cells to efficiently detach from the wells), and finally transferred to FACS tubes containing FACS buffer (2% FBS in PBS) before analysis by flow cytometry. A minimum of 10,000 events was collected for each sample. Statistical analysis. Statistical analysis was performed using Prism 3.0 software package (GraphPad). Categorical parameters in HMGB-1 nuclear translocation were analyzed using 2 by 3 Chi-square test. Comparisons between experimental groups for all other data was performed with an average of 3 to 5 normal donors per experiment by paired student t test. Differences were considered significant if p < 0.05. 44 Table 2-1. Outline of the thesis chapters including parameters investigated and the methodology used. THESIS CHAPT ER 1 2 OBJECTIVES AND AIMS PARAMETER ANALYSIZED METHODOLOGY EMPLOYED Introduction: background and purpose of study. Methodology, assay procedures, and materials used Characterization of y8 T cell influence on the inflammatory response in P B M C following TSST stimulation Role of HMGB-1 in TSST-1 induced inflammation Determination of possible mechanism by which y8 T cells modulate the immune response 1) Cytokine expression (Thl vs Th2 polarization of PBMC) 2) Apoptosis of immune cells Assessment of HMGB-1 expression 1) Analysis of the regulation of H M G B -1 expression by y8 T cells 2) Analysis of the expression of cell surface molecules involved with co-stimulation and differentiation 1) ELISA 2) Intracellular cytokine staining by flow cytometery 3) Analysis of Annexin V by flow cytometry 4) Direct cytolysis determined by the loss of CFSE staining 1) Translocation of nuclear HMGB-1 by fluorescent microscopy 2) Secretion of HMGB-1 by Western blot analysis 3) Flow cytometery analysis of surface HMGB-1 expression 1) Fluorescent microscopy of H M G B -1 expression 2) Western blot analysis for the secretion of HMGB-1 3) Flow cytometery analysis of surface expressed HMGB-1 4) Temporal analysis of the expression of co-stimulatory molecules expressed on APC and T cells by flow cytometery. Summary, conclusions, and future directions. 45 Chapter 3: Determining the influence of y5 T cells on TSST-1 induced inflammation and apoptosis. 3.1 Introduction. Toxic shock syndrome toxin-1 (TSST-1) is a bacterial superantigen (sAg) secreted by Staphylococcus aureus and is the major cause of toxic shock syndrome (129). Superantigens differ from conventional antigens by their ability to cross-link Vp-specific regions of the a P T cell receptor (TCR) and the class II major histocompatibility complex molecules (MHC II) on antigen presenting cells (APC) outside the peptide-binding groove (130). Superantigens have the capacity to activate between 5-30% of all T cells, whereas conventional antigens stimulate less than 0.01%. In addition to massive T cell proliferation, this tri-molecular interaction leads to the uncontrolled release of various pro-inflammatory cytokines, which are pivotal to the pathogenesis of TSS. In contrast to ap T cells, y8 T cells constitute only about 1-5% of human peripheral blood mononuclear cells (PBMC), but are more concentrated at mucosal sites such as the gastrointestinal and reproductive tracts, and the skin (131). In human P B M C , approximately 80% of y5 T cells co-express the Vy9 (alternatively named Vy2) and V82 TCR. These Vy9/V82 T cells (nomenclature according to the international ImMunoGeneTics database (132)) respond specifically to nonpeptidic antigens, such as alkylamines and alkylphosphates, that are ubiquitous in nature from microbes to plants and animal cells (34). Isopentyl pyrophosphate (IPP) is the prototypic antigenic alkylphosphate produced by multiple microbes including S. aureus, and is an activator of human Vy9/V82 T cells (133). yS T cells are known to prime macrophages to produce TNF-a in response to LPS (134). However, the roles of human yS T cells in modulating cytokine responses to 46 staphylococcal superantigens are still poorly understood. Thus, Part I of this study was to determine if IPP-primed human peripheral blood y8 T cells modulate the pro- and anti-inflammatory cytokine responses to TSST-1 in vitro. Figure 3-1 illustrates the basic hypothesis of how y5 T cells, as cells of the innate immune response, could modulate the subsequent inflammatory response to TSST-1 mediated by ap T cells. An intriguing corollary of the unconventional y8 T lymphocytes is that they induce potent pro-inflammatory responses with an effective Th l bias (135), but their absence is more often than not correlated with an exacerbated state of inflammation (10, 49, 50, 56, 136). This apparent discrepancy may be explained in part by the time-dependent and opposing activities of yS T cells, which are among the first responders to an immune system under stress, but then later down-regulate the same immune response by eliminating activated and/or infected cells using a multitude of cytolytic mechanisms including perforin/granzyme and Fas-Fas-ligand mediated killing (50, 137). Therefore, Part II of this study focused on investigating the potential of these same lymphocytes to also induce or augment apoptosis in TSST-1 activated cells. 47 Figure 3-1. The potential role of human yd T cells in modulating the inflammatory response to TSST-1. The basic hypothesis of the study is the premise that early stress-induced signals (such as IPP) present during infection would prime y8 T cells to exacerbate the subsequent immune response elicited by TSST-1 's superantigenic effect on aP T cells and monocytes. 48 3.2 Results: Part I - Human Peripheral yd T cells Potentiate the Early Pro-inflammatory Cytokine Response to Staphylococcal Toxic Shock Syndrome Toxin-1. IPP-primed human PBMC significantly augmented the pro-inflammatory cytokine response to TSST-1. The time course of EFN-y, TNF-a, EL-2 and IL-10 secretion in human P M B C with or without IPP treatment, and stimulated with either 1 nM TSST-1 or RPMI medium, are shown in Figure 3-2A. In the absence of EPP, TSST-1 induced all four cytokines in a time-dependent manner while treatment with RPMI medium or EPP alone produced only basal levels of each cytokine. The pro-inflammatory cytokines EFN-y, TNF-a and EL-2 were detected within 2-3 h after TSST-1 stimulation and reached a plateau at 48 h (Figure 3-2A, C, E). In contrast, the anti-inflammatory cytokine IL-10 was not detectable until 24 h after TSST-1 stimulation, and continued to escalate 72 h after stimulation (Figure 3-2G). Pre-treatment with EPP significantly augmented (greater than two-fold) the secretion of EFN-y, TNF-a and EL-2 as early as 2 h after TSST-1 stimulation. Maximal potentiation was reached within 4 h, and was barely detectable for TNF-a and no longer observed for EFN-y or IL-2 at 24 h. In contrast to the potentiation of EFN-y, TNF-a and EL-2, EPP treatment significantly suppressed the secretion of EL-10 (by approximately two-fold) at 48 h after TSST-1 stimulation (P <0.025, Figure 3-1G). This trend continued at 72 h when the suppression was of borderline significance (P =0.06). The modulating effect of IPP was completely abrogated following depletion of yd T cells. To establish that the augmentation of EFN-y, TNF-a or IL-2 and suppression of EL-10 induced by TSST-1 in EPP-primed vs. non-primed P B M C was mediated by y8 T cells, we repeated these same experiments with negatively selected P B M C depleted of yS T cells. As shown in Figure lb, depletion of y5 T cells completely abrogated both the 49 augmentation of EFN-y, TNF-a and IL-2 (Figure 3-2B, D and F, respectively), and the suppression of EL-10 (Figure 3-2H). 50 6 24 36 48 60 72 B 3500 3000-1 - o - RPMI (Y« Tcell depleted) - • - TSST-1 (Y6 Tcell depleted) IPP (y8 T cell depleted) - • - IPP+TSST-1 (78 Tcell depleted) 6 24 36 48 60 72 Time (hrs) Time (hrs) 6 24 36 48 60 72 6 24 36 48 60 72 Time (hrs) Time (hrs) Time (hrs) 3 4 5 6 24 36 48 60 72 Time (hrs) H 1500-Figure 3-2. Quantitation of cytokines (mean + S E M from 3 different donors) in culture supernatants from IPP-primed or non-primed human P B M C after stimulation with TSST-1, RPMI medium, or IPP alone at different time intervals: a) prior to y8 T cell depletion (left panel); b) after y5 T cell depletion (right panel). (A, B) IFNy; (C, D) TNFa; (E, F) EL-2; (G, H) EL-10. Statistically significant differences determined by paired Student t test in cytokine levels secreted in culture supernatants of EPP-primed ( A ) vs. non-primed (•) P B M C at different time intervals following TSST-1 stimulation are shown by asterisks (* P < 0.05; ** P<0.025). Stimulation of EPP-primed P B M C with TSST-1 resulted in significantly enhanced secretion of EFNy, T N F a and EL-2 within 4 h post-stimulation, and reduced production of EL-10 at 48 h, compared to nonprimed P B M C (left panel). Depletion of y8 T cells in P B M C completely abrogated this effect (right panel). 52 Confirming the role of yd T cells in potentiating the early IFNy response by intracellular staining. Intracellular cytokine staining determined that y5 T cells were largely responsible for the augmentation of IFNy secretion by human P B M C post-TSST-1 stimulation. Figure 3-3 shows that the proportion of IFNy producing y8 T cells 4 hours post-TSST-1 stimulation was significantly increased in PBMCs primed with IPP compared with that of cells without IPP priming (p<0.05, paired t test; A), while the proportion of a(3 T cells expressing intracellular IFNy in P B M C s primed with IPP was not significantly different from that of cells without IPP priming. Intracellular staining also confirmed that y5 T cells express IFNy in response to TSST-1 even in the absence of IPP. IPP priming appears to prepare y5 T cells to more readily respond to TSST-1 treatment, and IPP alone did not induce any significant level of IFNy above background controls (Figure 3-3 A). However, the level of IFNy expression per y8 T cells (as measured by relative mean fluorescence intensity) was not significantly increased after IPP priming (Figure 3-3B). In contast, priming yS T cells resulted in a small but significant increase in the mean expression of IFNy in a P T cells 4 h post-TSST-1 stimulation, compared with that of cells without IPP priming (24±6.9 vs. 22±7.2.; p<0.05, paried t test). This enhanced capacity to induce IFNy production is a well recognized property of y5 T cells (134). Thus, even a small increase in the number of y8 T cells producing IFNy post-TSST-1 stimulation has a much greater global impact on the level of IFNY secreted during the early inflammatory response to TSST-1. It should also be kept in mind that since intracellular staining involves blocking the export of cytokines from the cell, this technique may underestimate the secretion of cytokines by both y8 and 53 a(3 T cells. Despite this, we still found that the early augmentation of EFNy is attributed to the small population of primed y5 T cells present in PBMCs. 54 Figure 3 - 3 . Detection of IFNy by intracellular cytokine staining of y 8 and cd3 T cells in human PBMCs within 4 h after stimulation with TSST-1. Data are either the proportion (A) or relative mean flourescence (B) of T cells expressing intracellular IFNy (mean + S E M from 3 different donors). Statistically significant differences in IFNy expression between IPP-primed and nonprimed PBMCs after TSST-1 stimulatin are indicated (*p<0.05; paired t test). Stimulation with TSST-1 in the presence of IPP resulted in a discernable increase in the proportion of IFNy producing y8 T cells (from 55 21.7% to 32.1%). Priming of y8 T cells also resulted in a modest but significant increase in the mean expression of IFNy among TSST-1 responsive a(3 T cells, although a very small proportion (-1%) of oc(3T cells were producing this cytokine at 4 h after TSST-1 stimulation. 5 6 Taken together, the above data indicate that EPP-primed human peripheral y5 T cells augment the pro-inflammatory cytokine response and suppress the anti-inflammatory cytokine response to TSST-1. 3.21 Discussion. As a part of the innate immune system, y8 T cells have a limited TCR repertoire that enables them to respond to unprocessed, nonpeptidic antigens having conserved molecular patterns, most notably certain alkylamines and alkylphosphates, without any need of M H C class I or II presentation (34, 133). Human peripheral y8 T cells predominantly express the Vy9/VS2 TCR and respond specifically to EPP, an alkylphosphate that is an essential metabolite in the endogenous mevalonate pathway in eukaryotes as well as prokaryotes including S. aureus (133). As a precursor for the synthesis of a number of important molecules including steroids and cholesterol, EPP may function to boost the innate immune response by alerting Vy9/V82 T cells in times of stress. In the current study, we demonstrate that priming human peripheral y5 T cells with a relatively low stimulatory concentration of EPP significantly potentiated the secretion of the pro-inflammatory cytokines EFN-y, TNF-a, and IL-2 while suppressing the anti-inflammatory cytokine EL-10 in P B M C following TSST-1 stimulation. We found the augmentation of these pro-inflammatory cytokines was maximal at 4 h after TSST-1 stimulation, and was no longer present after 24 h. This affect is specific to TSST-1 's superantigenic properties since a recombinant null mutant of TSST-1, H135A, failed to induce any cytokine secretion in EPP primed PBMC's (unpublished data). However, other 57 bacterial products, notably LPS, have also been shown to elicit a pronounced inflammatory response from primed y8 T cells in human PBMCs (45, 134). It was demonstrated that primed y5 T cells induced greater mortality in a model of LPS-induced septic shock (134). Both the mechanism of action and the concentration of these ubiquitous antigens that are required to prime yS T cells in vivo remain to be defined. It was recently shown that the amount of y5 T cell specific antigen that is derived from regular tea was sufficient to prime Vy9V82 T cells in vivo resulting in a significantly augmented immune response to both LPS and bacteria. Similar to our results, the investigators from this study found that depletion of y5 T cells from the tea drinkers' P B M C abrogated the potentiated immune response (34). We determined that IPP primed PBMCs treated with TSST-1 not only resulted in a significantly greater proportion of y8 T cells that produced JPNy, but also a small but significant amplification in the level of EFNy expressed in TSST-1 activated aP T cells as early as 4 h post-stimulation. The augmentation of IL-2 secretion in EPP-primed P B M C is somewhat surprising, since yS T cells are not known to be strong secretors of IL-2 themselves (133). This further supports the possibility that IPP primed y5 T cells may directly or indirectly intensify Th l polarization of VP-specific ccP T cells in response to TSST-1. To our knowledge, the suppression of EL-10 secretion in EPP-primed human P B M C in response to TSST-1 has not been reported previously, but this may be part of the same immunoregulatory activity of y5 T cells to intensify TSST-1 induced Th l polarization. It has been reported that purified bovine y8 T cells were directly activated by TSST-1 stimulation (104). TSST-1 was found to stimulate the proliferation of purified 58 bovine y 8 T cells (which account for roughly 30% of bovine peripheral T cells) after 4 days, in a dose and time dependent manner. IFN-y, TNF-a and IL-12, but not IL-2, IL-4 or IL-10 mRNA expression was found to be transcriptionally activated after 48 h. These authors speculate that activation of y 8 T cells by TSST-1 may play an important role in the pathogenesis of bovine mastitis and other S. aureus infections. Both human y 8 T cells and TSST-1 are known to be vigorous inducers of macrophage migration inhibitory factor (MIF) in human P B M C (134, 139). MIF is a potent immune modulator which stimulates the release of various pro-inflammatory cytokines, including TNF-a and IFN-y, during experimental LPS or TSST-1 induced lethal shock. The administration of anti-MIF antibody significantly increased survival in murine models of lethal shock as late as 8 hours after the induction of sepsis (140). It remains to be determined whether the induction of MEF by activated human y 5 T cells is one of the underlying means for the observed potentiation of the pro-inflammatory cytokine response in IPP-primed P B M C following TSST-1 stimulation. Regardless of the mechanism, our observation of the early potentiation of the pro-inflammatory cytokine response, coupled by a suppression of the anti-inflammatory cytokine response by peripheral y 5 T cells could serve to further accelerate and exacerbate the systemic inflammatory response characteristic of TSS. 3.3 Results: Part II - Regulation of apoptosis by Vy9V52 T cells following stimulation with the staphylococcal superantigen, Toxic Shock Syndrome Toxin-1. Activated Vy9VS2 T cells mediate a small but significant increase in the early apoptosis of antigen presenting cells upon TSST-1 stimulation, yb T cells have been characterized as being both pro-inflammatory and cytotoxic T lymphocytes. We had 59 previously demonstrated that treating PBMCs with the Vy9V82 T cell antigen, JPP, significantly exacerbated the inflammatory response that ensued early upon TSST-1 stimulation (135). We therefore set out to determine whether these innate lymphocytes are also able to induce apoptosis in TSST-1 activated human PBMC. Apoptosis of TSST-1 stimulated P B M C was analyzed by flow cytometry following staining with both annexin V and 7ADD (Figure 3-4). JPP primed y5 T cells in P B M C induced a small but significant increase in cell death which was evident from 4 to 10 h following stimulation with TSST-1 (Figure 3-4A). Depletion of this small population of y5 T lymphocytes, which routinely constituted between 1-5% of all T cells, completely abrogated this phenomenon (Figures 3-4B). T cells did not appear to be targeted, since no difference in apoptosis was observed in CD3-gated T cells in the different groups after stimulation with TSST-1 for up to 10 h (Figure 3-6A). Additionally, IPP-primed P B M C did not induce a significantly higher rate of CD95 (fas) expression compared to nonprimed P B M C (Figure 3-6B). In contrast to enhanced apoptosis observed in EPP-primed P B M C at 4 and 10 h post-TSST-1 stimulation, the apoptotic index was significantly lower at 48 h post-TSST-1 stimulation compared to non-primed P B M C (p<0.05, paired t test, Figure 3-5A). This effect was not evident in y8 T cell depleted P B M C (Figure 3-5B). These data raised the possibility that priming with EPP may induce an anti-apoptotic effect at 48 h post-TSST-1 stimulation. Since the index of apoptosis was based on the ratio of cell death observed following TSST-1 stimulation over the background of either RPMI or IPP alone, the percentage of EPP-primed or nonprimed P B M C with apoptosis were separately analyzed over the same time intervals. No significant differences were observed among the different 60 treatment groups at different time intervals following TSST-1 stimulation, either before (Figure 3-5C) or after (Figure 3-5D) y§ T cell depletion. The biologic significance of these findings remains to be determined. 61 A) RPMI 103-in— 8.2% 102-J c 10"-d n— 3.6% r3 r4 87J%|r: 0.5% mrti—i—i M I H I B — i n u n — • i i i i m l fo< To 2 1b3 7-ADD B) IPP 103 rr 1 0 M | 10'-10°-J 9.0% r2— 4.0% r3 86.6% ir4 0.4% ? 0 ° i i n m—• ¥ 1I H M rb1 f02 TO3 7ADD C) T S S T - 1 10*-: 102-J > 1 101-J. 10°-J 7.3% Ir2 4.1%.;-.'' 88.4%:;' m |r4 0.2% rti—i i mm—i—• imm—i—• imill i'o9 ft1 To2 ib 7-ADD D) IPP + T S S T --i Ir1 1 0 . 4 % > Itf-J io1-4 10°-J Ir2 5 .4% 83.6%^ '" I ' T H l Ir4 0.5% •l— i iiim»—i i • H I M — i 1 1 in 7o° fb1 ft2 to 7-ADD Figure 3-4. Representative data from a single donor, showing the effect of IPP-activated yd T cells on apoptosis of PBMC following stimulation with 1 nM of T S S T -1 for 4 h. Apoptosis was assessed by flow cytometry after staining with both Annexin V (which detects early apoptosis) and 7-ADD (which detects dead or necrotic cells). JPP-primed P B M C had a higher percentage of dead cells (15.8%, D) following TSST-1 stimulation, compared to nonprimed P B M C (11.4%, C), or in P B M C treated with RPMI alone (11.8%, A) or IPP alone (13%, B). 62 P B M C P B M C 3.0n I ** o a 2.0-o a < 1.5-O S 1-«H •a S 0.54 0.0' - I — l — l — I 1—I I I 1 1 1 <s ^ * <b % s * <w^ > 4> $> «$> Time (h) 12.5-. 1 1 0 I 5 I 2 1 0 0.0 T RPMI —*— TSST1 —*— IPP — • - IPP + TSST1 Time (h) —i i 3.0-1 .2 2.5-ii) a a 2.0-o a < La-'s x 1.0-s •a = 0.5-0.0 y5 T ce l l dep le ted — i — i — i — i — 1 1 — i — i — i — i Time (h) « i o o n 1 "S 5.0-1 I I 2.5-^ 0.0 y5 T ce l l d e p l e t e d - i — i — i i i i i i O \ * <b <b N<S N T ^ i # #> Time (h) Figure 3-5. Effect of IPP-activated y8 T cells on apoptosis of TSST-1 stimulated PBMCs. PBMCs with or without Vy9V82 T cells were treated overnight with IPP or RPMI culture medium and subsequently stimulated with 1 nM of TSST-1 for 48 h. The index of apoptosis (calculated by the ratio of percent apoptosis after TSST-1 stimulation over those treated with RPMI or IPP alone) was compared between IPP-primed and nonprimed PBMC (A), and in y§ T cell depleted PBMC (B). The apoptotic index in IPP-primed PBMC was significantly higher at 4 h and 10 h, but lower at 48 h, following TSST-1 stimulation (p<0.05, paired t test from 3 separate donors). These effects were completely 63 abrogated following y8 T cell depletion. The percentage of apoptosis for each group also shown before (C) and after (D) y8 T cell depletion. 64 10 h Post-TSST-1 stimulation CD95 expression B) 551 5<H T RPMI TSST IPP IPP+TSST Figure 3-6. IPP-activated y5 T cells did not induce apoptosis of TSST-1 reactive ap T cells or upregulated CD95 (fas) expression in PBMC following TSST-1 stimulation. A) Index of apoptosis for of T cells at 10 h post-TSST-1 stimulation was evaluated by FACS in both EPP-pretreated and untreated P B M C before or after y8 T cell depletion. No significant differences were observed among the different groups. B) The surface expression of CD95 in P B M C at 48 h post-TSST-1 stimulation was also not significantly upregulated in IPP-primed PBMC. 65 Expanded yd T cells can specifically lyse TSST-1 activated monocytes. It had previously been shown that monocytes stimulated with TSST-1 were resistant to apoptosis (141), and this lack of AICD is thought to contribute to systemic inflammatory response syndrome culminating in toxic shock (142). However, since we found that EPP-activated yS T cells did enhance apoptosis of P B M C within 4 and 10 h post-TSST-1 stimulation (Figure 3-5), and since Vb2-specific ab T cells were not affected (Figure 3-6A), we postulated that antigen presenting cells such as monocytes or B cells might undergo apoptosis when stimulated by TSST-1 in the presence of EPP-activated y8 T cells. To address whether human monocytes could undergo apoptosis when stimulated by TSST-1 in the presence of y8 T cells, we expanded Vy9V82 T lymphocytes present in P B M C by culturing in the presence of m M of isobutylamine (EBA) in the presence of 20 U/ml of IL-2 as previously described (see Materials and Methods in Chapter 2). Expaned y8 T cells were co-cultured with autologous monocytes (1:1 ratio), and subsequently analyzed their potential for inducing cytolytic activity following TSST-1 treatment. yS T cell mediated cytotoxicity was determined using a modified version of the fluorometric method described previously (127) which utilizes CFSE stained monocytes as target cells and PKH-26 stained y8 T cells as effector cells (see Materials and Methods). Percent specific-lysis is determined by the loss of CFSE-positive cells. The results shown in Figure 3-7 verify that y8 T cells do actively target TSST-1 stimulated monocytes. The proportion of live monocytes in RMPI treated controls (57% of RPMI-treated cells, Figure 3-7A) was reduced by ~2.5-fold (20.5% of TSST-1 stimulated cells, Figure 3-7B) following stimutation with 1 nM TSST-1 for 24 h. Monocytes cultured in the absence of gd T cells did not undergo apoptosis following TSST-1 stimulation (Figure 3-7C) when 66 compared to treatment with RPMI alone (Figure 3-7C). In three separate experiments, the mean reduction of live monocytes following TSST-1 treatment in the presence of y8 T cells was 2.4-fold (52.2% ± 9.9 following RPMI treatment vs. 21.6% ± 7.9 following TSST-1 stimulation, mean ± S E M ; p<0.025, 2-tailed paired t test). 67 A) W U *P4 Monocyte and y6 T cell coculture RPMI B) T S S T - l RPMI monocyte* 6T.S% i n IF StSf/ 1 T B I Monocytes alone D) TSST-1 Forward scatter monocyte* mm • ' * * * * * '1023 Forward scatter Figure 3-7. Direct cytolysis of TSST-1 stimulated autologous monocytes by expanded yd T cells. Purified monocytes were stained with the cell permeable dye, CFSE, and expanded yd T cells were labelled with the membrane lipophyllic dye, P K H -26, to differentiate the two cell populations. Monocytes (106 per well) were co-cultured with y8 T cells (1:1 ratio) for 24 h in the presence of RPMI culture medium (A) or 1 nM TSST-1 (B). Lysed monocytes were identified by the loss of CFSE and assessed by flow cytometry (as described in Materials and Methods, Chapter 2). Monocytes alone in the absence of y8 T cells did not undergo enhanced apoptosis following TSST-1 stimulation 68 (D) as compared to RPMI controls (C). Cells were gated for live cells. Data was representative of 3 separate experiments. 3.31 Discussion. Deleterious inflammatory responses become critical when there is a failure to down-regulate activated immune cells. TSST-1 is a staphylococcal superantigen and a major cause of TSS. TSST-1 has unique features distinct from other staphylococcal enterotoxins such as, SEA and SEB, which are major causes of food poisoning (75). For example, TSST-1 is able to activate y8 T cells associated with innate immunity that are present in the mucosal epithelia where the toxin first makes its entry (143). TSST-1 is also able to transverse the vaginal mucosal barrier and cause an influx of lymphocytes into the upper layers of the tissue (143). Thus, y8 T cells are likely among the first lymphocytes to encounter pathogenic TSST-1-secreting S. aureus. We previously demonstrated that these innate lymphocytes could significantly augment the inflammatory response to TSST-1 during the early course of immune activation (135). Apart from their central role in recruiting and arming the cells of the immune response, y8 T cells are also critical in subsequently down-regulation of the inflammatory response by killing off activated macrophages and mediating wound healing (6, 10, 49, 50, 144). This study confirmed that y8 T cells can induce early apoptosis in TSST-1 activated monocytes, which, like a(3 T cells, are otherwise resistant to early apoptosis upon stimulation with TSST-1. The means by which y8 T cells mediate their cytolytic activity towards superantigen-stimulated monocytes is currently unknown, but we speculate that binding of this superantigen to M H C class II molecules on monocytes/macrophages may induce the up-regulation of stress induced molecules, such as M I C A or MICB, that are 69 recognized by activated y8 T cells. This means of recognition of stressed cells have been demonstrated to occur either directly through the y8 TCR (145) or via the NKG2D receptor born by many circulating y8 T cells (40, 41, 45). Support for this scenario comes from a study demonstrating that SEA mediated cytotoxicity was enhanced in macrophages by the surface expression of M I C A (41). These findings raises the possibility that y8 T cells stimulated by TSST-1 and present abundantly in mucosal tissues, may delay the clearance of S. aureus locally by inducing apoptosis of monocytes and other antigen-presenting cells, and thereby functions as a virulence factor (45). Figure 3-8 summarizes the major findings of the studies contained within this chapter. We hope further elucidation of the role of y8 T cell in TSST-1 mediated pathogenesis would yield a better understanding of the mechanism leading to the progression of such serious inflammatory disorders and allow for the development of more effective modes of treatment. 70 J Gamma-delta T cells (early response) >lFNy >TNFoc >IL-2 7i INFLAMMATORY CYTOKINES > apoptosis of monocytes > I L - 1 0 IMMUNOSUPPRESSIVE CYTOKINE (late response) Anti-apoptosis Figure 3-8. Summary of the modulatory effect of IPP-activated y8 T cells on TSST-1 induced proinflammatory cytokines and apoptosis in human P B M C . IPP activated yS T cells were found to significantly augment the early inflammatory cytokine response to TSST-1 which peaked between 3 to 6 hours post toxin treatment. A small, but significant, increase in apoptotic activity was also noted during this time, and both these effects were abrogated by the removal of y8 T cells from P B M C . In contrast to the up-regulation of the early inflammatory response to TSST-1, P B M C s with IPP-treated yS T cells were found to have a dampened anti-inflammatory response as measured by the reduced secretion of EL-10. IPP-activated y8 T cells also induced apoptotic cell death in P B M C within 4-10 h following TSST-1 stimuation, but appeared to be anti-apoptotic at 48 h 71 after TSST-1 stimulation. EPP-activated y§ T cells did not induce apoptosis in T cells, but appear to target monocytes when co-cultured in the presence of expanded y5 T cells. The mechanisms for these activities remains unclear and warrants further study. 72 Chapter 4: Regulation of HMGB-1 expression by staphylococcal Toxic Shock Syndrome Toxin-1 4.1 Introduction As mentioned previously, toxic shock syndrome toxin-1 (TSST-1) is a supernantigen (sAg) secreted by some strains of S. aureus, and this exotoxin was determined to be a major causative agent of toxic shock syndrome (129). In contrast to LPS-induced sepsis, which is primarily monocyte-mediated, sepsis induced by sAgs requires the cross-linking of Vfi-specific regions of the a P T cell receptor (TCR) to class II major histocompatibility complex molecules (MHC II) on antigen presenting cells (APC) (63). In addition to massive T cell proliferation, this tri-molecular interaction leads to the uncontrolled release of various pro-inflammatory cytokines (149), which are pivotal to the pathogenesis of TSS. The work presented in Chapter 3 established that activated y5 T cells have the potential to significantly potentiate the early inflammatory response to TSST-1; however, the downstream consequence of this phenomenon is still unclear. High mobility group box-1 protein (HMGB-1) is seen as a useful tool to address this issue of the downstream severity of inflammation, as illustrated in Figure 4-1. High mobility group-1 protein (HMGB-1) is a 30 kDa non-histone nuclear D N A binding protein recently found to have an extracellular role in inflammation, cell differentiation, adherence, and motility (150, 151, 152). Extracellular HMGB-1 was initially identified as amphoterin, a heparin-binding protein promoting neurite outgrowth in the perinatal rat brain (153). This ubiquitous protein was also known as p30, sulphoglucoronyl carbohydrate binding protein-1 (SBP-1), and differentiating enhancing factor (DEF) (152). However, HMGB-1 was the established designation following recent revision of the nomenclature of H M G family of proteins (154). HMGB-1 is now regarded 73 as an endogenous danger signal that is passively released by necrotic cells or actively secreted by stimulated cells (155). Activated macrophages and monocytes were shown to secrete this inflammatory molecule by a process requiring acetylation of the protein, which permits its translocation from the nucleus to secretory lysosomes (156). Wang et al discovered that this pivotal protein was a late mediator of endotoxic shock (112); whereas TNFa and EFNy appeared within the first 4-6 h post-LPS treatment in mice, HMGB-1 serum levels rose between 16-32 h after LPS administration. Importantly, neutralizing antibodies directed at HMGB-1 rescued mice from lethal endotoxemia even when administered 24 h after sepsis initiation (112, 157). Thus, HMGB-1 has been viewed as an attractive therapeutic target for various inflammatory disorders including endotoxic shock (115). The role and expression of HMGB-1 has not been previously studied in the context of superantigens, or TSST-1 in particular. In the present study, we sought to determine whether HMGB-1 also plays a central role in TSST-1-induced hyper-inflammatory responses. 74 HMGB-1 jo=4» TSST-1 ? y 5 T cells potentiate the early inflammatory response to TSST -1 - what are the downstream consequences? Anorrocia / Weight lose Taat© avaroion Pain Neurtto outgrowth Neutrophil KNMnflM Endothela activation Inflammation Edama Injury t Nrtnc OXKJO synthase Loss of opithefia barrier fundior Bactenal translocation Flalanaa of Hvaranzyma ft II t i n iiMlTim In ® (•) ' Macrophage activation Monocyte actrvatjon i Nautrochil activation Endothelial activation T t-PA/PAl Figure 4-1. The potential role of HMGB-1 in TSST-1 induced pathology. As discussed in Chapter 1, H M G B - 1 was determined to be the late mediator of septic shock and lethality, and this nuclear protein is pleiotropic in its effect on various tissues and organs associated with disorders stemming from excessive inflammation. Toxic shock syndrome triggered by TSST -1 shares the same pattern of pathology induced by H M G B -1, suggesting that this pivotal inflammatory cytokine may also mediate T S S . The aims of the present study are: 1) to confirm a causal relationship between TSST -1 and H M G B - 1 ; and 2) to determine whether the potentiation of the proinflammatory response to TSST-1 induced by the activation of y5 T cells is mediated by H M G B - 1 . 75 4.2 Results. 4.2.1 Translocation of nuclear HMGB-1 in human PBMC 10 h post-TSST-1 stimulation. The intracellular regulation of HMGB-1 in the context of TSST-1-induced inflammatory response has not been investigated previously. Unlike LPS, TSST-1-induced immune activation requires the participation of both APC and T cells (149). To investigate the intracellular regulation of HMGB-1 upon TSST-1 stimulation, human P B M C were treated with 1 nM of TSST-1 for 10 h, and subsequently evaluated for the translocation of nuclear HMGB-1 by fluorescence microscopy (Figure 4-1). As reported previously (158), HMGB-1 in resting P B M C was localized primarily in the nucleus (Figure 4-2A). However, 10 h after treatment with 1 nM TSST-1, a significant proportion of P B M C had translocated their HMGB-1 from the nucleus into the cytosol (Figure 4-2B). It was verified that translocation of nuclear HMGB-1 was due to the superantigenie properties of TSST-1, since a recombinant null mutant of TSST-1, H135A, had no effect (Figure 4-2C). The distribution of P B M C demonstrating nuclear translocation of H M G B -1 was significantly higher in donor cells following TSST-1 stimulation compared to RPMI medium control (p<0.001; two by three Chi-Square analysis; n=4) (Fig. 4-2D). Furthermore, this change in nuclear HMGB-1 expression was not restricted to macrophages as was the case when stimulated with LPS or the inflammatory cytokines IFNy and T N F a (113) (123), but was also observed in T cells for the first time (Figure 4-3). 76 A) Resting PBMC B) TSST-1 stimulated PBMC | \ | C) H13SA (TSST-1 null mutant) stimulated PBMC D) Distribution of cells showing translocation of nuclear HMGB-1 o *E m QL *-o c o t o O RPMI 1.00 0.75 0.50 0.25 n nn JL X X o m Q_ *-O c o o Q. o 0L TSST-1 0.754 0.254 n nn. -T NONE INCOMPLETE COMPLETE NONE INCOMPLETE COMPLETE 77 Figure 4-2. Nuclear translocation of HMGB-1 in human PBMC following TSST-1 stimulation. Human P B M C from healthy donors were treated with either 1 nM TSST-1 or RPMI medium for 10 h, and the intracellular expression of HMGB-1 was detected by confocal fluorescent microscopy. Close-up views from representative fields are depicted in the right panels. A) In resting or RPMI treated PBMC, intracellular HMGB-1 detected with anti-HMGB-1 antibodies conjugated to Alexa488 (green) was seen to be localized primarily in the nucleus stained with the Hoechst 3342 nuclear dye (blue). B) At 10 h following stimulation with 1 nM TSST-1, HMGB-1 was seen to translocate into the cytosol. C) HI35A, a null mutant of TSST-1, served as a control to ensure that the nuclear translocation of HI35A following toxin stimulation was a result of the superantigenic properties of TSST-1. D) Box plot analysis of the translocation of nuclear HMG-1 in human P B M C from four different donors 10 h following TSST-1 stimulation. A minimum of 50 cells per donor were evaluated by confocal microscopy for the nuclear expression of HMGB-1 following TSST-1 stimulation (hatched bars) in comparison to donor cells stimulated with the RPMI medium control (open bars). Based on the localization of HMGB-1 , cells were categorized as demonstrating no nuclear translocation, incomplete translocation, or complete translocation into the cytosol. The proportion of P B M C that demonstrated either incomplete or complete nuclear translocation of HMGB-1 were significantly higher following stimulation with TSST-1 compared to RPMI medium control (p<0.001; two by three Chi-square analysis). 78 A) Resting T cell B) TSST-1 stimulated T cells F i g u r e 4-3. N u c l e a r t r a n s l o c a t i o n o f HMGB-1 i n T cells f o l l o w i n g TSST-1 s t i m u l a t i o n . Human P B M C from healthy donors were treated with either 1 nM TSST-1 or RPMI medium for 10 h, and the intracellular expression of HMGB-1 in T cells was detected by confocal fluorescent microscopy. T cells were detected by staining with anti-human C D 3 antibodies followed by anti-mouse IgG conjugated to Alexa594 (red). Translocation of HMGB-1 (green) out of the nucleus (blue) is clearly visualized. 79 4.2.2 Secretion of translocated nuclear HMGB-1 from the surface of differentiated and adherent cells 24 h post-TSST-1 stimulation. Some differentiated as well as adherent cells express HMGB-1 on the cell surface, and this cell membrane-associated HMGB-1 has been referred to as amphoterin to distinguish it from intracellular HMGB-1 (159). Extracellular amphoterin expression was confirmed by subcellular fractionation studies, immunogold electronmicroscopy, and mRNA localization (160). We examined the surface expression of HMGB-1 (amphoterin) in human P B M C 48 h after culture in 96-well U-bottom plates in RPMI medium. We identified a population of cells that expressed high levels of HMGB-1 even in the absence of TSST-1 stimulation (Figure 4-4A). Cytosolic or extracellular HMGB-1 was expressed primarily on adherent cells, since T cells identified by anti-CD3 and Alexa594-conjugated anti-mouse IgG (red) did not express amphoterin. The upregulation of cell surface-associated HMGB-1 took place over the course of time that human P B M C were cultured and became adherent, while freshly purified P B M C did not express amphoterin (Figure 4-5). Extracellular HMGB-1 was also found to be tightly associated with tubular processes that extend into the extracellular milieu (Figure 4-4). This was similar to the finding of Rouhiainen et al (160) who reported the accumulation of amphoterin in the extracellular space of cells bearing process extensions, and that amphoterin surface expression was inhibited in cells without extending processes. Following TSST-1 stimulation, there was significant loss of amphoterin from the cell surface, as observed by fluorescent microscopy (Figures 4-4A and 4B), and by flow cytometric analysis (Figure 4-4C). Flow cytometry also confirmed that the high-level surface expression of amphoterin was confined to a subpopulation of P B M C having a high forward scatter corresponding to the monocyte/macrophage 80 population (estimated to be between 3 - 12 % of resting P B M C among the donors tested). More than half of these amphoterin-expressing cells released HMGB-1 upon TSST-1 stimulation (7.3 ± 3.69 % prior to stimulation vs. 3.3 ± 1.32 % post- TSST-1 treatment; p<0.05, paired student's t test from 4 different donors). Flow cytometric analysis, to our knowledge, has not been widely utilized previously for quantifying the surface expression of HMGB-1 , and was found to be a useful tool. Finally, we verified by Western blot analysis that HMGB-1 was secreted into the culture supernatant when collected 24 h post-TSST-1 treatment (Figure 4-7). In contrast to the leading edges of motile and adherent cells, we did not detect T cells as having stably associated HMGB-1 on the cell surface. We observed (by fluorescent microscopy) only transient surface association of this nuclear protein on T cells following TSST-1 stimulation, presumably in the process leading to its secretion into the extracellular milieu (Figure 4-3). Of interest, we observed that expression of extracellular HMGB-1 was often at the interface of monocyte-T cell interaction following TSST-1 stimulation (Figure 4-4B). 81 A) Resting PBMC 82 Figure 4-4. Secretion of membrane-associated HMGB-1 in the extracellular milieu following TSST-1 treatment. Human P B M C from healthy donors were cultured in 96-well U-bottomed plates in RPMI medium for 24 h, and then treated with either 1 nM TSST-1 or RPMI medium for an additional 24 h. The secretion of extracellular HMGB-1 was detected on the cell surface by confocal fluorescent microscopy, and in the culture supernatant by immunoblot. Extracellular expression of HMGB-1 on adherent P B M C treated with RPMI medium (A) or TSST-1 (B). Anti-HMGB-1: green fluorescence (Alexa488); nucleus: blue (Hoechst 3342 nuclear dye); anti-CD3: red (Alexa594). Close-up views from representative fields are depicted in the left panels. (C) Flow cytometry analysis of surface expressed HMGB-1 in P B M C 24 h following TSST-1 stimulation (representative of 4 separate donors). Left panel, resting or P B M C treated; right panel, 24 h after TSST-1 stimulation. Surface HMGB-1 expression significantly decreased after TSST-1 stimulation (12.0% vs. 4.3% in the donor shown; 7.3 ± 3.69 % prior to stimulation vs. 3.3 ± 1.32 % post-TSST-1 treatment from 4 different donors; p<0.05, paired student's t test). 83 OVERNIGHT Figure 4-5. Flow cytometery analysis of the increase of cell surface expressed HMGB-1 on PBMCs cultured overtime. As shown above, HMGB-1 is not expressed on the cell surface on freshly purified cells in suspension, and surface expression increases with time in culture as adherent cells (such as macrophages) differentiate. A) after overnight culture; B) after 48 h. 84 4.2.3 Requirement of activated T cells in TSST-1-induced nuclear translocation and secretion of HMGB-1 in human PBMC. Previous studies have determined that non-activated (undifferentiated) and non-adherent monocytes in suspension do not express cell surface-associated HMGB-1 . However, monocytes actively transport this molecule to their cell surface following activation, leading to its secretion into the extracellular milieu (159). Furthermore, T cells were not found to undergo nuclear translocation of HMGB-1 following stimulation with LPS or the proinflammatory cytokines, IFNy and TNFa. To further investigate the requirement of T cells for nuclear translocation and secretion of HMGB-1 following TSST-1 stimulation, we studied the response of the human monocytic cell line, THP-1, to treatment with either TSST-1 (1 nM), LPS (500 ng/ml) or RPMI medium. THP-1 cells express basal levels of both M H C Class II required for TSST-1 activation, and CD14 required for LPS-induced activation (161). As shown in Figure 4-6, TSST-1 treatment failed to induce surface expression of HMGB-1 on undifferentiated THP-1 cells, in contrast to stimulation with LPS (Figure 4-6A). To further ascertain that T cells were required for HMGB-1 secretion, human P B M C were depleted of their T cell pool (removing over 60% of CD3+ T cells by magnetic separation) and were subsequently stimulated with either TSST-1 or LPS. Figure 4-6B shows that this procedure resulted in undetectable surface expression of HMGB-1, in contrast to P B M C from the same donor without T cell depletion (Figure 4-4B). Western blot analysis also verified that whereas T cell-depleted P B M C or THP1 cells secreted HMGB-1 upon LPS stimulation, they did not secrete HMGB-1 following TSST-1 treatment (Figures 4-7B and 4-7C). Collectively, these results are consistent with our previous observations which established that both monocytes and T cells are required for 85 the secretion of T N F a and I L - i p from human P B M C stimulated with highly purified TSST-1 (149). 86 A) THP-1 calls RPMI TSST-1 LPS B) T call depleted PBMC RPMI TSST-1 LPS Figure 4-6. Requirement of T cells for HMGB-1 secretion following TSST-stimulation. THP-1 cells (A) or T cell-depleted human P B M C (B) were stimulated with either TSST-1 (1 nM), LPS (500 ng/ml), or RPMI medium for 24 h, and surface expression of HMGB-1 was examined by fluorescence microscopy (representative of 3 different donors). Surface expression of HMGB-1 was detected after LPS treatment (green), but not stimulation with TSST-1 or medium control in the absence of T cells. S7 A) PBMC rHMGBI RPMI TSST1 B ) T cell depleted PBMC T S S T 1 LPS rHMGBI I THP1 C) RPMI LPS TSST1 Figure 4-7. T cells are required for the detection of H M G B - 1 in culture supernatants following TSST-1 stimulation but not L P S . P B M C without T cells depletion (A), T cell depleted PBMC (B), or THP-1 monocytic cells (C) were treated with either RPMI medium, TSST-1 (1 nM), or LPS (500 ng/ml) for 24 h. Culture supernatants were collected and analysed for the presence of HMGB-1 antibody (see Materials and Methods in Chapter 2), rHMGB-1 (15 ng) was used as a reference standard in (B) and 100 ng (A). HMGB-1 was detected in culture supernatants when PBMC without T cell depletion was stimulated with TSST-1 (A), but not T cell depleted P B M C (B) from the same donor, or the THP-1 monocytic cell line in the absence of T cells (C). In contrast, HMGB-1 could still be detected in culture supernatants when T cell-depleted P B M C (B) or THP-1 cells alone (C) were stimulated with LPS. 88 4.3 Discussion Various cell types have recently been identified as contributing to the extracellular pool of HMGB-1 , including human umbilical vein endothelial cells (162), platelets (160), pituicytes, and macrophages (152). HMGB-1 was shown to induce monocytes to differentiate into dendritic cells that specifically polarized T cells to give a Th l response (158), a feature characteristic of the effect of TSST-1 stimulation on human PBMC. We previously demonstrated that the cytokine secretion and co-stimulatory molecule expression by human P B M C following TSST-1 stimulation (125) follows a bimodal pattern, with the first phase peaking at ~3 h post- stimulation, and a second burst at -24 h post-stimulation. In light of the secretion of HMGB-1 into the extracellular milieu at this later time point, it will be worthwhile to determine whether the second inflammatory burst and the up-regulation of co-stimulatory molecules, such as CD86, CD40, as well as H L A - D R (which peaked at 48 h) (125), is in direct response to the secretion of HMGB-1 that is considered the late mediator of sepsis (163). The best studied ligand for extracellular HMGB-1 is R A G E (receptor of advanced glycation end products) which is up-regulated on activated macrophages as well as endothelial cells (164). Previous studies have aimed to determine how superantigen-activated T cells adhere to vascular endothelial cells and induce vascular injury (165). It was also shown that rHMGB-1 elicited pro-inflammatory responses on endothelial cells (164). Thus, it would be of interest to further investigate the role of HMGB-1 on TSST-1-activated monocytes and T cells, and to determine the role of R A G E in the inflammatory response and vascular endothelial injury mediated by TSST-1. It is anticipated that the model of TSST-1-induced inflammation, which necessitates the bridging of Vp2-specific 89 T cells with M H C class II molecules on antigen presenting cells, will provide a useful means to study the role of HMGB-1 in T cell-monocyte interactions, Th l polarization, and the ensuing immune response leading to tissue injury. In summary, our results demonstrate for the first time that TSST-1 mediates the translocation and subsequent secretion of HMGB-1 from the nucleus of resting human PBMC. Unlike previous studies which found that HMGB-1 was released by monocytes but not T cells when stimulated with LPS or TNFa (112, 113, 123), we observed the the secretion of HMGB-1 induced by TSST-1 was dependent upon the cooperative interaction of both T cells and monocytes, and that both these cell types mobilized HMGB-1 upon TSST-1 treatment. Finally, it is proposed that TSST-1 could provide a useful model to study the role of HMGB-1 in T cell-monocyte interaction, Thl polarization, and the ensuing immune response (158). 90 Chapter 5: A means to an end - Human Vy9V52 T cells enhance the inflammatory response to TSST-1 by up-regulating HMGB-1 and CD40 expression. 5.1 Introduction The central role of y8 T cells in regulating immune homeostasis at both steady states and under physiological stress has recently gained a greater appreciation. This subset of highly conserved innate lymphocytes, however, still remains enigmatic in regards to function, mechanism of antigen recognition, and their plasticity in response to both infectious and non-infectious stimuli. Despite their relative lack of receptor diversity in comparison to their ap T cell counterparts, much less is known about their ontogeny and evolutionary chronology. Indeed, y8 T cells have been implicated for both instigating over-zealous immune responses (134, 135, 166) and downregulating inflammation by cytolytic killing of activated macrophages, as well as promoting wound healing (16, 56). In humans, the main group of peripheral blood y8 T cells usually comprise between 1-5 % of circulating T cells and bear the canonical Vy9V82 T cell receptor. These peripheral blood y8 T cells respond to endogenous and exogenous stress-induced molecules which are nonpeptidic in nature and preclude the requirement for processing or antigen presentation by classical M H C molecules (34, 167). Isopentylpyrophosphate (IPP) is the prototypical phosphoantigen recognized by Vy9V82 T cells that was first isolated from lysates of mycobacteria (27, 168) and was later determined to be a metabolite in the melavonate pathway of cholesterol metabolism in both eukaryotic and prokaryotic cells (133). We had previously demonstrated that the numerically diminutive pool of circulating human peripheral blood y8 T cells markedly augmented the early pro-91 inflammatory cytokine responses to Toxic Shock Syndrome Toxin-1 (TSST-1) after overnight priming of P B M C cultures with EPP. TSST-1 is a staphylococcal superantigen that exerts a potent inflammatory response by bridging together V02 specific a(3 T cells and M H C class II molecules of antigen presenting cells outside the peptide binding groove (130). Our previous investigation revealed that activated y8 T cell potentiate the proinflammatory response to TSST-1 mainly within the first 6 hours post-stimulation (135). The first aim of the present study was to ascertain the mechanisms by which this relatively small number of activated y8 T cells could provoke such a potent augmentation of the proinflammatory cytokine response to the staphylococcal superantigen, TSST-1. Specifically, we wish to determine whether this potentiating effect by y8 T cells could be mediated through the nuclear translocation and secretion of HMGB-1 , a recognized late mediator of sepsis and septic shock (112, 113). Additionally, we wish to examine the effect of y8 T cells on surface expression of various co-stimulatory molecules known to be important in the proinflammatory cytokine response to TSST-1. HMGB-1 is a highly conserved non-histone DNA-binding protein found in virtually all nucleated cells. Activated monocytes were shown to secrete HMGB-1 after exposure to endotoxin (163); and this inflammatory cytokine can be detected in the serum of septic patients as late as 24 h following the onset of the systemic inflammatory response. Furthermore, neutralizing the effects of HMGB-1, either with anti-HMGB-1 antibodies or other antagonists, rescued mice from lethal endotoxic shock even when administered 8 to 32 h following endotoxin exposure (112). More importantly, we established that HMGB-1 is also secreted by both T cells and monocytes following TSST-1 stimulation (see Chapter 92 4). HMGB -1 was found to undergo translocation from the nucleus into the cytosol following TSST-1 stimulaton, and was subsequently released extracellularly into the culture supernatant. Thus, we hypothesized that the potentiating effect of yd T cells on TSST-1 induced proinflammatory cytokine responses could also be mediated by H M G B -1. 5.2 Results 5.2.1 Activated yd T cells significantly potentiate the translocation and secretion of nuclear HMGB-1 following TSST-1 stimulation. We recently established that nuclear HMGB -1 is translocated and secreted by human P B M C following TSST-1 stimulation, and that this phenomenon took place in both activated monocytes and T cells (Chapter 4). In these studies, loss of nuclear HMGB -1 could be observed by fluorescent microscopy within 10 hours following TSST-1 stimulation, and extracellular secretion of HMGB -1 could be detected by immuoblot of culture supernatants at 24 h following TSST-1 stimulation. In this Chapter, we evaluated both the nuclear translocation and subsequent secretion of HMGB -1 in IPP primed and unprimed P B M C cultures following TSST-1 stimulation. As shown in Figure 5-1 A , incubating human P B M C with IPP overnight significantly augmented the translocation of nuclear HMGB -1 following TSST-1 stimulation. IPP treatment alone had a variable affect that appeared to be primarily donor-dependent. This is the first demonstration that IPP-primed yd T cells in P B M C could influence the change in nuclear expression of HMGB -1 . 93 f ^ r ^ RPMI TSST-1 IPP IPP * TSSTI Figure 5-1. Translocation and secretion of nuclear HMGB-1 from IPP pretreated or untreated P B M C following TSST-1 stimulation. A) N u c l e a r e x p r e s s i o n o f H M G B - 1 i n P B M C f o l l o w i n g d i f f e r e n t e x p e r i m e n t a l t r e a t m e n t s w a s e v a l u a t e d b y f l u o r e s c e n c e m i c r o s c o p y (see M a t e r i a l a n d M e t h o d s ) . T r a n s l o c a t i o n o f i n t r a c e l l u l a r H M G B - 1 w a s e n u m e r a t e d f r o m 4 d i f f e r e n t d o n o r s at 10 h p o s t - T S S T - 1 s t i m u l a t i o n ( m i n i m u m o f 50 w e r e c o u n t e d p e r d o n o r a n d e x p e r i m e n t a l g r o u p ) . B a s e d o n the m i c r o s c o p i c l o c a l i z a t i o n o f n u c l e a r H M G B - 1 ( i l l u s t r a t e d i n B a n d C ) , e a c h c e l l w a s c a t e g o r i z e d e i t h e r as: a) " n o n e " , d e f i n e d as " b a s a l " l e v e l s s e e n i n m o s t r e s t i n g c e l l s ( s o l i d b a r s ) ; b ) " i n c o m p l e t e " , d e f i n e d as i n c o m p l e t e l o s s o f n u c l e a r H M G B - 1 ( s t r i p e d b a r s ) ; o r " c o m p l e t e " , d e f i n e d as n o o b s e r v a b l e n u c l e a r H M G B - 1 ( o p e n b a r s ) . T h e 94 proportion of P B M C that demonstrated either incomplete or complete nuclear translocation of HMGB-1 was significantly higher following stimulation with TSST-1 compared to RPMI medium control (p<0.001, two by three chi-square analysis). Similarly, the proportion of P B M C with incomplete or complete nuclear translocation following treatment with EPP + TSST-1 was significantly higher than that following treatment with TSST-1 alone (p ^ 0.01). B) Representative appearance by fluorescence microscopy of resting P B M C treated with RPMI medium, illustrating co-localization of HMGB-1 (green) in the nucleus (blue). C) Representative appearance by fluorescence microscopy of P B M C following treatment with TSST-1 alone, showing an even distribution of all three categories of cells based on their nuclear translocation of HMGB-1 . 95 5 . 2 . 2 yd T cells regulate the maturation of antigen presenting cells and the cell surface expression of HMGB-1 (amphoterin). Rauvala et al. (159) reported that only monocytes that undergo differentiation and become adherent export HMGB-1 to the cell surface, where it serves a function for process extension, cell migration, and cell-cell interaction. We also observed that only adherent monocytes in cultured P B M C demonstrate surface expressed HMGB-1 , which is subsequently released into the culture supernatant cells are stimulated with TSST-1 (Chapter 4). Given the direct influence of EPP-primed y5 T cells on the activation and maturation of monocytes in culture, we examined whether EPP pretreatment of PBMCs would influence the surface expression of HMGB-1 on monocytes, and its subsequent release upon TSST-1 stimulation. Figure 5-2 displays the change in surface-expressed HMGB-1 on cultured and adherent monocytes in P B M C following treatment with IPP + TSST-1 and visualized by fluorescence microscopy. Resting adherent cells have HMGB-1 (stained with Alexa488 in green) localized at focal points around the perimeter in vesicle-like pockets (Figure 5-2A). T cells (stained with Alexa594 and identified in red), which are non-adherent in cell suspension, do not express HMGB-1 on the surface in this manner. Stimulation with TSST-1 led to a release of HMGB-1 from these pocketed foci of HMGB-1 (Figure 5-2D). This secreted HMGB-1 could be detected around the extensions of activated macrophages and even spotted on T cells, which could represent either the release of this molecule from activated T lymphocytes, or binding of released HMGB-1 to R A G E receptors expressed on activated T cells. In fact, the secretion of HMGB-1 by antigen-presenting dendritic cells was recently described as being necessary for the clonal expansion, survival and functional Th l polarization of naive T cells (121). Figure 5-2C illustrates the change in HMGB-1 96 expression in P B M C cultures that had been treated with IPP alone. The macrophages appeared to take on a spindle-like form and increase their adherence capacity after incubating with IPP in the presence of y5 T cells, where the distribution of HMGB-1 around their perimeter appeared less concentrated in vesicle-like foci and became more dispersed. Treatment with IPP followed by TSST-1 resulted in a significant loss of HMGB-1 from the cell surface (Figures 5-4D and 5-1A). 97 98 Figure 5-2. Investigation of the change in cell surface expressed HMGB-1 (amphoterin) on cultured adherent and differentiated monocytes in PBMC following IPP and TSST-1 treatment by fluorescence microscopy. HMGB-1 is shown as green; T cells are shown as red; and the nucleus is shown as blue. A) Resting P B M C cultured on cover slips for 48 h were stained for surface expressed HMGB-1 (see materials and methods). B) Surface expression of HMGB-1 at 24 h post-TSST-1 treatment. C) Surface expression of HMGB-1 48 h after IPP treatment. D) Surface expression of P B M C that had been treated overnight with EPP and subsequently treated with TSST-1 for 24 h. E) Unstimulated y8 T cell depleted P B M C were relatively deficient in the surface expression of HMGB-1 . 9 9 The change in surface expressed HMGB-1 was further assessed by flow cytometery (Figure 5-3) which enabled us to both confirm and quantify the release of HMGB-1 from the cell surface. Based on flow cytometry, surface expression of HMGB-1 was significantly lower in EPP-pretreated P B M C that were subsequently stimulated with TSST-1, compared to all other groups (p<0.05, paired student t test from 4 different donors; Figure 5-4A). Immunoblot analysis of their culture supernatants, on the other hand, revealed the highest level of extracellular HMGB-1 (Figure 5-4B). Taken together, these data revealed that -7.5% of resting P B M C in culture medium exhibit HMGB-1 on their cell surface. However, following treatment with EPP or TSST-1, particularly in the presence of EPP + TSST-1, most of the surface-expressed HMGB-1 is released into the extracellular milieu. However, it remains unclear whether the translocation of HMGB-1 from the nucleus to the cell surface and the extracellular milieu is unidirectional or bidirectional, and the signaling pathways that regular this nuclear translocation and release from the cell surface. What was both striking and unexpected was the discovery that simply depleting y8 T cells from P B M C , independent from any exogenous EPP stimulation, impaired the cell surface expression of HMGB-1 (Figures 5-3). We confirmed that this was not due to an artifact caused by our experimental procedure of depleting the y8 T cells. Prior to plating and incubating at 37°C, both the y8 T cell-depleted and undepleted P B M C fractions had little or no surface-expressed HMGB-1 when examined in overnight cultures. However, the surface expression of HMGB-1 gradually increased over time, particularly on adherent and differentiated macrophages when examined at 48 h (Figure 100 5-5). This phenomenon supports the notion that y8 T cells play a major role in differentiating monocytes, and may, as a result, enable these antigen presenting cells to efficiently mobilize and express HMGB-1 on their cell surface. 101 Figure 5-3. Quantification of surface expressed HMGB-1 by flow cytometry in PBMC with or without y 8 T cell depletion. P B M C were cultured overnight in the presence or absence of IPP before stimulation with 1 nM of TSST-1 or RPMI growth medium. Surface expression of HMGB-1 was highest in resting P B M C without y8 T cell depletion, and treated with RPMI growth medium alone (12%, panel A), lowest when treated with IPP + TSST-1 (2.2%), and intermediate in P B M C treated with IPP alone (3.4%) or TSST-1 alone (4.3%). In contrast, P B M C with depletion of y8 exhibited very low levels of surface-expressed HMGB-1 regardless of treatment with RPMI, IPP or TSST-1 (1.9%, 1.2%, 0.9%, and 1.1%, respectively; panel B). 103 9.5-t 9.0-8.5 -RPMI TSST-1 IPP IPP+TSST B RPMI TSST1 IPP+TSST1 IPP m m - « Figure 5-4. Quantifying the level of H M G B - 1 surface expression in P B M C by flow cytometery. Freshly isolated P B M C from four different donors were cultured overnight in the presence or absence of IPP before stimulation with 1 nM of TSST-1 or RPMI medium. A . Surface expression of HMGB-1 was significantly higher in P B M C treated with RPMI alone, compared to all other groups (p < 0.05, paired student t test). P B M C pretreated with IPP and stimulated with TSST-1 had the lowest level of surface expressed HMGB-1 which was significantly lower than P B M C treated with TSST-1 alone (p<0.05). B . Immunoblot revealed that HMGB-1 in culture supernatants was highest from P B M C pretreated with IPP + TSST-1. 104 PBMC with y5 T cells A)io»-10N •a S 10*J I v5 T cell depleted PBMC overnight B)ioS HMGB-1 0.4% ; '1023 Forward Scatter 10S 101-! 10*. 48 h D) HMGB-1 0.1% '1023 Forward Scatter 1023 Forward Scatter 10H 10"-r<jt&t&xk- HMGB-1 . . ^ p y ^ ^ ^ i ^ ? ' . ; 3.6% -I tf" '1023 Forward Scatter Figure 5-5. Upregulation of surface expressed H M G B - 1 in cultured P B M C over time. PBMC were cultured in complete growth medium either with or without y5 T cell depletion, and subsequently surface stained for the detection of HMGB-1 by flow cytometyr. Surface expression of HMGB-1 was minimal in overnight cultures of P B M C with or without y8 T cell depletion (0.1% vs. 0.4%, respectively). After 48 h, the expression of HMGB-1 in P B M C with y8 T cells increased to 7.1%, while that in yo depleted P B M C was markedly delayed (only 3.6%). 105 5 . 2 . 3 IPP-primed yS T cells induced the up-regulation of CD40 on antigen presenting cells. PBMCs cultured overnight in complete growth medium at 37°C established a population of antigen presenting cells of monocytic lineage that expressed basal levels of CD86, CD80, and HLA-DR, but very modest levels of CD40 on the cell surface (Figure 5-6, A-D). This finding was consistent with other studies (169). In the currant study, cultures that had been pretreated with IPP overnight had a significant up-regulation of CD40 compared to the RPMI control (p ^ 0.05, paired t test; Figure 5-6A). Stimulation with TSST-1 further augmented CD40 expression (p < 0.05, TSST-1 vs. TSST-1 + IPP at 3 and 6 h post-stimulation). After this time, CD40 expression was lower in TSST-1 or IPP treated P B M C than their counterparts, likely due to binding with its ligand, CD40L (CD 154), which is known to be expressed on T cells within 3 h after TSST-1 stimulation (169). The enhanced expression of CD40 in IPP-pretreated P B M C at 3 and 6 h was attributed solely to the presence of activated yd T cells, since depletion of this small subset (routinely estimated to comprise -1-5% of the total T cell pool among the donors tested) completely abrogated this effect (Figure 5-6E). In contrast to CD40, we found that overnight treatment with IPP resulted in a significant down-regulation of HLA-DR, CD86, and to a lesser extent, CD80 compared to that in untreated P B M C (Figure 5-6, B-D). This cellular phenotype exhibited by APCs present in IPP pretreated overnight cultures with activated yd T cells (characterized by rapid endocytosis of HLA-DR, and low levels of CD80/86 co-stimulatory molecules) is remarkably similar to that described for immature dendritic cells (iDC) (170). 106 A), m K. I PBMC with 76 T cells CD40 expression > hr M hr 18 hr CO 86 expression ! hW(h) ; 4 h r r 3 o S 10 F) 28 I « co a. I yS T cell depleted PBMC CD 40 expression 5hr "1 - t ; 5 h r time (hfhr «^  GD(-)IPP+TSST| &- G D(-)TS ST B- GD(-)RPMI Shr ?4hr IS hr CD86 expression C) 25-s S «i ai a 5-0-D) 2 5 H 0. 10-CD80 expression 15 —t— 20 10 time (h) HLA-DR expression —1 25 1 24 hr G) 25-20-£ a a. is -S m-1— aj a s' 0-H) 30 ^ 20 •g 154 CD80 expression Q--10 IS time (h) • 20 25 HLA-DR expression 3 hr time (h) 6 hr 24 hr time (h) 1 0 7 Figure 5 - 6 . Modulation of co-stimulatory molecules on APCs by activated y 5 T cells. P B M C were cultured overnight in the presence or absence of IPP before stimulation with 1 nM of TSST-1. The surface expression of CD40, CD80, CD86 and H L A - D R were monitored by flow cytometry from 3 to 48 h following TSST-1 stimulation. To confirm that the differences seen in co-stimulatory molecule expression were attributable to the presence of activated y5 T cells, PBMCs from the same donor which had been depleted of y8 T cells were similarly treated and studied in parallel (depicted in the right panel). A. The percentage of cells expressing CD40 were significantly up-regulated on EPP treated P B M C compared to untreated cells at 3 h and 6 h post-stimulation (EPP vs. RPMI, p < 0.05; IPP+TSST-1 vs. TSST-1, p < 0.05). B-D. In contrast to CD40 on APCs, the surface expression of CD80, CD86 and H L A -DR were significantly down-regulated on IPP treated P B M C compared to untreated cells within 3-6 h post-stimulation (EPP vs. RPMI, p < 0.05; EPP+TSST-1 vs. TSST-1, p < 0.005). E-H. There was no significant difference in the expression of these co-stimulatory molecules in y5 T cell depleted P B M C in EPP-treated or untreated cultures. • , RPMI; • , TSST-1; • , EPP + TSST-1; • , EPP; • , RPMI (y5 depleted); A , TSST-1 (y5 depleted); 0 , EPP + TSST-1 (y5 T cell depleted). 108 5.2.4 IPP activated yd T cells do not appear to directly affect aft T cells. In contrast to this direct affect of EPP primed y8 T cells on monocyte maturation and function as described above, there was little influence by these cells on ap1 T cells as evaluated by the expression of CD25 and CD28, which remained largely unchanged in response to EPP pretreatment (Figure 5-7, A and C). However, it was noted that the expression of CD28, which is constitutively expressed on T cells and further up-regulated following activation (171), was slightly enhanced in EPP primed cultures. This observation was most marked when compared to the y5 T cell depleted counterparts (Figure 5-7 C and D). We speculate that this small change in CD28 expression may be attributable to the up-regulation of this costimulatory molecule on y5 T cells themselves. 109 A) PBMC with 76 T cells CD25 expression y8 T cell depleted PBMC B) • RPMI •TSST-1 - IPP+TSST1 -IPP 1 1 1 1— 10 15 20 25 time (h) CD28 expression 1 1 1— 10 15 20 time (h) 25 —I 30 15 i 10 S D) y 70 ca a. « GO 8 CL 50 m CD25 expression 10 15 20 time (h) 25 - o - GD(-)RPMI - D - GD(-)TSST1 GD(-)IPP+TSST1 - 7 - GD(-)IPP 1 1 1 1 1 10 15 20 25 30 time (h) CD28 expression __5 30 Figure 5-7. Activated 78 T cells had minimal effects on the expression of T cell co-stimulatory surface markers, CD25 and CD28, after IPP and TSST-1 treatment. PBMC with or without y8 T cell depletion were similarly analyzed for the surface expression of CD25 (A-B) or CD28 (C-D) by flow cytometry and monitored over 5 - 24 h following TSST-1 stimulation. Only the expression of CD28 was slightly enhanced in IPP pretreated PBMC, and these effects were abrogated in y8 T cell-depleted PBMC. 110 5.3 Discussion We have previously demonstrated that the relatively small population of y8 T cells packed a powerful punch in the early phase of the inflammatory response following TSST-1 stimulation (135). In vivo evidence also indicate that y8 T cells may play a role in superantigen induced diseases. In bovine mastitis, a serious inflammatory disorder caused by staphylococcal infection and associated superantigens, it is believed that y8 T cells are activated and recruited to the area of pathology (103, 104, 105). Two different murine models of sepsis also demonstrated that y8 T cells may indeed promote a detrimental inflammatory response. In one model, a hu-SCID chimera in which human P B M C with activated y8 T cells were reconstituted in mice and subsequently challenged with LPS (44). In the other, a caecal ligation and puncture (CLP) model of sepsis was used to demonstrate the detrimental effect of activated y8 T cells (166). However, the means by which this small population of innate lymphocytes accomplishes such a feat remains uncertain. Our previous work suggested that it was unlikely that direct recognition of TSST-1 by y8 T cells led to an exacerbation of the inflammatory cascade. Rather, it appears that their effect on the reactivity of superantigen-responsive cells is primarily responsible for the aggravated outcome. The present study confirmed that overnight priming of P B M C with IPP, a specific antigen for y8 T cells, activated this innate faction of T cells to upregulate the expression of CD40 on monocytes in preparation for the subsequent stimulation with TSST-1. The expression of CD40L on T cells is upregulated within the first 3 h following TSST-1 stimulation (169). In the absence of IPP-primed y8 T cells, P B M C do not express CD40 until after 24 h following TSST-1 stimulation. In fact, we found that blocking the CD40 - CD40L interaction (but not the CD80 or CD86 111 interaction) completely abrogated the TSST-1-induced inflammatory response (169). Thus, the significant early priming of the inflammatory cascade mediated by activated yS T cells is at least partly mediated through their induction of the early expression of this critical costimulatory molecule. In contrast to CD40, CD86 and to a lesser extent, CD80, were perceptibly down-regulated on monocytes/macrophages after overnight priming with IPP in the presence of y5 T cells. This, taken together with the observation that H L A - D R was more rapidly down-regulated or internalized after TSST-1 stimulation in IPP-primed P B M C (compared to unprimed PBMC) reflects a phenotypic change in the maturation of monocytes described as "immature dendritic cells" in which cycling of the H L A - D R molecule is rapid and levels of CD86 and CD80 are relatively low (170). Further corroboration of the change in the maturation state of monocytes by IPP-activated y5 T cells is shown by the significantly higher mean fluorescence intensity (MFI) of CD86 expression in P B M C cultured over time in the presence of IPP alone and left unchallenged by TSST-1 (Figure 5-ID). These results are in agreement with recent demonstrations by other groups that y5 T cells induce dendritic cell maturation and share an intimate relationship with APCs (172, 173, 174). However, unlike many of these previous studies that used either expanded y8 T cell lines or a large number of these cells in co-culture conditions, the conditions used in our studies were more physiological in that human PBMCs were only briefly exposed to IPP, thus precluding the possibility of any expansion of yS T cells present. The most intriguing discovery, however, was the demonstration of the influence of y5 T cells on the expression and regulation of HMGB-1 . We first demonstrated that nuclear translocation and secretion of HMGB-1 was significantly enhanced in IPP-112 primed PBMC. This finding further supports our earlier observation that activated yS T cells could markedly potentiate the proinflammatory cytokine response of P B M C to TSST-1. This finding was not entirely unexpected since activated y8 T cells induced between 3 to 4-fold higher concentrations of TNFa and EFNy in P B M C within the first 6 h post-TSST-1 stimulation (135). Both of these early cytokines are known to bring about the subsequent release of HMGB-1 from activated monocytes (123), leading to septic shock. It has been established that HMGB-1 is an endogenous signal for dendritic cell maturation and Th l polarization (158). Both these functions have also been attributed to y5 T cells, leading us to speculate that the effect of y5 T cells in potentiating the proinflammatory cytokine response to TSST-1 may be mediated by the nuclear translocation and release of HMGB-1 from both T cells and monocytes. The surprising finding that just the presence of activated y8 T cells under resting culture conditions was sufficient to induce surface expression of HMGB-1 on monocytes in vitro, without the presence of other activating stimulus such as LPS or TSST-1, supports the notion that y8 T cells and HMGB-1 are linked as endogenous adjuvants. Our findings also resonate with previous work showing that mice that lacked y8 T cells had deficient TNFa production from monocytes in response to LPS (53). How yS T cells may induce this regulating effect is currently unknown, but it has been shown that these T cells continuously produce TNFa and EFNy in an on/off cycle (44). Ln addition to this, y8 T cells also produce specific growth factors, such K G F (6), IGF-1 (10), and FGF (175), serving a vital homeostatic function for the cells in the surrounding environment. Taken all together, yS T cells are clearly emerging as a key player in the innate immune system that orchestrates an intricate balance between innate 113 and adaptive immunity. Consequently, y8 T cells paradoxically hold the potential to trigger potent homeostatic as well as deleterious inflammatory responses. 114 Chapter 6: Summary, Discussion and Conclusions. 6.1. Summary of findings. 1) Characterization and influence of activated Vy9V52 T cells in the cytokine response of P B M C following TSST-1 treatment. > Activated Vy9V82 T cells significantly potentiated the early secretion (encompassing the first 6 hr following toxin stimulation) of Thl inflammatory cytokines (TNFa, IFNy, and IL-2). o This augmentation was not evident after 24 hr. > In contrast to the exacerbation of the early inflammatory cytokines, activated Vy9V82 T cells downregulate the later appearance of the anti-inflammatory cytokine, IL-10 at 48 hr following TSST-1 stimulation. 2. Regulation of early and late apoptosis by Vy9V82 T cells following TSST-1 treatment. > Activated Vy9V82 T cells induced a small but significant increase in early apoptosis of P B M C on antigen presenting cells (largely of monocytic lineage) within 6 hr following TSST-1 stimulation. > The majority of peripheral yS T cells underwent AICD at later time points following TSST-1 stimulation. 3) Induction of HMGB-1 as a late cytokine following TSST-1 stimulation. > HMGB-1 was translocated from the nucleus and secreted from both activated T cells and monocytes following TSST-1 treatment. 4) Regulation of HMGB-1 expression by y8 T cells in TSST-1 induced inflammation. > The very presence of y8 T cells enhanced the surface expression of HMGB-1 on adherent cells in culture under steady state conditions. 115 > Activated y8 T cells further potentiated the level of nuclear translocation and subsequent secretion of HMGB-1 from both monocytes and T cells following TSST-1 stimulation. 5) y5 T cells in propagating the inflammatory response: Upregulation of cell surface expression of co-stimulatory molecules. > IPP stimulated y5 T cells in P B M C induced the upregulation of CD40 on monocytes. > IPP pretreament of yS T cells in P B M C also induced a phenotypic change in the maturation of monocytes to those resembling "immature" dendritic cells as assessed by the surface expression of CD86, CD80, and HLA-DR. > IPP activated yS T cells did not induce any significant change in the expression of activation markers and co-stimulatory molecules (i.e. CD25 and CD28) on aP T cells. 6.2. Discussion. The superantigenie and pro-inflammatory properties of TSST-1 have long been known to be attributed to its ability to bypass the regulatory checkpoints on aP T cells and monocyte activation. However, exactly what factors influence the downstream consequence of this interaction is still not fully elucidated. Toxic shock syndrome, which is the most serious pathology caused by TSST-1, is a cytokine mediated disease that is driven by the distortion of the highly regulated relationship between the adaptive and the innate immune system that is represented by the aP T cells and the A P C s respectively. It is within the immediate immunological micro-environment in which the host-microbe interaction takes place that determines the course of immune regulation - be it ultimately 116 protective or deleterious in nature. A great deal is still unknown about what leads some people to develop TSS when exposed to TSST-1 and others to acquire protective antibodies thereby avoiding a hyperactive inflammatory response. This thesis was initiated with the aim to investigate the possible involvement of yS T cells, an unconventional subset of lymphocytes thought to link adaptive and innate immunity (28, 37, 176), in setting the stage for either immune protection or corruption. The results of this study unambiguously position y5 T cells as powerful positive modifiers of the hyper-inflammatory response to TSST-1. As illustrated in Figure 6-1, activated Vy9V82 T cells can significantly augment the very early response to TSST-1. Furthermore, activated y8 T cells also have the ability to augment the nuclear translocation and subsequent secretion of HMGB-1 , a recently recognized late mediator of septic shock (112, 113). Intracellular cytokine staining revealed that IPP pretreated y8 T cells were not the sole source of the early enhanced inflammatory cytokines following TSST-1 stimulation; the expression of these cytokines were also upregulated in ap T cells - but only in the presence of activated Vy9V82 T cells. This concept of "uncoventional" T cells regulating and augmenting the activity of "conventional" M H C restricted CD4+ and CD8+ aP T cells is just coming to the forefront of immunology (177). What has been lacking is the mechanism by which this regulation occurs. The experimental protocol that was employed to study the role of y8 T cells in TSST-1 mediated pathogenesis ensured that both the number of these innate lymphocytes as well as the other constituents of the immune system reflected the in vivo situation. Investigations have often studied the activity of human y8 T cells by isolating and expanding these cells in vitro and measuring certain aspects of activation, such as proliferation, to decipher their function. This 117 approach would fail to reveal how y5 T cells actually regulate the response and activity of other cells - which may be one of the prominent functions of yS T cells in vivo. 118 Figure 6-1. A uniform hypothesis for the role of y5 T cells in TSST-1 induced inflammation. Activated Vy9V82 T cells can significantly augment the very early proinflammatory response to TSST-1, particularly the secretion of EFNy, T N F a and EL-2, within the first 4 to 6 h after TSST-1 stimulation. This effect may well be mediated in part by the upregulation of the co-stimulatory molecule, CD40, in TSST-1 activated monocytes. Additionally, y8T cells down-regulate the anti-inflammatory cytokine, EL-10, at a later stage 48 h after TSST-1 stimulation, further skewing to a Thl proinflammatory bias. Finally, activated y5 T cells also have the ability to augment the nuclear translocation and subsequent secretion of HMGB-1 , a recently recognized late mediator of septic shock. Finally, activated y§ T cells also induce apoptosis in monocytes/macrophages as they undergo maturation and differentiation. They could be a 119 homeostatic mechanism that eventually lead to downregulation of the hyperimmune response and tissue recovery. 120 The mechanism by which yd T cells enhance the immune response is still largely ambiguous. Arguably, the most pivotal finding of this investigation was serendipitous in nature. The decision to utilize HMGB-1 as a tool to assess the downstream consequence of the early influence of yd T cells in superantigen activation was a fortuitous one. The regulation of HMGB-1 expression has never before been studied in the context of y8 T cells or in superantigen-mediated toxic shock. It was surprising how closely related these two immune modulators (i.e. y8 T cells and HMGB-1) turned out to be. In parallel experiments in which y5 T cells had been depleted from peripheral blood, monocytes in culture actually became noticeably impaired in differentiating into adherent cells and expressing the surface localized HMGB-1 (also called amphoterin) under steady state (or resting) conditions. It was ascertained that this phenomenon was not an artifact caused by the experimental procedure for y5 T cell depletion, since both populations of P B M C s (those with or without yS T cells) did not express HMGB-1 on the cell surface when initially purified, and it was the cell-cell interactions within the culture conditions that led to the differentiation of monocytes and subsequent surface expression of HMGB-1. The cumulative evidence suggests that the means by which y5 T cells augmented TSST-1 induced inflammation may be mediated by their role in differentiating and maturing monocytes into a more antigen-receptive state. This conclusion resonates with recent findings from other investigators demonstrating that these enigmatic lymphocytes play a major role in differentiating dendritic cells (43, 172). More interestingly, unrelated studies have established HMGB-1 as a key endogenous molecule for macrophage differentiation and dendritic cell maturation (158). The release of HMGB-1 from dendritic cells was recently shown to polarize activated T cells towards a Th l bias via 121 binding to R A G E on various endothelial and epithelial cells (121). Figure 6-2 illustrates the current concept and hypothesized model of the mechanism by which y8 T cells and HMGB-1 intersect in the regulation of TSST-1 induced stimulation of ap T cells and monocytes. Our data suggests the possibility that y5 T cells potentiate the proinflammatory response to TSST-1 in part through differentiating monocytes, and this effect is likely mediated by the mobilization and secretion of HMGB-1 and the upregulation of CD40. Thus, this investigation, which focused on delineating whether y5 T cells can regulate the immune response to TSST-1, unexpectedly provides the missing link uniting these two disconnected models of APC differentiation (i.e. the previously dissociated and distinct functions of HMGB-1 and y5 T cells in monocyte maturation). It should be noted that a recent study (8) also found that yS T cells play an active role in TSST-1 induced immune activation of aP T cells, but these investigators suggest that y8 T cells are potent antigen presenting cells themselves. Indeed, especially in the murine system where dendritic epithelial T cells even have a morphological similaritary to professional antigen presenting cells (12, 16), y8 T cells appear to have the capacity to present antigens to ap T cells. However, given the paucity of y8 T cells in human peripheral blood, and the evident change observed in the effector capabilities of macrophages in cultures with EPP-activated y8 T cells, it seems unlikely that the potentiation of the inflammatory response to TSST-1 observed in our studies can be attributed to the professional APC capabilities of peripheral Vy9VS2 T cells. The hypothesis that y8 T cells enhance immune function via their effect on monocytes was further supported by the finding that CD40 was significantly upregulated in cultures with IPP pretreated y8 T cells. CD40 was previously shown to be a pivotal 122 costimulatory molecule in TSST-1 induced inflammation by our group. Blocking the interaction between CD40 on A P C with its ligand (CD40L) on TSST-1 activated a P T cells completely abrogates the immune response to TSST-1 (169). In the absence of stimulated yd T cells, CD40 does not get expressed on APC until the later phase of the inflammatory response to TSST-1 (169). Therefore, the very early up-regulation of this molecule on monocytes by activated yd T cells is likely one of the key factors leading to the significant early enhancement of the Th l cytokines observed following TSST-1 exposure. In contrast to the direct influence of activated yd T cells on monocyte function, none of the parameters measuring the early priming of a[3 T cells were significantly affected by IPP stimulated y5 T cells in the absence of TSST-1 stimulation. It appears that the change of state in monocyte maturation by activated yd T cells is the primary driving force for the enhanced activation of a P T cells by providing more potent signals via costimulatory moleucles. Figure 6-3 provides the amalgamated model of the role of y8 T cells in TSST-1 pathogenesis. 123 A) RESTING STATE yd T cells) i Induce differentiation & surface expression of H M G B - 1 B) IPP (infectious or non-infectious stress) RAGE \ J*~~HMGB-1 C) IPP (infectious or noninfectious stress) + TSST-1 RAGE TSST-1 124 Figure 6-2. Proposed model of how yd T cells and HMGB-1 intersect in the regulation of TSST-1 pathogenesis. A . Under resting conditions, y8 T cells regulate monocyte maturation leading to the eventual surface expression of HMGB-1 on adherent macrophages. B. In the presence of either endogenous or exogenous IPP, y5 T cells induce the release of HMGB-1 into the local extracellular milieu which acts as a feedback loop leading to further differentiation via R A G E and up-regulation of CD40 on maturing monocytes. In the absence of TSST-1, a(3 T cells don't express either R A G E or CD40L; thus, there is no proliferation or massive cytokine secretion. C. In the presence of both IPP and TSST-1, ap T cells get activated and express R A G E and CD40L; leading to massive proliferation and Th l polarization. 125 Figure 6-3. A model illustrating how human peripheral blood yS T cells regulate the immune response to TSST-1. Under infectious stress, activated yS T cells directly enhance the maturation, differentiation and responsiveness of cells in the monocytic lineage by upregulating C D 4 0 and mobil izing the endogenous differentiating factor, H M G B - 1 . This results in a potent early proinflammatory cytokine response to TSST-1 , and the subsequent exacerbation of the downstream systemic inflammatory response, leading to further release of H M G B - 1 from activated monocytes and T cells. This exacerbated inflammatory response is not a dysfunction of y5 T cells. Rather, corruption of the immune response is due to the nature of the antigen, T S S T - 1 , which bypasses normal immunoregulatory checkpoints, leading to an uncontrolled activation of aP T cells and monocytes. 126 6.3. Considerations, potential significance, and future endeavours. As with all in vitro studies, there has to be caution when extrapolating experimental in vitro data to what occurs in vivo. Future work will have to validate both the role of y8 T cells and HMGB-1 in clinical cases of TSS. There is new data from patients suffering from severe systemic inflammatory response syndrome (SIRS) that seems to support some key findings of this study (148). Early activation in comparison to a P T cells, and subsequent loss of peripheral y8 T cells by activation induced cell death (AICD) was also observed in patients recovering from severe SIRS or sepsis resulting from acute injury in which peripheral yS T cells were significantly lower than in healthy controls within 48 h of the insult. The authors concluded that y5 T cells have a distinct role as early responders in severe injuries leading to SIRS (148). It is acknowledged that only the peripheral blood Vy9V82 T cells, which have been characterized as being "pro-inflammatory" in nature (44, 45, 134), were investigated in this study, and the effect of tissue localized V51 T cells may be entirely different in function. It is plausible that the predominant subset of y8 T cells in the environment in which staphylococcal TSST-1 is encountered could determine the nature of the immune response rendered. These questions may be challenging to study in the murine system due to the species differences in y8 T cell subsets, and the inherent lack of susceptibility of mice to TSS. Careful consideration regarding the animal model and the relevance to human disease is critical in further studies. An interesting matter that has not been fully explored and warrants further study is the question of gender bias in yS T cell response and activity. It has been recognized for a long time that y8 T cells play a unique role in reproductive immunobiology (178, 127 179). In fact, there is a very specialized invariant subset of y8 T cells that line the reproductive tracts in female mice (178). y8 T cells and other innate lymphocytes dominate during pregnancy to provide the first line of defense since the adaptive immune system could prove deleterious for the growing embryo by causing rejection of the foreign body (180). The observation that TSST-1 has a predilection for causing hyper-inflammatory responses in females (143) and in inducing pathology of the female reproductive organs including those of cattle and sheep should perhaps be further studied in regards to the repertoire of innate T cells and the influence of hormones regulating immunity. The most immediate clinical application from the results of our studies is the potential use of HMGB-1 inhibitors for the treatment of superantigen induced TSS, as it is currently being assessed for sepsis (157). If proven successful, this would certainly save both time and resources for the identification and treatment of various systemic inflammatory disorders including septic or toxic shock, and other inflammatory disorders induced by the specific bacterial superantigens. Another intriguing relationship that requires elucidation is that between y8 T cells and the regulation of HMGB-1 expression in myeloied cell differentiation. 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