Staphylococcus aureus enterotoxin mediated specific non-responsiveness of human T cells

Immunology Letters, 30 (1991) 165 - 170 Elsevier IMLET 01683

Staphylococcus aureus enterotoxin mediated specific nonresponsiveness of human T cells Robyn E. O ' H e h i r 1, Roland Buelow 2, Hans YsseP and Jonathan R. L a m b 1 1Department of Immunology, St. Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London, U.K., 2ImmuLogic Pharmaceutical Corporation, Palo Alto, CA, U.S.A. and 3DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA, U.S.A. (Received 21 June 1991; accepted 25 June 1991)

1. Summary Exotoxins produced by certain strains of Staphylococcus aureus are able to both stimulate and induce non-responsiveness in T cells expressing specific T cell antigen receptor V/3 gene elements. The exposure of human CD4 ÷ T cells to the appropriate enterotoxin rendered them anergic to restimulation with their natural ligand, although responsiveness to exogenous IL-2 remained intact. The loss of antigen-dependent proliferation was associated with the down-regulation of the TCR complex that was paralleled by enhanced cell surface CD2 and CD25. Further analysis of the phenotypic changes revealed that membrane levels of CD28 were increased only on activation, suggesting a differential expression of this protein on activated and anergic T cells. During the induction of anergy it was observed that the synthesis of the lymphokines IL-2, IL-4 and IFN-7 was differentially regulated. IL-4 and IFN-3,, but not IL-2 were detected in the supernatants of overnight cultures of T cells exposed to tolerising concentrations of toxin. Transcription of IL-4, as determined by polymerase chain reaction at selected intervals, was elevated during the inducKey words: Superantigen; T cell anergy; Lymphokine regulation

Correspondence to: Robyn E. O'Hehir, Department of Immunology, St Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, Norfolk Place, London, W2 1PG, U.K.

tion of anergy and accounted for the presence of the protein in the supernatants. In contrast, no tight coupling was observed between protein and mRNA levels for IL-2, suggesting post-translational regulation. 2. Introduction T cell recognition of the Staphylococcus aureus enterotoxins, like that of nominal antigen, requires the presence of class II major histocompatibility (MHC) bearing antigen presenting cells [1, 2]. However, further similarities between the interaction of the S. aureus enterotoxins and nominal antigen with either MHC class II or the T cell antigen receptor (TCR) molecules are limited. The bacterial toxins appear to require minimal processing prior to presentation [3, 4], and bind to a non-polymorphic external domain of the class II MHC ce chain [5]. In contrast, nominal antigen, as peptide fragments, occupies the putative antigen combining cleft created by the oel and/~1 domains [6]. Antigenic complexes of peptides and MHC class II molecules form specific interactions with the variable elements of both the oe and ¢3 chains of TCRs [7], whereas T cell recognition of the staphylococcal enterotoxins is determined by only TCR-V~3 gene products that they express [8, 9]. The binding site for these toxins on the TCR-V/3 gene elements appears to be located on the exposed surface of chain involving residues 67 - 76 [10]. The toxins are able to stimulate powerful polyclonal responses,

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and characteristically interact with TCRs bearing common V~ gene elements, irrespective of antigen and restriction specificity, which categorises them in the family of protein antigens termed the "superantigens" [11]. Nevertheless, despite obvious differences in the structural requirements of their functional domains the S. aureus enterotoxins, similar to nominal antigen, are able to induce T cell anergy in vivo [12, 13]. The ability of S. aureus enterotoxins to inhibit the functional activity of selected families of T cells theoretically offers an alternative approach of downregulating harmful immune responses. This hypothesis prompted us to investigate the potential of bacterial toxins to tolerise human T cells expressing different TCR-VB gene products. In order to establish the generality of this approach in the development of specific anergy a variety of populations of cloned T cells were examined independently of their antigen and M H C restriction specificities [14, 15]. In this paper we summarise our observations on the cellular and molecular basis of enterotoxin-induced T cell non-responsiveness. 4. Results and Discussion 4.1.

The induction o f enterotoxin-induced specific clonal anergy

The ability of S. aureus enterotoxins to induce clonal expansion or anergy of mature peripheral antigen-reactive human T cells expressing the appropriate TCR-V/~ gene elements is influenced by the presence or absence of antigen presenting cells (APC), as well as the concentration and duration of exposure to the toxins [14]. Stimulation of T cells with supraoptimal concentrations of enterotoxin, in the absence of APCs, rendered the T ceils nonresponsive to an immunogenic challenge with their natural ligand of specific antigen complexed with M H C class II molecules. Enterotoxin-mediated anergy was examined in house dust mite-reactive human T cells expressing different TCR-V/~ gene elements, and with different antigen and restriction specificities [15]. The results of this study demonstrated that this form of anergy is a general phenomenon and not unique to selected bacterial toxins or human T cells. Despite the loss of antigendependent proliferation the toxin-treated T cells re166

mained responsive to IL-2, demonstrating that anergy was not the result of cytolysis. It is important to emphasize, particularly in consideration of any potential practical application in vivo, that it is possible to induce anergy with the bacterial superantigens in the presence of APCs, but higher concentrations of toxin are required to achieve the same level of efficacy. Although after 3 h exposure to toxin the T cells were partially anergised complete nonresponsiveness was not observed until 6 h. The kinetics of toxin-mediated anergy are identical to that observed with nominal antigen presented in the form of peptides [15]. These observations suggest that the tolerogenic signals are inducing biochemical or molecular changes within the T cells and that T cell anergy is not simply the result of receptor blockade. Determination of the longevity of nonresponsiveness of human T cells is hard to assess, primarily due to difficulties in establishing culture conditions in vitro that represent the environment that the tolerant cells would encounter in the intact host. However, after exposure to tolerising concentrations of toxin the T cells are returned to culture in IL-2-supplemented medium, and then assayed for antigen-dependent proliferation at selected time intervals. The restoration of function that was observed by 5 - 7 days may be due to the effects of IL-2 which has been demonstrated in vitro [16] and in vivo [17] models both to prevent the induction of and to break established tolerance. However, it is also possible that the T ceils responding to antigen in these cultures are the progeny of the anergic T cells as they would not inherit tolerance. The results of recent in vivo experiments have indicated that superantigens are able to induce T cell anergy in the adult animal [12, 13], and, at least in the case of Mls, the induced anergy is long-lasting. 4.2. Identification o f immunologically site(s) in S. aureus enterotoxin B

active

Bacterial superantigens, in contrast to nominal antigen, interact with the external domains on both M H C class II and TCR molecules and, therefore, it is likely that they have different physical characteristics as regards their molecular mass and conformational dependence. Studies designed to identify the immunologically active site(s) in the bacterial enterotoxins have relied on the use of en-

zymatic cleavage fragments [3, 18]. The results of these experiments were inconsistent and, in order to avoid the problems of this approach recombinant SEB proteins in which sequences had been deleted were examined for their ability to induce T cell proliferation and/or anergy. The full-length recombinant protein was able to induce proliferation although it was approximately 100-fold less potent than the intact native protein. Removal of the amino terminal 30 residues resulted in the failure of SEB both to activate and induce non-responsiveness in cloned V~3.1 ÷ T cells. However, the response of unfractionated peripheral T cells to residues 30 - 239 suggests that ceils expressing TCRV~3 receptors of other specificities known to interact with SEB, such as 8 and 17, are interacting with different regions of the enterotoxin. Several re: ports, although not all, using tryptic fragments [19] or synthetic peptides [20] are in agreement that mitogenic activity resides in the amino terminal portions of these molecules. Moreover, inconsistency in the observations made by the different laboratories examining this issue was not due to the use of different serotypes of enterotoxin. It would also appear that the immunological and emetic properties may be dissociated from one another by enzymatic digestion or chemical modification, such as carboxymethylation [19, 21]. The recombinant SEB fusion protein with residues 131- 239 of the carboxyl terminus deleted was functionally inactive. However, constructs containing residues 1 - 138 had comparable potency as compared to the full-length recombinant protein, indicating that regions of the molecule additional to amino acids 1 - 30 contribute to T cell recognition. Alternatively, the loss of recognition of the recombinant protein with selected mutations may result from minor modifications in conformation, even though their reactivity on Western blots was apparently unaffected. From their primary amino acid sequence the bacterial enterotoxins may be aligned on the basis of two cysteine residues located in the central region [22]. Both the disulphide loop formed by these residues and the presence of a conserved motif located immediately carboxy-terminal to this loop have been analysed for their contribution to the mitogenic activity of the toxins [3, 23]. It was observed that disruption of the disulphide loop by

reduction and alkylation of the cysteines resulted in the loss of mitogenic activity of both SEA and SEB, but without abrogation of their ability to bind to MHC class II molecules [23]. These findings contrasted with earlier reports in which reduction and alkylation appeared to have no effect on the activity of the toxins [3] and, perhaps, the difference was attributable to contamination of the toxin preparations with unmodified protein. The results of our experiments demonstrating that residues 1 - 138 are required for the activation of V/33.1 + T cells are consistent with the reports that the immunological activity maps to the central region of these molecules. It was proposed that an intact disulphide loop is necessary for interacting with TCRs, while those residues in the conserved motif determine binding to MHC class II molecules. However, the association of function with these regions of the toxins may not be so clearly defined. For example, toxic shock syndrome toxin-1 (TSST-1) has no disulphide loop and, despite minimal sequence homology in the putative motif, is able to compete with SEB for binding to MHC class II molecules. Furthermore, haplotype specificity in the interaction of the bacterial toxins with both human and murine MHC class II molecules has been demonstrated, suggesting polymorphic residues may contribute to the interaction [11]. Thus, the possibility exists that different enterotoxins may use selected regions for binding to either TCR and/or MHC class II molecules.

4.3. Phenotypic modulation In order to determine the molecular basis of enterotoxin-induced non-responsiveness the T cells were initially examined for changes in phenotype [14, 15]. Only those concentrations of toxin able to abrogate proliferation to an immunogenic challenge induced alterations in the phenotype of T cells expressing the relevant TCR-V/3 gene products, as compared to the control cultures. The Ti-CD3 antigen receptor complex was modulated from the T cell surface after pretreatment with toxin in a dosedependent manner as determined by fluorescence flow cytometry with both anti-CD3 and anti-TCR antibodies [14]. However, concentrations of toxin that reduced membrane expression of Ti-CD3 by only approximately 50°7o resulted in complete func167

tional inactivation of the T cells. Concomitant with down-regulation of Ti-CD3 expression, CD25 was up-regulated accounting for the enhanced responsiveness of the T cells to exogenous IL-2 [14, 15]. Coprecipitation studies have demonstrated that approximately 40% of membrane CD2 is physically associated with Ti-CD3 [24]; therefore, the enhanced expression of CD2 in toxin-induced anergy was unexpected. The relationship between these molecules is complex with CD2 possibly operating with different functional roles in T cell activation depending upon its association with the Ti-CD3 complex. For the majority of T cell clones, expression of CD4 was unaffected by exposure to the toxins; however, for some T cells CD4 was comodulated with Ti-CD3, suggesting that it may be a physical component of the T cell antigen recognition structure [25]. The examination of additional membrane proteins demonstrated that C D l l a / C D 1 8 and CD45 were up-regulated in both activated and nonresponsive T cells. Indeed, the pattern of phenotypic modulation observed for the membrane proteins described above was identical in both T cell activation and anergy. However, while cell surface CD28 on the tolerised T cells was partially but reproducibly down-regulated (approx. 2 0 - 3 0 % ) , on those cells that had been activated expression of CD28 was markedly increased [14, 15]. It has been postulated that CD28 may be the receptor for costimulatory activity, the engagement of which determines the outcome of anergy or clonal expansion following occupancy of the TCR in peptide-mediated non-responsiveness [26]. The phenotype of the anergic T cells is fully restored to normal by 60 h after the initial exposure to tolerising concentrations of toxin, yet the cells remain non-responsive to rechallenge for up to 7 days. Therefore, the loss of antigen-specific responsiveness does not arise simply as the result of the modulation of the Ti-CD3 receptor complex from the cell surface. Since it has been reported that murine Th 1 cells rendered non-responsive by stimulation with antigen presented by chemically modified APCs were unable to produce IL-2 we examined lymphokine regulation in T cell anergy.

4.4. Regulation of lymphokine production Specialisation of the function of murine CD4 + T 168

cells is accompanied by their ability to produce only certain lymphokines [27], such that the TH2 cells, able to provide B cell help, no longer secrete IL-2, whereas those T cells that mediate delayed-type hypersensitivity produce IL-2 and IFN-~,, but not IL4. However, for human T cells the segregation of function with lymphokine profile is not so clearly delineated and, although quantitative differences in the levels of lymphokines secreted are often marked, they appear to be more similar to the TH0 population of cells first described in the mouse [28]. This characteristic of human T cells allowed us to examine the regulatory effects of toxin-induced anergy on several different lymphokines produced by a single population of T cells. The results of our experiments analysing the supernatants of overnight cultures revealed that IL-2 secretion induced after pretreatment with the appropriate S. aureus enterotoxins under conditions that inhibit antigen-dependent T cell proliferation, was minimal compared to that produced in response to activation signals [29]. Although IL-2 receptor expression is similar in both the tolerised and activated T cells [14], it is possible that the failure to detect IL-2 in the supernatants was due to differential rates of consumption. Therefore, in order to address this issue and to examine the kinetics of lymphokine production, mRNA levels were quantirated by polymerase chain reaction during the induction of anergy. The transcripts for IL-2 were initially increased in both activated and tolerised T cells but by 6 h had returned to steady-state levels. The finding that increased IL-2 mRNA was not accompanied by detectable amounts of protein implies that post-translational modification may regulate IL-2 synthesis in the anergic T cells. As both the mRNA and protein levels were enhanced in activation the mechanism of IL-2 regulation may differ from that operational in anergy. The lack of IL-2 in the supernatants of murine TH1 cells, made unresponsive by antigen complexed with MHC class II molecules on chemically modified APCs, was accompanied by a 95°?o reduction in IL-2 mRNA as compared to the control of activated normal T cells [26]. The activity of several lymphokines is inhibited in non-responsive T cells; nevertheless, from in vitro cell culture systems it is apparent that the regulation of IL-2 synthesis is critical in the development of T cell anergy, and that IL-2 may also con-

tribute to the reversal of non-responsiveness [16]. Furthermore, the demonstration that T cells from adult mice rendered tolerant to Mls-1 in vivo fail to produce IL-2 on restimulation [12], combined with the ability of IL-2 to break neonatal tolerance in graft rejection [17] supports a central role for IL-2 in activation and non-responsiveness. Pretreatment of the T ceils with the S. aureus enterotoxins under tolerising conditions resulted in the secretion of IL-4 and IFN-3, into the culture supernatants in quantities which equalled or exceeded that produced during T cell activation. Kinetic analysis demonstrated that mRNA for IL-4, during both activation and anergy, was increased between 2 and 6 h after stimulation. Therefore, for IL-4 production of the protein was paralleled by enhanced levels of transcripts. The exposure of murine TH0 ceils to fixed APCs inhibited the production of IL-2 but not IL-4 [30]. However, it is not possible from these experiments to determine whether non-responsiveness under these conditions was due to fixation altering APC-derived signals [31] or that a negative signal was being delivered directly through the TCR. It would appear from the findings reported here that the presentation of the bacterial enterotoxins in a non-immunogenic form has differential effects on the secretion of lymphokines during the induction of T cell anergy. The ability of S. aureus enterotoxins to induce non-responsiveness in human T cells, as described here, together with the results of in vivo experiments in murine model systems raises the possibility of using superantigens to inactivate subpopulations of T cells that express TCR with common features. This approach may be of potential therapeutic value in diseases where the diversity of TCRs is restricted.

Acknowledgements This research was. supported by programme grants from the Wellcome Trust and the Medical Research Council. R.E. O'H. is the recipient of a Wellcome Senior Research Fellowship in the Clinical Sciences. DNAX Research Institute is supported by Schering Plough.

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