Memory T Cell Tolerance to Superantigens is not due to Increased Susceptibility to Apoptosis

Journal of Autoimmunity (1997) 10, 357–365

Memory T Cell Tolerance to Superantigens is not due to Increased Susceptibility to Apoptosis William T. Lee, Jayanthi Padmanabhan and Jocelyn Cole-Calkins Laboratory of Immunology, Wadsworth Center for Laboratories and Research, P.O. Box 22002, Albany, NY 12201-2002, USA Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY, USA

Received 24 January 1997 Accepted 1 April 1997 Key words: immunological memory, anergy, superantigens, apoptosis, tolerance

Naive (virgin) and memory T lymphocytes differ markedly in their response to superantigens (SAg). When cultured with the SAg staphylococcal enterotoxin B (SEB), virgin but not memory CD4 + T cells proliferate and secrete lymphokines. Memory cells do express increased levels of activation markers after interaction with SEB, which suggests that the cells are not ignorant of the SAg. In this report, we have considered whether SEB, rather than activating memory cells, promotes their death by apoptosis. Our results indicate that while in vivo exposure to SEB induces apoptosis, there is no greater level of cell death in the memory cell population relative to virgin cells. Further, elimination of the Fas-mediated cell death pathway does not permit memory cells to be stimulated by SEB. Memory T cells from either Fas-expressing or Fasdeficient (MRL-lpr/lpr) mice are hyporesponsive to SEB. Blockade of Fas/Fasligand interactions by a Fas-Fc chimeric protein does not permit BALB/c memory cells to proliferate upon culture with SEB. These results provide evidence that the failure of memory T cells to respond to SEB is not due to cell death and that inactivation (anergy) is the likely fate of these cells when they encounter SEB. © 1997 Academic Press Limited

Introduction

SAg, staphylococcal enterotoxin B (SEB), into MRL lpr/lpr mice results in deletion after T cell expansion, as in wild-type mice; however, the process exhibits delayed kinetics [11]. As with normal mice, the remaining bulk CD4 + T cells from lpr mice are anergic to in vitro restimulation with SEB [11]. As a consequence of activation by conventional or SAg, either ‘effector’ or memory cells develop. There are numerous differences which distinguish TM cells from their precursor virgin (TV) cells. Among these are differences in phenotype, homing properties, activation requirements, and repertoires of secreted lymphokines (LK) (reviewed in [12]). Thus, preferential responses by either TV or TM cells may have two distinct functional outcomes. We have previously examined the relationship between immunological memory and SAg-mediated anergy. In BALB/c mice, bulk CD4 + T cell anergy in response to SEB correlated with an increased proportion of SEB-specific (Vâ8 + ) TM cells, perhaps arising from activated TV cells [13]. While normal TV cells (from non-SEB-treated mice) responded vigorously to in vitro stimulation with SEB, TM cells failed to proliferate or secrete LKs [13]. Thus, the activation of CD4 + T cells with SEB reflects the expansion of TV cells; TM cells appear to be nonresponsive. As an earlier study had shown that CD95 was preferentially expressed on peripheral TM cells [14], we considered whether SEB caused TM cells to die, rather than proliferate. If the mechanism of TM

Superantigens (SAgs) have been used as models to examine central and peripheral tolerance (reviewed in [1]). With respect to peripheral tolerance, SAgs promote activation of CD4 + T cells bearing specific Vâ-containing T cell receptors (TcR) [1]. In vivo, expansion of the specific T cells is followed by their disappearance [2, 3]. Interestingly, the remaining SAgspecific T cells fail to be restimulated by the SAg, and are thought to be anergic. Hence, bacterial or retrovirally derived SAgs may influence the immune repertoire and the T cell response during infection. An important mechanism of peripheral deletion involves activation-induced cell death (AICD) (reviewed in [4]). In hybridomas and in normal T cells, triggering through the TcR can result in apoptosis, predominantly mediated by the Fas (CD95) receptor interaction with its ligand [5]. Recent studies have shown that this process is caused by Fas-ligand (Fas-L) that is induced on the surface of T cells and may suggest that death occurs in an autocrine fashion [6–8]. Mice carrying the lpr mutation are deficient in Fas [5] and mature T cells from lpr mice have impaired ability to undergo AICD [9, 10]. Administration of the Correspondence to: Dr William T. Lee, David Axelrod Institute for Public Health, Wadsworth Center for Laboratories and Research, P.O. Box 22002, Albany, NY 12201-2002, USA. E-mail: [email protected]. 357 0896-8411/97/040357+09 $25.00/0/au970146

© 1997 Academic Press Limited

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non-responsiveness to SEB was due to Fas-mediated apoptosis, then the absence of Fas/Fas-ligand interactions should promote TM cell proliferation. The present study was designed to test this hypothesis through the examination of the response to SEB by TM cells from lpr mice and by inhibition of Fas/Fas-ligand interactions on BALB/c TM cells. Our data provide evidence that TM cell tolerance to SEB is independent of a Fas-deletion pathway. In addition, we have directly compared AICD in the T cell population which proliferates in response to SEB (TV cells) with the population which does not proliferate (TM cells). We show that the levels of AICD in both populations are comparable, hence deletion cannot account for the difference in reactivity to SEB of TV and TM cells. These data support the hypothesis that the failure of TM cells to respond to SEB is primarily due to a state of inactivation rather than apoptosis.

Materials and Methods Animals Female BALB/c mice were bred and maintained at the Griffin Laboratories, New York Department of Health. These mice were used at 6–8 weeks of age. Female MRL-lpr/lpr mice were purchased from Jackson Laboratories (Bar Harbor, ME). Young mice were used at 6–8 weeks of age; older MRL-lpr/lpr mice, showing obvious signs of lymphoproliferative disease, were used at 16 weeks of age. In these experiments, only data from MRL-lpr/lpr mice are shown. However, comparable experiments were performed using lpr/ lpr mice on a C57BL/6 background. Similar results were obtained from these experiments. Further, control experiments were also performed on MRL-+/+ mice to preclude background influences. These mice behaved similarly to the BALB/c controls. In some experiments, mice were immunized by i.p. injection of 20 ìg of SEB in PBS, at indicated time points prior to analysis.

Lymphokines and antibodies Human recombinant IL-2 (rIL-2) (Amgen Biologicals, Thousand Oaks, CA), PE-anti murine Fas (Jo-2) [15] (Pharmingen, San Diego, CA), and SEB (Sigma Chemicals, St Louis, MO) were purchased. mAbs 23G2 (rat anti-murine CD45RB) [16], GK1.5 (anti-CD4) [17], MARK-1 (mouse anti-rat ê chain) [18] and F23.1 (mouse anti-murine Vâ8.1,8.2,8.3) [19] were purified from hybridoma cell supernatants (SNs). Purified human Fas-Fc chimeric protein, derived from recombinant baculovirus [7], was kindly provided by Drs T. Brunner and D. Green (La Jolla Institute for Allergy and Immunology). The amounts of soluble Fas-Fc used in this study were sufficient to block 80% of activation-induced apoptosis of A1.1 murine T hybridoma cells (T. Brunner, pers. comm.) and DO.11.10 murine T hybridoma cells (10 ìg/ml blocked >50% of

the death caused by immobilized anti-CD3; WTL, unpubl. obs).

Immunofluorescence staining and analysis Where indicated, mAbs were directly labeled with FITC (Sigma Chemical, St Louis, MO) or Cy-5 (Biological Detection Systems, Pittsburgh, PA). Alternatively, mAbs were biotinylated using sulfo-NHSbiotin (Pierce Chemical, Rockford, IL) and were detected using either PE-streptavidin (PE-Av) (Tago, Inc., Burlingame, CA), Red-670-streptavidin (GIBCO, Grand Island, NY) or Cy5-streptavidin (Cy5-Av) (Biological Detection Systems, Pittsburgh, PA). Unless otherwise indicated, spleens from two mice per group were pooled and CD4 + T cells were purified prior to staining. All flow cytometry experiments were performed a minimum of three times; representative experiments are shown. Fluorescence staining was performed at 4°C in 100 ìl containing 1×106 cells and a pre-determined optimal amount of primary antibody in balanced salt solution (BSS) containing 2% fetal bovine serum (FBS) (GIBCO, Grand Island, NY), 20 mM HEPES, and 0.1% NaN3. Staining with a secondary reagent was performed in a similar manner after washing the cells. For measurements of apoptosis, cells were labeled with 0.1 ìg/ml of the lipophilic dye, merocyanine-540 (MC-540) (Molecular Probes, Eugene, OR) [20, 21], 10 min before analysis. Preliminary experiments on either ã-irradiated or dexamethasone-treated murine thymocytes showed that increased MC-540 staining correlated with both decreased forward light scatter (by FACS) and DNA laddering on agarose gels (data not shown). Flow cytometric analyses of stained cells were performed using either a BD-FACScan or a FACS-Vantage (Becton Dickinson, Mountain View, CA) (for Cy5containing samples).

Preparation of cells In all experiments, enriched populations of CD4 + cells were prepared by negative selection, as previously described [22]. Using these procedures, cells from control and young MRL-lpr/lpr mice were 90–95% CD4 + (assessed by staining with FITC-GK1.5) as determined by flow cytometric analyses. Purified populations of TM or TV cells were prepared from the CD4 + T cells by sorting using mAb 23G2 and FITCMARK-1 to separate the CD45RBhi (TV) and CD45RBlo (TM) T cells. Both populations contain similar numbers of Vâ8 + cells, as indicated by flow cytometry [13]. An alternative procedure was used to isolate subpopulations of lymphocytes from older MRL-lpr/lpr mice. Following enrichment for CD4 + T cells by negative selection, the cells were stained with FITCGK1.5 and biotin-23G2 plus Cy5-Av. The cells were then separated by cell sorting and collecting the CD4 + CD45RBhi (TV) and CD4 + CD45RBlo (TM) cell fractions. Cell sorting was carried out using a FACSVantage; cells other than small lymphocytes were

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Results Previous studies have shown that in vivo administration of the superantigen, SEB, induces an expansion of reactive (largely Vâ8 + ) CD4 + T cells, followed by deletion and anergy [3]. Further analysis indicated that the mechanism of deletion of the stimulated cells was apoptosis [23]. Within the Vâ8 + population, not all of the cells respond similarly. We have previously reported that murine memory, but not virgin, T cells are unresponsive to SEB treatment as measured by proliferation, lymphokine secretion, and B cell helper function [13, 24]. Several distinct mechanisms may account for the failure of TM cells to proliferate when cultured with SEB, including SAg-induced anergy and apoptosis. Since a major mechanism of AICD is Fasmediated apoptosis, we wished to determine the extent of Fas involvement in the TM response to SEB. In the present report, we show that the failure of TM cells to be activated by SEB is independent of Fasmediated apoptosis.

Fas expression on CD4 cells from SEB-treated mice Previous studies have shown that Fas expression is upregulated on activated CD4 + T cells, including activation induced by SEB [25, 26]. As heightened Fas expression might be a prelude to apoptosis of TM cells, we measured the expression of Fas after injection of SEB into BALB/c mice. Different groups of mice were

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Unless otherwise indicated, TV or TM cells (1×105/ well) were cultured in 96-well U-bottom clusters (Falcon Labware, Oxnard, CA) with 1×105/well APCs in 0.2 ml RPMI-1640 medium supplemented with 10% FCS, 50 ìM 2-mercaptoethanol, 100 U/ml penicillin, 100 ìg/ml streptomycin, and 2 mM glutamine. Where indicated, SEB (10 ìg/ml), rIL-2 (20 U/ml), or human Fas-Fc chimeric protein (10 ìg/ml) [7] was added into the cultures. Titration experiments demonstrated that these were saturating concentrations. The cells were cultured in duplicate wells for 3 days followed by a 12 h pulse with [3H]TdR (1 ìCi/well). Cells were harvested using a 96-well automated harvester and radioactivity was measured using a BetaPlate (Wallac, Inc., Gaithersburg, MD). Unless otherwise indicated, the presented proliferation data represent average cpm from duplicate wells from a representative experiment of multiple (over four) experiments.

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Figure 1. SEB induces changes in CD4 + Vâ8 + T cells and in Vâ8 + TV and TM cells. BALB/c mice (two per group) were injected with SEB. At the indicated times, CD4 + cells were prepared from pooled splenocytes and were stained with mAbs reactive with CD4 (Cy-5), Vâ8 (FITC), and CD45RB (PE-Av). (A) Levels of Vâ8 + cells plotted as a percentage of total CD4 + cells. (B) After gating on the CD4 + Vâ8 + population, CD45RB expression was examined. The data are presented as the percentage of Vâ8 + TM (CD45TBlo) cells in total Vâ8 + cells.

injected with SEB spaced at 24 h intervals. Seven days after the initial injection, spleens were obtained and CD4 + T cells were prepared and examined by flow cytometry. Consistent with previous findings, SEB stimulated a marked expansion of Vâ8 + CD4 + cells with cell numbers peaking and declining over the first 4 days (Figure 1A). Within the Vâ8 + population, the ratio of TM:TV cells increased (Figure 1B). This is likely to reflect the differentiation of SEB-reactive TV cells into TM cells [13, 24]. Our analysis also showed that SEB caused an elevation in Fas expression on the Vâ8 + cells relative to the Vâ8 population ([Figure 2A and B). Within 48 h, most of the Vâ8 + cells (54%) expressed elevated Fas, which was increased approximately two-fold (Figure 3). The increase in Fas expression was rapid and transient. A similar experiment, measuring Fas levels after injections with SEB, spaced 12 h apart, showed that peak expression occurred 36 h after injection (data not shown). Hence, upregulated Fas levels occurred early in the Vâ8 expansion phase and declined as the cells were deleted. We next examined Fas expression on TV (CD45RBhi) and TM (CD45RBlo) cells. In non-injected mice, the levels of Fas were comparable on TV and TM cells (Figure 2C), regardless of Vâ8 expression. As increased Fas expression was limited to the Vâ8 + population, we examined gated Vâ8 + TV and TM cells. Injection with SEB caused an increase in Fas

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Figure 2. Fas expression increases on CD4 + Vâ8 + and in Vâ8 + TV and TM cells after SEB treatment. CD4 + cells were prepared from spleens of non-injected (A, C) BALB/c mice or mice injected 1 day previously (B, D) (two per group) with SEB. Cells were stained with mAbs reactive with Vâ8 (FITC), CD45RB (Red 670-Av), and Fas (PE). (A, B) Expression of Fas on non-injected CD4 + cells. (C, D) Expression of Fas on TV and TM cells after gating on Vâ8 + cells. Negative controls stained in the first decade of all parameters.

expression on both subpopulations, although the effect was more pronounced on TV cells (Figures 2D & 3). Hence, Fas is present on TM cells and is upregulated in response to SEB. However, there is neither accelerated nor enhanced upregulation relative to TV cells. Fas involvement in SEB-induced anergy We next wished to determine whether Fas plays a role in the failure of TM cells to proliferate in response to SEB, i.e. could SEB induce apoptosis of TM cells before the cells are able to proliferate? To test this possibility, we analysed whether purified TM cells would proliferate in response to SEB under conditions where Fas-mediated cell death could not occur. This was done in two ways: analysis of the response to SEB by TM cells from Fas-deficient mice and direct inhibition of Fas/Fas-L interactions. Fas-deficient lpr/lpr (MRL/lpr or C57BL/6/lpr) mice have been studied with respect to their response to SAgs. Treatment of adult lpr mice with SEB resulted in impaired deletion of CD4 + Vâ8 + T cells when compared with control mice [11, 25]. As in control mice, the remaining lpr T cells were anergic in response to restimulation with SEB [11]. Other reports, using cloned T cell lines generated from lpr mice, showed that a Fas deficiency led to resistance to SAg and anti-CD3-induced anergy and apoptosis [9, 10]. In the present study, we found that Fas-deficient MRL-lpr/lpr mice possess higher numbers of CD4 +

TM cells, as determined by CD45RB expression (50% TM cells, as compared to 20% TM cells in controls; Figure 4). In addition, there are lower levels of CD4 + T cell proliferation in response to in vitro culture with SEB, as compared with controls ([11] and Figure 5A). Because the decreased proliferation of CD4 + T cells after SEB priming (anergy) also correlated with increased numbers of TM cells [13], we examined TM responses directly. In these experiments, CD4 + T cells from MRL-lpr/lpr mice were separated into TV and TM cells by FACS sorting based upon CD45RB expression. The isolated cell populations were cultured in the presence of APCs from wild-type mice and were stimulated with SEB. As indicated, TM cells from non-SEB-treated lpr mice failed to proliferate in response to SEB stimulation (Figure 5B). As with our previous studies using BALB/c mice [13], the addition of IL-2 into the cultures restored proliferation (Figure 5B). It should be noted that in this experiment we used cells that were obtained from young (8-weekold) MRL-lpr/lpr mice. These mice were free of lymphoproliferative disease and contained few CD4 − CD8 − T cells. We have also examined TM cells from older (4-month-old) mice that were undergoing active lymphoproliferative disease. These mice contained a significant number of CD4 − CD8 − T cells, so we compared CD4 + TV and TM cells purified by FACS sorting on the basis of both CD4 and CD45RB expression. Consistent with the data obtained from young mice, TM cells failed to respond to SEB, whereas TV cells proliferated well (Figure 5C). Indeed, purified populations of TV cells proliferated markedly better

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Figure 3. SEB induces changes in Fas expression on CD4 + Vâ8 + TV and TM cells. BALB/c mice (two per group) were injected with SEB. At the indicated times, CD4 + cells were prepared from pooled splenocytes and were stained with mAbs reactive with Vâ8 (FITC), CD45RB (Red 670-Av), and Fas (PE). Changes in Fas expression indicated by (A) percentage of cells with increased levels of Fas (compared to non-injected mice) within the indicated population or (B) as mean fluorescence channels of the entire population of indicated cells. (m), Vâ8 + CD4 + cells; (e), Vâ8 + CD4 − cells; (●), gated Vâ8 + TV cells; (■), gated Vâ8 + TM cells.

in response to SEB stimulation than unfractionated T cells (containing single and double-positive cells) (Figure 5C). The previous experiments suggested that TM cell unresponsiveness to SEB was not due to Fasmediated apoptosis. To confirm these results and to extend them to a non-autoimmune model, we examined the involvement of Fas in the proliferative response of BALB/c TM cells to SEB. Recent studies have shown that anti-CD3-induced AICD could be blocked by interfering with the interaction between Fas and its ligand [7, 8]. In those studies, the addition of a soluble Fas-Fc chimeric protein prevented cell death and permitted T cell proliferation in response to immobilized anti-CD3 [7]. In our model, if TM cells were to undergo Fas-dependent cell death upon exposure to SEB, then the inhibition of Fas-mediated events should permit activation, leading to cell proliferation. Hence, purified TV and TM cells from BALB/c mice were cultured in the presence of SEB. As we have shown previously [13], TV cells proliferated vigorously, whereas TM cells responded poorly. When soluble Fas-Fc was added into the cultures, there was a reproducible increase in the level of proliferation of TV cells in response to SEB (Figure 6). In contrast, Fas-Fc did not promote a proliferative response to SEB by TM cells (Figure 6).

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Figure 4. MRL-lpr/lpr mice have increased numbers of TM cells. (A) CD4 + T cells from pooled 10-week-old MRLlpr/lpr mice were examined for CD45RB expression by flow cytometry. (B) Typical CD45RB expression staining pattern on BALB/c splenic CD4 + cells. In multiple experiments, TM cells comprised >50% of CD4 + splenic T cells in MRL mice, compared to <30% in BALB/c mice.

Apoptosis of Vâ8 + CD4 cells from SEB-treated mice Although the previous results suggested that the failure of TM cells to proliferate in response to SEB was independent of Fas, other mechanisms of AICD could be involved. Hence, we wished to measure directly apoptosis in TM cells after interaction with SEB. In these experiments, BALB/c mice were injected with SEB and at different times, CD4 + T cells were prepared and analysed by flow cytometry. Apoptosis was determined by alterations in plasma membrane phospholipids, as assessed by increased labeling with MC-540. Among the points to emerge from this analysis is that resting TM cells, regardless of their Vâ phenotype, bind slightly higher amounts of MC-540 (mean fluorescence of 24.8 vs. 17.5 on TV cells (Figure 7; data shown for Vâ8 + cells only). We also note that activated cells have an increased MC-540 fluorescence relative to resting cells (activated TM: 43.6; activated TV: 47.3) (Figure 7). Apoptotic cells were easily visualized as a discrete population with a mean fluorescence >650 after exposure to SEB (Figure 7). In the unfractionated Vâ8 + population, apoptotic cells were visualized within 1 day of priming (16–30%) and in three out of four experiments the percentage of apoptotic cells

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Figure 6. Inhibition of Fas/Fas-ligand interactions does not permit BALB/c CD4 + TM cells to proliferate in response to SEB. CD4 + TV and TM cells (1×105/well) from BALB/c mice were cultured with exogenous APCs (1×105/well) for 3 days. Where indicated, SEB (10 ìg/ml) or human Fas-Fc chimeric protein (10 ìg/ml) was added into the cultures. Proliferation analyses of TV ([) and TM (■) cells.

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Figure 5. CD4 + TM cells from MRL-lpr/lpr mice fail to proliferate in response to SEB. T cells (1×105/well) were cultured with exogenous mitomycin C-treated APCs (1×105/well). After 3 days the wells were pulsed with [3H]-TdR and harvested 12 h later. Where indicated, SEB (10 ìg/ml) or human rIL-2 (20 U/ml) was added into the cultures. (A) Proliferation analyses of unfractionated CD4 + T cells from BALB/c ( ) vs. MRL-lpr/lpr ( ) mice; (B) proliferation analyses of purified TV ( ) vs. TM ( ) cells from 8-week-old MRL-lpr/lpr mice. These mice were disease-free and contained few CD4 − CD8 − T cells, hence purified CD4 + T cells were sorted into TV and TM subpopulations; (C) proliferation analyses of T cells from 4-monthold MRL-lpr/lpr mice—these mice were undergoing lymphoproliferative disease and contained significant levels of CD4 − CD8 − T cells, hence cells were sorted based upon both CD4 and CD45RB expression to remove the double-negative cells. Data from purified TM ( ) and TM ( ) cells vs. unfractionated (B cell and CD8-depleted) splenocytes ( ) are shown. In all panels, in the absence of T cells, <1000 cpm/well were incorporated.

remained constant through the 5–7 days postinjection (data not shown). In the fourth experiment there was a gradual increase from day 1 (16.7%) to day 5 (21.6%) (data not shown). In all experiments, there was a higher baseline of apoptotic TM cells in the non-immune population (Figure 7). After injection with SEB, both the Vâ8 + TV and TM subpopulations contained apoptotic cells. The percentages of these cells were similar in both populations (after correcting for the initial difference) in the first 2 days. Beyond this time point, the TM cell fraction

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Figure 7. SEB induces apoptosis in BALB/c TV and TM cells. BALB/c mice were injected with SEB and enriched populations of CD4 + cells were isolated on the indicated days. The cells were stained to measure the expression of Vâ8 (FITC) and CD45RB (Red 670-Av). The cells were then labeled with MC-540, as described in Materials and Methods. Shown are the amounts of MC-540 incorporated on cells after gating on both Vâ8 and CD45RB expression.

contained cells which were originally TV cells but which had down-regulated CD45RB expression. We would conclude that although some AICD does occur, most of the Vâ8 + cells are not apoptotic.

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Further, deletion cannot account for the difference in SEB-mediated proliferation between TM and TV cells, as SEB does not preferentially induce TV cells to undergo apoptosis.

Discussion Both T cell deletion and anergy are important mechanisms for maintaining central and peripheral tolerance. In the periphery, accumulated evidence suggests that different subsets of mature T cells have distinct regulatory requirements [12, 27, 28] and it is reasonable to assume that conditions which induce activation of one subset might induce tolerance in another. Indeed, our own data show that SAgs stimulate only TV cells, while TM cells are not activated [13, 24]. Hence, previous studies of SEB-induced tolerance, which have documented the in vivo expansion, deletion, and anergy of Vâ8 + CD4 + T cells, have been influenced by the differentiation into a subset (TM cells) that is naturally refractory. In order to relate the TM cells’ hyporesponsiveness to described mechanisms of tolerance, we have extended our previous studies to demonstrate that the failure of TM cells to respond to SAgs is not due to elevated Fas expression or increased susceptibility to Fas-mediated apoptosis. Further, our data indicate that Fas-independent elimination of SAg-reactive TM cells is only a minor component of tolerance to SEB and cannot account for the lack of proliferation or LK secretion. As previous studies of human peripheral blood CD4 + T cells had shown that Fas was preferentially expressed on the CD45RO (TM) subset [14], the simplest explanation for the lack of response to SEB was that AICD occurred before proliferation and LK secretion were induced. However, unlike in humans, murine CD4 + cells (which are largely comprised of TV cells) constitutively express Fas [25, 26]. Still, in the light of the previous studies it was somewhat surprising that TM cells did not express higher levels, as compared to TV cells (Figure 2). This may relate to the activation states of the human and murine TM cells. Fas expression is increased upon activation and it is worth noting that human, but not murine, TM cells also express the activation Ag, CD25 [29, 30]. It may be that circulating TM cells have been recently activated whereas splenic TM cells are closer to resting. However, our data are in agreement with previous results that suggested that splenic TM cells are not completely in a resting state but are in a slightly higher activation state than are TV cells [31]. In our study, the level of MC-540 incorporation was increased in TM cells relative to TV cells, and further increased upon stimulation (Figure 7). We have previously reported that TM cells are not completely non-responsive to SEB. Although deficient in major functions, such as the capacity to proliferate, secrete LKs, and help B cells, TM cells respond to SEB by expressing CD25, CD40L, and CD28 ([24] and unpubl. obs.). In the present study, we have observed that Fas is also induced by SEB exposure. This pro-

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vides further evidence that TM cells are not ignorant of SEB. However, Fas induction does not account for reduced proliferation, as there was no faster induction than was seen on TV cells. Indeed, Fas expression was elevated on a higher number of TV cells throughout the entire examination period. This does not preclude a role for Fas in SEB-induced tolerance. In studies with lpr mice in vivo, SEB-induced cell deletion still occurs due to other mechanisms, such as via a TNF-mediated pathway [32]. However, deletion is slowed, which suggests that Fas does play a prominent role in the response to SEB. In our study, we have not attempted to identify elements which might confer greater sensitivity to a Fas-mediated signal, such as elevated expression of Fas-L. At present, we believe that, regardless of the levels of Fas-L, if Fas/Fas-L played a role in the TM response to SEB, proliferation would have occurred when the inhibitory Fas-Fc fusion protein was added into the cultures (Figure 6). With regard to the latter, our data do point to a role for Fas in the response to SEB by TV cells. As we observed increased in vitro TV cell proliferation in the presence of soluble Fas-Fc protein, we would suggest that Fas contributes to in vivo TV cell deletion after exhaustive proliferation. This would correlate well with the slowed kinetics of CD4 + cell deletion in lpr mice. Based upon the data described in this report, we suggest that SEB fails to induce Fas-mediated AICD in TM cells. Although the Fas/Fas-L pathway is one of several mechanisms of AICD, we have no evidence that increased apoptosis in the TM population causes decreased proliferation in response to SEB. Using the loss of membrane phospholipid asymmetry [20, 21] determined by MC-540 staining, to indicate early apoptotic cells, our data suggest that TM cells may have a higher propensity for apoptosis (higher levels of apoptotic cells in non-treated TM cells as compared to TV cells). However, at any point after exposure to SEB the majority of Vâ8 + TM cells are non-apoptotic. Further, there is no greater increase in apoptosis relative to TV cells after exposure to SEB (Figure 7), even though only TV cells proliferate in response to the SAg. In additional experiments, we determined apoptosis by examination of cells with reduced forward light scatter [33]. When measuring this parameter, we did not observe accumulation of apoptotic cells until 3–4 days after SEB injection, reflecting that membrane alterations are early events relative to DNA degradation. Still, when measuring forward scatter the percentages of apoptotic TM cells were not increased relative to TV cells (data not shown). This is somewhat surprising as, by 2–3 days, activated TV cells might be expected to have downregulated CD45RB levels and resemble TM cells (WTL, unpubl. obs.). This suggests that many of the SEB-stimulated TV cells which undergo apoptosis die before or soon after they acquire a CD45RBlo phenotype. Hence, at all time points, most of the apoptotic cells have the phenotype of TV cells. As we have previously observed that TM cells do not proliferate when exposed to SEB, the results of this study fit well with experiments described by Renno et al. [34], which showed that only cells that had

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proliferated in response to SEB underwent apoptosis. In a different study, Hayden et al. [35] examined cells that had previously proliferated and incorporated BrdU in response to SAg (Mlsa). They found that a significant proportion of the cells had a memory phenotype. Taking these studies together, our results are consistent with a model where SEB induces anergy in TM cells while promoting activation and expansion of TV cells followed by either their deletion or their differentiation into non-reactive TM cells. Further, we would suggest that deletion occurs before or very soon after the acquisition of a TM cell phenotype. We would conclude that the elevated levels of TM cells that we have observed after SEB priming reflect reduction of TV cells due to deletion, differentiation of activated TV cells, and maintenance of the pre-existing TM population. Hence, it is the accumulation of nonresponsive TM cells which describes anergy in the bulk CD4 + Vâ8 + population. As in previous studies, we have used SEB-induced tolerance as a model for self-tolerance. In this context, we might speculate that the failure to delete TM cells after inappropriate stimulation allows for future recovery and maintenance of antigen-specific memory responses. Similarly, after an encounter with SAgproducing pathogens, the deleterious consequences of unregulated broad LK production would be prevented while sustaining memory. As TV cells are limited in the LKs which they secrete, the consequences of polyclonal activation are not as grave. From the perspective of the pathogen, specific tolerance of TM cells might facilitate survival as protective LKs would not be produced. Although the beneficial effects of unregulated cell TV expansion are not known, perhaps SAgs stimulate large numbers of cells which are not pathogen-specific clones.

Acknowledgements We thank Drs T. Brunner and D. Green for their kind gift of the Fas-Fc fusion protein. We also thank Dr C. F. Ware (University of California, Riverside), whose group made the Fas-Fc construct, and Mr R. Dilwith and the Wadsworth Center Flow Cytometry Core Facility for assistance in sorting on the FACS-Vantage. We gratefully acknowledge J. Mittler for his assistance in developing the MC-540 staining analyses. We acknowledge Drs G. Winslow and D. Murphy for helpful discussions during the course of this work and D. Murphy for critical review of this manuscript. This study was supported by the National Institutes of Health grant AI-35583.

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