APO-1) Signaling in Mouse Thymocyte Death

CELLULAR IMMUNOLOGY ARTICLE NO.

169, 99–106 (1996)

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Synergy between T Cell Receptor and Fas (CD95/APO-1) Signaling in Mouse Thymocyte Death GALEN H. FISHER, MICHAEL J. LENARDO,

AND

JUAN CARLOS ZU´N˜IGA-PFLU¨CKER*

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892; and *Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada Received August 23, 1995; accepted December 23, 1995

signals through other receptors, such as CD28, are required for efficient induction of thymocyte apoptosis (12–14). Fas is expressed on a variety of tissues including all a/b-TCR-bearing thymocytes (15, 16). Fas/Fas ligand interactions play an important role in peripheral lymphocyte homeostasis by inducing apoptotic cell death (17–19). However, the role of Fas in the thymus is still unresolved. Studies of TCR transgenes in lpr/lpr mice, which are genetically deficient in Fas, demonstrate that Fas is not essential for antigen-induced negative selection (20, 21). However, the TCR repertoire in thymuses of MRL lpr/lpr mice shows an increased percentage of potentially autoreactive Vb6 and Vb8.1 receptors compared to control MRL /// mice (22). Recent data show that in vivo administration of agonistic antiFas mAb induces mouse thymocyte apoptosis, thus suggesting a potential role for Fas in the thymus (15). In vitro, however, anti-Fas alone failed to induce high levels of thymocyte apoptosis unless cycloheximide was coadministered (15). This suggests that Fas requires additional signals in order to cause thymocyte death. In this paper, we demonstrate that signals delivered by Fas and TCR/CD3e act synergistically to induce thymocyte apoptosis in vitro. Our data indicate that the TCR/CD3e and Fas signals must occur simultaneously and that this synergy does not act through Fas upregulation. We therefore suggest that Fas is capable of inducing thymocyte death only if the TCR is coengaged and discuss the relevance of this observation to models of thymic negative selection.

Administration of anti-TCR/CD3e antibody in vivo or in thymic organ culture results in the apoptotic death of CD4//CD8/ thymocytes. In contrast, purified thymocytes in suspension culture are resistant to TCR/CD3einduced apoptotic death. We show that induction of thymocyte death, in suspension culture, can be induced by the combination of TCR/CD3e and Fas (CD95/ Apo-1) signaling. No significant thymocyte death was observed after in vitro Fas cross-linking unless TCR/ CD3e was simultaneously co-cross-linked or metabolic inhibitors such as actinomycin D were added. Furthermore, TCR/CD3e and Fas synergy did not operate through upregulation of Fas but by facilitation of the Fas-mediated death signal. Both TCRmid/lo/HSAhi/CD4// CD8/ (double positive) and TCRhi/HSAlo/CD4//CD80 or CD40/CD8/ (single positive) thymocytes were susceptible to death induced by co-cross-linking of TCR/CD3e and Fas. Our results reveal a signaling synergy between the Fas and TCR/CD3e complex that has important implications for our understanding of in vivo vs in vitro models of thymocyte deletion. q 1996 Academic Press, Inc.

INTRODUCTION Maintenance of self-tolerance is largely mediated by the process of negative selection during thymocyte development (1–3). From studies of mice bearing transgenic TCR, it is apparent that antigen specificity of the TCR determines the fate of a developing thymocyte (4). This process has been modeled by the in vivo administration of agonistic antibodies directed against TCR/CD3e, which results in the deletion of CD4//CD8/ immature thymocytes (5–7). Negative selection of thymocytes by antigen (8–10) or TCR/CD3e stimulation occurs by a process of programmed cell death or apoptosis (11). Recently several authors have provided data which suggest that in addition to TCR stimulus,

MATERIALS AND METHODS Mice. Female B10.A, MRL/MPJ ///, and MRL/ MPJ lpr/lpr were obtained from The Jackson Laboratory (Bar Harbor, ME). Preparation of thymocytes for in vitro deletion assays. Freshly prepared adult thymus single-cell suspensions 99

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0008-8749/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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were filtered twice through 70-mm nylon mesh cell strainers (Falcon, Franklin Lakes, NJ), in 10 ml media (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 50 mM b-mercaptoethanol, penicillin, and streptomycin (Biofluids, Rockville, MD)). After one wash the thymocytes were gently resuspended in 10 ml of media and gently refiltered through nylon cell strainers. Refiltration is essential to avoid deleting elements found in the thymic stromal matrix. If this step is omitted the administration of either mAb anti-Fas or anti-TCR/ CD3e causes significant deletion. After preparation cell viability was ú99% by trypan blue assay. Of the recovered cells, 7–11% were CD4//CD80 TCRhi, 1.5–4% were CD40/CD8/ TCRhi, and 85–90% were CD8//CD4/ TCRmid/lo. Typical cell yield was 90–120 1 106 cells/ thymus. In vitro thymocyte deletion assays with immobilized mAb. Antibodies used for in vitro stimulation assays were anti-Fas (Jo2, hamster IgG; Pharmingen, San Diego, CA), without azide, low endotoxin. The antimouse CD3e mAb 145-2C11 was isolated from ascites using protein A column chromatography by standard procedures (6). Anti-H-2Dd, clone 34.5.8 (23), in PBS was a gift of Dr. David Margulies (NIAID). Ninetysix-well flat-bottom polystyrene plates (Falcon) were coated with mAb as previously described (24). For sequential mAb treatments, thymocytes were harvested from the first treatment after 6 hr (10 mg/ml anti-CD3e, 10 mg/ml anti-Fas, or no antibody), using a wide-bore 200-ml pipet tip. The pretreated cells were immediately transferred to the treatment well (10 mg/ml anti-Fas, 10 mg/ml anti-CD3e, 10 mg/ml anti-Fas / 10 mg/ml antiCD3e, or no mAb), centrifuged for 3 min, and incubated at 377C with 5% CO2 until harvesting 18 hr later. Actinomycin D (Sigma, St. Louis, MO) was resuspended in 100% ethanol at a stock concentration of 5000 mg/ml and used at final concentrations of 100 and 500 ng/ml. Staining for flow cytometry. Except for the staining of surface Fas and CD3e, all staining was done using primary-conjugated antibodies (Pharmingen) as described previously (25). To stain either Fas or CD3e, the thymocytes were harvested, washed in FACS buffer (11 PBS, pH 7.4 (without Mg 2/ and Ca2/ (Biofluids), with 1% BSA and 0.1% sodium azide (Sigma)), and incubated 20 min with unconjugated goat anti-hamster antibodies at 50 mg/ml in FACS buffer (to block any residual immobilized mAb). Thymocytes were washed, incubated 20 min with 25 mg/ml of either Jo2 or 2C11, in FACS buffer, washed again, incubated with FITCconjugated polyclonal goat anti-hamster antibodies, washed twice, and analyzed by flow cytometry on a FACScan instrument (Beckton–Dickinson, Mountain View, CA). Control staining showed that the preincubation with unconjugated goat anti-hamster could block subsequent FITC-conjugated goat anti-hamster bind-

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ing to both 2C11 and anti-Fas, but did not interfere with subsequent binding of 2C11 and anti-Fas. Preparation of HSAlo 1 TCRhi thymocytes. Thymocytes were filtered and washed as above and then resuspended on ice for 15 min at 1 1 107/ml in the culture supernatant of clone J11d.2, anti-mouse HSA (26). Then, rabbit complement was added (Cedar Lane, Ontario, Canada) and the cells were incubated for 45 min at 377C. Viable cells were then isolated using Lympholyte M density centrifugation (Cedar Lane), washed twice in medium, counted, and resuspended at 50,000 cells/ml. A sample was stained for CD4/CD8 and TCR before and after HSA-mediated killing to confirm specific cytotoxicity (Fig. 5a). Multiparameter flow cytometric analysis and quantitation of viable cells. Quantitation of viable cells was performed as detailed previously (27). Briefly, cells were harvested, after culture, using a wide-bore 200ml pipet tip with vigorous pipetting to dislodge and resuspend the thymocytes. Each well was subsequently washed with 200 ml of PBS. Six wells were pooled per sample. Cells were pelleted and resuspended in 400 ml of FACS buffer with propidium iodide (PI) (30 mg/ml final concentration). Samples of equal volume were collected using constant flow for 2 min on a FACScan instrument. All events were collected without live gating. Postacquisition, forward scatter profile (FSC), and PI exclusion were used to determine the absolute number of live cells collected in 2 min from each sample. RESULTS Co-cross-linking of Fas (CD95/Apo-1) and TCR/ CD3e causes a synergistic decrease in the recovery of viable mouse thymocytes. To investigate the role of TCR stimulation in mouse thymocyte deletion, we used an in vitro assay system in which disaggregated thymocytes were incubated at a low density, 104 cells per well, on plastic dishes coated with immobilized antibodies. Quantitation of viable cells recovered after stimulation was carried out using multiparameter flow cytometry analysis (27, 28). In repeated experiments, we found that stimulation of thymocytes with up to 10 mg/ml plate-bound anti-CD3e (mAb 145-2C11) caused only slight (10–20%) thymocyte loss compared to control antibody or media control cultures (Fig. 1). These data agree with previous in vitro tests of anti-CD3e stimulation on thymocytes (12, 13, 28), but present a paradox because anti-TCR/CD3e mAb has strong mitogenic and apoptosis-inducing effects on mature T cells and po1 Abbreviations used: HSA, heat-stable antigen; PI, propidium iodide; FSC, forward light scatter; RT-PCR, reverse transcriptase polymerase chain reaction; DP, double positive; SP, single positive; fu, fluorescence units.

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sults indicate that the combination of anti-CD3e and anti-Fas induces programmed thymocyte death, which could not be achieved by treatment with either antibody alone. Furthermore, to demonstrate that the synergy between anti-Fas and anti-CD3e was specific, we used a control anti-H-2Dd antibody that binds to thymocytes of B10.A mice (29). Co-cross-linking of H-2Dd with either Fas or CD3e failed to augment cell loss (data not shown). This suggests that the synergy for death induction requires specific ligation of the Fas and TCR/CD3e molecules.

FIG. 1. Loss of viable mouse thymocytes after in vitro crosslinking of CD3e and Fas. Shown is loss of the viable cells in 5 independent experiments in which thymocytes from 2- to 3-month-old B10.A female mice were incubated for 24 hr with plate-bound anti-CD3e, anti-Fas, or both antibodies together. All mAb were plated at 10 mg/ ml final concentration. Viable cell counts were determined by flow cytometry as described previously (27). The percentage loss in viable cells is calculated as [1 0 ((number of PI-negative viable cells recovered from wells with plate-bound antibodies) 4 (number recovered from control wells without plate-bound antibodies))] 1 100. The data are representative of 10 independent experiments.

tently deletes immature CD4//CD8/ thymocytes when administered in vivo or in fetal thymic organ culture (5–7). To understand the lack of deletion seen in thymocyte single-cell suspensions in vitro by anti-CD3e, we investigated the role of Fas (CD95/APO-1) in thymocyte apoptosis. Fas, which is highly expressed on developing a/b-TCR/ thymocytes, is a transmembrane receptor that can induce apoptosis of activated peripheral T cells when engaged by Fas ligand (16, 17). Furthermore, a recent report demonstrates that anti-Fas administered in vivo induces thymocyte apoptosis (15). Surprisingly, in vitro treatment of immature CD4// CD8/ thymocytes with agonistic anti-Fas mAb failed to induce significant apoptosis (Fig. 1). In contrast, in vitro co-cross-linking with both anti-CD3e and anti-Fas resulted in an up to 60–70% decrease of viable thymocytes (Fig. 1). Combinations of these mAbs at lower concentrations or incubations for shorter time periods yielded less than the maximal cell loss shown here (data not shown). To ensure that thymocyte loss reflected the induction of cell death, we carried out two analyses. Trypan blue dye exclusion assay and two-parameter flow cytometry for cell size (FSC) and PI incorporation analysis showed that treatment with anti-CD3e and anti-Fas together decreased the number of large viable trypan blue- or PI-excluding cells and increased the number of dead, trypan blue-including, small apoptotic bodies that stain strongly with PI (data not shown). Together, these re-

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Simultaneous cross-linking of TCR/CD3e and Fas is required for the induction of thymocyte death. To determine whether the signals from TCR/CD3e and Fas could be given sequentially, we pretreated thymocytes with each individual immobilized mAb for 6 hr, followed immediately by 18 hr of treatment. We chose 6 hr of pretreatment based on previous studies showing that 6 hr of anti-CD3e was sufficient to commit T cell hybridomas to apoptosis (G.H.F. and M.J.L., unpublished results). Our results show that mAb pretreatment does not prime for deletion by either anti-Fas or anti-CD3e and does not alter the effect of co-cross-linking during the final 18 hr (Fig. 2). Thus, the signals from Fas or CD3e are incapable of mediating deletion unless given simultaneously. Cellular effects of TCR/CD3e and Fas cross-linking. Surface staining of thymocytes showed that immobilized anti-Fas alone did not affect the surface expression of CD4 and CD8 (Fig. 3). However, immobilized anti-CD3e alone, and to a greater extent when combined with anti-Fas, caused a shift in the frequency of

FIG. 2. Simultaneous co-cross-linking of CD3e and Fas is required for synergistic deletion. Thymocytes were pretreated with the first plate-bound antibody for 6 hr and subsequently treated for 18 hr with the indicated plate-bound antibody(ies). All mAb were plated at 10 mg/ml final concentration. Viable cell counts were determined by flow cytometry as in Fig. 1. Percentage cell loss equals the number of cells recovered from wells with plate-bound antibodies for the 18hr period divided by control cells which had the same 6-hr pretreatment but no plate-bound antibody for the 18-hr period. Data are the averages of two independent experiments, each done in duplicate.

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PCR on total RNA isolated from in vitro cultured thymocytes. RT-PCR analysis showed that anti-CD3e failed to alter the expression level of Fas mRNA (data not shown). Anti-CD3e-stimulated thymocytes also showed a 75% decrease in TCRhi expression (Fig. 4d). To confirm that this apparent downregulation of the TCR/CD3e complex was not due to blockade of staining by antibodies removed from the plate, we used secondary staining with unlabeled 145-2C11 (anti-CD3e) followed by FITC-conjugated goat anti-hamster antibodies. Secondary antibody staining confirmed the 75% decrease in TCRhi thymocytes (data not shown).

FIG. 3. Effect of plate-bound anti-CD3e and anti-Fas on the surface expression of CD4 and CD8. Gate shows the CD4dull/CD8dull thymocytes as previously reported (14, 30). Cells were live gated (FSC vs PI). The percentage of cells within the gate, CD4dull/CD8dull, is shown.

CD4dull/CD8dull thymocytes (Fig. 3). These ‘‘double-dull’’ thymocytes have been previously reported to be in the process of negative selection (14, 30, 31). To confirm that thymocytes exposed to immobilized anti-CD3e underwent an ‘‘activation’’ response, we analyzed the surface expression of the thymocyte activation markers CD69 and CD5 (32–35). After 24 hr on immobilized anti-CD3e alone, thymocytes significantly downregulated TCR expression and upregulated both CD69 and CD5 (Figs. 4a, 4b, and 4d). Neither anti-Fas nor antiCD3e alone dramatically changed Fas surface expression but co-cross-linking of TCR/CD3e and Fas enriched for Fas dull thymocytes (Fig. 4c). Thus, the synergy observed between anti-Fas and anti-CD3e could not be explained by anti-CD3e-induced Fas upregulation. To further assess the possibility that anti-CD3e induced Fas upregulation, we performed semiquantitative RT-

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The effect of anti-CD3e and anti-Fas on single positive thymocytes. As seen in Fig. 4d, anti-CD3e treatment decreased the proportion of TCRhi thymocytes. Because a 10–20% cell loss (approximate percentage of TCRhi thymocytes) was consistently seen when thymocytes were stimulated with anti-CD3e alone (Fig. 1), we hypothesized that the TCRhi cells may be directly deleted by immobilized anti-CD3e. To study the TCRhi thymocytes, we used anti-HSA plus complement to delete HSAhi TCRmid/lo immature thymocytes (26). After HSA depletion, CD4//CD8/ (double positive, DP) thymocytes comprised 1.1% of the recovered population, CD4//CD80 and CD40/CD8/ (single positive, SP) thymocytes comprised 87.8%, and the mean level of TCR surface expression was substantially increased (Fig. 5a). Contrary to our hypothesis, anti-CD3e alone did not delete of HSAlo TCRhi thymocytes. However, as was seen with total thymic suspensions, co-cross-linking of CD3e and Fas caused a 50–60% deletion of HSAlo TCRhi thymocytes (Fig. 5b). To confirm that anti-CD3e induces downmodulation, and not death, of TCR in TCRhi thymocytes, we stained TCR/CD3e using secondary staining with unlabeled 2C11 (anti-CD3e) followed by FITC-conjugated goat anti-hamster cocktail. Our data demonstrate that immobilized anti-CD3e causes a marked downregulation of TCR/CD3e surface expression (Fig. 5c). Taken together, our findings indicate that both DP and SP are susceptible to cell death mediated by co-cross-linking of Fas and TCR/CD3e, whereas anti-CD3e alone suffices to cause TCR downmodulation but not significant cell death. Actinomycin D potentiates thymocyte death by antiFas alone. It has been previously reported that actinomycin D potentiates Fas-mediated cell death in mouse fibroblasts (36). We therefore tested whether actinomycin D also facilitated Fas-induced death in thymocytes. Concentrations of actinomycin D, which block [3H]uridine incorporation and have minimal toxicity, strongly promoted deletion by anti-Fas, in the presence or absence of anti-CD3e (Fig. 6). Actinomycin D was unable to enhance deletion by immobilized antiCD3e alone, although it increased the deletion seen

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FIG. 4. Effects of plate-bound antibody treatment on the expression of TCR, CD95 (Fas/Apo-1), CD5, and CD69. (a, b) Histogram overlays of surface staining with PE-conjugated CD5 and FITC-conjugated CD69, respectively, after incubation for 24 hr with and without plate-bound antibodies. Mean, median, and peak channel are given in fluorescence units (fu). (c) Histogram overlay plots comparing Fas surface staining intensity of thymocytes incubated 24 hr with and without plate-bound antibodies. For staining of Fas surface expression, cells were blocked with unconjugated, polyclonal, goat anti-hamster (to bind residual 2C11), washed and incubated with unconjugated antiFas, and washed and subsequently incubated with FITC-conjugated goat anti-hamster Ig. (d) Histogram overlay of thymocytes, stained with FITC-conjugated anti-mouse TCR, after 24 hr in vitro incubation with and without plate-bound antibodies as indicated; all mAb were plated at 10 mg/ml final concentration. The region R1 shows the percentage TCRhi thymocytes.

with co-cross-linking of Fas and CD3e. Cycloheximide also potentiated Fas-mediated deletion with and without anti-CD3e (data not shown) (15).

MPJ lpr/lpr mice abrogated thymocyte deletion induced by coimmobilized anti-CD3e and anti-Fas (Fig. 7).

MRL lpr/lpr mice are not susceptible to deletion induced by co-cross-linking of CD3e and Fas. Because MRL lpr/lpr mice have a deficit in Fas surface expression (17), we tested the ability of anti-CD3e and anti-Fas to cause thymocyte deletion in MRL lpr/lpr and control MRL /// mice. We found that the lpr mutation in MRL

DISCUSSION

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Previous work has shown that treatment with agonistic anti-CD3e causes significant deletion of DP thymocytes only in the context of an intact thymus and not when thymocytes are disaggregated in culture (5–

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FIG. 5. Co-cross-linking of Fas and CD3e induces synergistic deletion of HSAlo, single-positive thymocytes. (a) Complement plus antiHSA enriches the single-positive TCRhi thymocytes. Quadrant statistics are shown for the CD4 by CD8 profile. Histogram overlay shows the level of TCR surface expression in the thymocyte population before and after HSA / complement-mediated cytotoxicity. (b) Treatment of HSAlo single-positive thymocytes with anti-CD3e and anti-Fas. The experimental setup and viable cell quantitation were carried out as in Fig. 1. (c) Histogram overlays of TCR staining; staining was performed using unlabeled anti-CD3e followed by a secondary FITCconjugated goat anti-hamster.

7, 12, 13). These observations imply that in vitro stimulation of thymocytes with anti-TCR/CD3e requires additional signals to induce apoptosis. Furthermore, recent work has demonstrated that administration of an agonistic anti-Fas mAb induces thymocyte apoptosis in the context of a whole thymus but not in disaggregated thymocyte cultures (15). It was reported that, in suspension culture, the addition of a metabolic inhibitor (cycloheximide) was required for Fas-mediated deletion of thymocytes (15). Thus, either anti-TCR/CD3e or antiFas alone can mediate thymocyte deletion in vivo, yet neither is able to mediate significant deletion in disaggregated thymocyte cultures. Our findings show that in vitro coengagement of Fas and CD3e causes substantial deletion of mouse thymocytes. The in vitro cooperative effect of Fas and TCR stimulation on thymocyte death suggests that Fas may participate in TCR-induced negative selection. To date,

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evidence exists for and against this possibility (37, 38). Studies with human thymocytes show that Fas is required for superantigen-mediated deletion (37). Moreover, thymuses of MRL lpr/lpr mice have increased percentages of potentially autoreactive Vb6 and Vb8.1 thymocytes relative to control MRL /// mice (22). In contrast, recent experiments using TCR transgenic mice with lpr mutations show that Fas does not appear to play an obligatory role in thymic negative selection (20, 21). Furthermore, we were unable to demonstrate an effect of Fas:fc on superantigen deletion in mouse fetal thymic organ culture (G.H.F., unpublished observations). Thus, Fas has the potential to participate in thymocyte selection, but Fas does not appear to be an obligate participant. Processes, potentially involving other members of the TNF/NGF family, may be more prominent mediators of thymocyte negative selection. Because Fas is highly expressed on all a/b-TCR/ thy-

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mocytes (16), its ability to induce apoptosis is likely to be tightly regulated. Regulation of Fas-mediated apoptosis could be achieved by inhibiting the Fas signal transduction pathway, limiting the thymic localization of Fas ligand (FasL), or both. Our observations that either TCR stimulation or metabolic inhibitors (actinomycin D and cycloheximide) strongly potentiate Fasmediated deletion of thymocytes suggest that this process is constitutively inhibited in mouse thymocytes. Consistent with this idea is the recent characterization of a putative inhibitory domain in the intracellular portion of the Fas molecule (39) and the identification of a Fas-associated protein tyrosine phosphatase (FAP-1) that inhibits Fas signal transduction (40). The localization of FasL, in the thymus, could also regulate Fas-mediated apoptosis. Using RT-PCR, we detected low levels of FasL mRNA in total thymus, thymic stroma preparations, and CD4/ TCRhi thymocyte mRNA (data not shown). However, we were unable to find FasL RNA expression in sorted DP and DN thymocytes (data not shown). The absence of FasL mRNA in DP, but its presence in total thymus and thymic stromal elements raises the interesting notion that cells constituting the thymic matrix could mediate negative selection by the presentation of a TCR stimulus (MHC/antigen) together with molecular ligands that mediate apoptosis. Our finding that the TCR/CD3e complex and Fas must be stimulated simultaneously to achieve death lends support to this idea. This simultaneous activation by TCR/CD3e and Fas not only may serve to remove the constitutive Fas signaling inhibition but may also activate the recently identified molecules (RIP, FADD/MORT-1, and TRADD) that interact with the Fas death domain resulting in thymocyte apoptosis (41–44). The parsimonious FasL mRNA expression in the thy-

FIG. 6. Actinomycin D potentiates cell loss induced by anti-Fas. All mAb were plated at 10 mg/ml final concentration. Viable cell counts were determined by flow cytometry as in Fig. 1. The percentage loss in viable cells was calculated as [1 0 ((number of viable cells in treated sample) 4 (number of control thymocytes that had been incubated without actinomycin D and without plate-bound antibodies))] 1 100. Data are the averages of two independent experiments.

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FIG. 7. MRL/MPJ lpr/lpr mice are not susceptible to deletion by plate-bound antibodies against CD3e and Fas. The experimental setup and quantitation of viable cell were carried out as in Fig. 1. Data are the averages of two independent experiments.

mus, together with the requirement for accessory stimulation (TCR/CD3e) raises the question as to what proportion of thymocytes could be deleted by Fas and TCR costimulation. Previous work has suggested that most DP thymocytes never receive strong TCR stimulation and die due to neglect (45). In the absence of stimulation, we observed a 20–30% spontaneous death of thymocytes after 24 hr in vitro culture (28, 31). Our data suggest that Fas-mediated deletion would not contribute to thymocyte death by neglect. Thus, only a small population of DP thymocytes, and SP thymocytes, could be the target of Fas- and TCR-stimulated death. Which processes of thymocyte development might be mediated by simultaneous costimulation by Fas and TCR? To answer this question it is necessary to elucidate which subsets of thymocytes are most sensitive to apoptosis, induced by in vivo anti-Fas administration. Recent work by Ogasawara et al. demonstrated that anti-Fas antibody could mediate thymocyte deletion in vivo, but the analysis did not provide information about which thymocyte subsets were affected (15). In vitro cultures with intact thymic organs treated with anti-Fas antibody alone showed a fourfold reduction in DP thymocytes, while a similar treatment of thymuses derived from MHC class I- and class II-deficient mice showed only a slight reduction in DP thymocytes (G.H.F., unpublished observations). These preliminary observations support the notion that thymocyte death resulting from in vivo engagement of Fas may require costimulation with TCR/CD3e that is actively engaged with self-MHC. Our studies show that coengagement of Fas and the TCR/CD3e complex is capable of causing the death of HSAlo TCRhi SP thymocytes despite the fact that these cells have lower Fas surface expression compared to DP thymocytes (16). This finding is particularly interesting in light of the observation, by Ogasawara et al., that cycloheximide could allow in vitro Fas-mediated

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deletion of DP but not SP (15). Thus, TCR/CD3e engagement may provide a more complete signal for death than metabolic inhibitors. We note that our findings are consistent with previous reports that negative selection can occur at all a/b-TCR/ stages of thymocyte development (2, 46, 47) and with the observation that Fas-mediated apoptosis of activated peripheral T cells is potentiated by TCR coengagement (J. Russell, personal communication). In summary, the striking parallels between our in vitro model of thymocyte death and the known features of negative selection suggest that deleting TCR/CD3e signals may require additional input from Fas or several other related molecules to induce thymocyte death in vivo. ACKNOWLEDGMENTS The authors thank R. N. Germain, L. X. Zheng, S. A. Boehme, and M. H. Julius for helpful discussions and technical assistance, Lisa Boyd and David Margulies for providing anti-H-2Dd, and Shirley Starnes for editorial assistance. J.C.Z.P. is supported by an MRC grant. G.H.F. is supported by the Howard Hughes Medical Institute– National Institutes of Health–Research Scholars Program.

REFERENCES 1. Kappler, J. W., Roehm, N., and Marrack, P., Cell 49, 273, 1987. 2. Fowlkes, B. J., Schwartz, R., and Pardoll, D. M., Nature 334, 620, 1988. 3. Finkel, T. H., Cambier, J. C., Kubo, R. T., Born, W. K., Marrack, P., and Kappler, J. W., Cell 58, 1047, 1989. 4. Nossal, G. J. V., Cell 76, 229, 1994. 5. Smith, C., Williams, G., Kingston, R., Jenkins, E., and Owen, J. J. T., Nature 337, 2175, 1989. 6. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., and Bluestone, J. A., Proc. Natl. Acad. Sci. USA 84, 1374, 1987. 7. Shi, Y., Bissonnette, R. P., Parfrey, N., Szalay, M., Kubo, R. T., and Green, D. R., J. Immunol. 146, 3340, 1991. 8. Hogquist, K. A., Jameson, S. C., Heath, W. R., Howard, J. L., Bevan, M. J., and Carbone, F. R., Cell 76, 17, 1994. 9. Ashton-Rickardt, P. G., and Tonegawa, S., Immunol. Today 115, 362, 1994. 10. Iwabuchi, K., Nakayama, K., McCoy, R. L., Wang, F., Nishimura, T., Habu, S., Murphy, K. M., and Loh, D. Y., Proc. Natl. Acad. Sci. USA 89, 9000, 1992. 11. Surh, C. D., and Sprent, J., Nature 372, 100, 1994. 12. Punt, J. A., Osborne, B. A., Takahama, Y., Sharrow, S. O., and Singer, A., J. Exp. Med. 179, 709, 1994. 13. Page, D. M., Kane, L. P., Allison, J. P., and Hedrick, S. M., J. Immunol. 151, 1868, 1993. 14. Vasquez, N. J., Kane, L. P., and Hedrick, S. M., Immunity 1, 45, 1994. 15. Ogasawara, J., Suda, T., and Nagata, S., J. Exp. Med. 181, 485, 1995. 16. Andjelic, S., Drappa, J., Lacy, E., Elkon, K. B., and NikolicZugic, J., Int. Immunol. 6, 73, 1994. 17. Nagata, S., Semin. Immunol. 6, 3, 1994.

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03-08-96 11:06:25

cias

18. Crispe, I. N., Immunity 1, 347, 1994. 19. Alderson, M. R., Tough, T. W., Davis-Smith, T., Braddy, S., Falk, B., Schooley, K. A., Goodwin, R. G., Smith, C., Ramsdell, F., and Lynch, D. H., J. Exp. Med. 181, 71, 1995. 20. Singer, G. G., and Abbas, A. K., Immunity 1, 365, 1994. 21. Zhou, T., Bluethmann, H., Eldridge, J., Brockhaus, M., Berry, K., and Mountz, J. D., J. Immunol. 147, 466, 1991. 22. Mountz, J. D., Smith, T. M., and Toth, K. S., J. Immunol. 144, 2159, 1990. 23. Ozato, K., Mayer, N. M., and Sachs, D. H., Transplantation 34, 113, 1982. 24. Lenardo, M. J., Nature 353, 858, 1991. 25. Zu´n˜iga-Pflu¨cker, J. C., Schwartz, H. L., and Lenardo, M. J., J. Exp. Med. 178, 1139, 1993. 26. Crispe, I. N., and Bevan, M. J., J. Immunol. 138, 2013, 1987. 27. Boehme, S. A., and Lenardo, M. J., Leukemia 7(Suppl.), S45, 1993. 28. Curnow, S. J., Barad, M., Brun-Rouberreau, N., and SchmittVerhulst, A.-M., Cytometry 16, 41, 1994. 29. Klien, J., Bontrop, R. E., Dawkins, R. L., Erlich, H. A., Gyllensten, U. B., Heise, E. R., Jones, P. P., Parham, P., Wake, E. K., and Watkins, D. I., Immunogenetics 31, 217, 1990. 30. Swat, W., Ignatowicz, L., von Boehmer, H., and Kisielow, P., Nature 351, 150, 1991. 31. Kishimoto, H., Surh, C. D., and Sprent, J., J. Exp. Med. 181, 649, 1995. 32. Swat, W., Dessing, M., von Boehmer, H., and Kisielow, P., Eur. J. Immunol. 23, 739, 1993. 33. Bendelac, A., Matzinger, P., Seder, R. A., Paul, W. E., and Schwartz, R. H., J. Exp. Med. 175, 731, 1992. 34. Fowlkes, B. J., Edison, L., Mathieson, B. J., and Chused, T. M., J. Exp. Med. 162, 802, 1985. 35. Lanier, L. L., Allison, J. P., and Phillips, J. H., J. Immunol. 137, 2501, 1986. 36. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S., Sameshima, M., Hase, A., Seto, Y., and Nagata, S., Cell 66, 233, 1991. 37. Yonehara, S., Nishimura, Y., Kishil, S., Yonehara, M., Takazawa, K., Tamatani, T., and Ishii, A., Int. Immunol. 6, 1849, 1994. 38. Alderson, M. R., Armitage, R. J., Maraskovsky, E., Tough, W. T., Roux, E., Schooley, K., Ramsdell, F., and Lynch, D. E., J. Exp. Med. 198, 2231, 1993. 39. Itoh, N., and Nagata, S., J. Biol. Chem. 268, 10932, 1993. 40. Sato, T., Irie, S., Kitada, S., and Reed, J. C., Science 268, 411, 1995. 41. Stanger, B. Z., Leder, P., Lee, T. H., Kim, E., and Seed, B., Cell 81, 513, 1995. 42. Chinnaiyan, A. M., O’Rourke, K., Tewari, M., and Dixit, V. M., Cell 81, 505, 1995. 43. Boldin, M. P., Mett, I. L., Varfolomeev, E. E., Chumakov, I., Shemer-Avni, Y., Camonis, J. H., and Wallach, D., J. Biol. Chem. 270, 387, 1995. 44. Hsu, H., Xiong, J., and Goeddel, D. V., Cell 81, 495, 1995. 45. Huesman, M., Scott, B., Kisielow, P., and von Boehmer, H., Cell 66, 533, 1991. 46. Ramsdell, F., and Fowlkes, B. J., Science 248, 1342, 1990. 47. Guidos, C. J., Danska, J. S., Fathman, C. G., and Weissman, I. L., J. Exp. Med. 172, 835, 1990.

AP: Cell Immuno