In VitroThymocyte Maturation Is Associated with Reduced Cellular Susceptibility to Fas-Mediated Apoptosis

CELLULAR IMMUNOLOGY ARTICLE NO.

185, 134 –145 (1998)

CI981280

In Vitro Thymocyte Maturation Is Associated with Reduced Cellular Susceptibility to Fas-Mediated Apoptosis1 Pamela A. Hershberger,*,2 Huiling He,*,3 and Susan A. McCarthy*,† *Department of Surgery, and †Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 Received October 20, 1997; accepted March 10, 1998

We have developed a novel system in which the susceptibility of murine thymocytes to Fas-mediated apoptosis can be modulated. Thymocyte susceptibility to Fas decreases under in vitro culture conditions that promote aspects of thymocyte maturation. The hyporesponsive state is specific for the Fas pathway, since cellular susceptibility to other apoptotic stimuli is not reduced. Hyporesponsiveness is not associated with alterations in the thymocyte subset distribution, decreased expression of full-length Fas protein, or alterations in FADD, Bcl-2, or Bcl-XL expression. Hyporesponsiveness is overcome by increasing the strength of the Fas cross-linking stimulus, leading us to propose that reduced thymocyte susceptibility to apoptosis results from altered Fas signaling. The block in Fas signaling resides proximal to ceramide generation, since Fas-hyporesponsive thymocytes are susceptible to ceramide-induced apoptosis. Further characterization of Fas signaling in these in vitro cultured thymocytes may facilitate the identification of factors regulating the susceptibility of wild-type lymphocytes to Fas. © 1998 Academic Press

INTRODUCTION Fas (APO-1/CD95) is a transmembrane, cell surface protein belonging to the tumor necrosis factor receptor family, which also includes TNF-R1, TNF-R2, OX40, CD30, CD27, and CD40 (reviewed in 1). Ligation of cell surface Fas either by its natural ligand, FasL, or by agonistic antibodies leads to the apoptotic death of a variety of cell types including thymocytes (2, 3), acti1 This work was supported by NIH Grant AI-32554 to S.A.M, by NIH fellowship AI-09634-01 to P.A.H., and by grants to P.A.H. from the American Cancer Society (IRG-58-35) and the Arthritis Foundation, Western Pennsylvania Chapter. 2 Current address: Department of Pharmacology, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15213. 3 Current address: Department of Medicine, Case Western Reserve University, Cleveland, OH 44106.

0008-8749/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

vated T and B lymphocytes (4, 5), and activated macrophages (6). A reduction in lymphocyte susceptibility to Fas-mediated apoptosis due to deletion or inactivation of Fas (7–9), or its ligand (10), is associated with the development of autoimmune syndromes in both humans (9) and mice (7, 8, 10), indicating a crucial role for Fasmediated apoptosis in shaping the immune repertoire and maintaining homeostasis within the immune system. Recent studies indicate that immune defects also result from excessive deletion of lymphocytes via the Fas pathway. For example, the induction of Fas-mediated apoptosis in tumor-infiltrating lymphocytes by FasL1 tumors may contribute to tumor evasion of immune-mediated destruction (11–14). Taken together these studies suggest that modulation of lymphocyte susceptibility to Fas represents an important therapeutic target in the treatment of human disease. However, several studies have documented that no simple correlation exists between the quantitative level of Fas expression and cellular susceptibility to Fas-mediated apoptosis (2– 6). The design of successful Fas-based therapies thus relies upon identification of the critical determinants of susceptibility. Candidate regulators of Fas-mediated apoptosis include molecules implicated in the Fas signal transduction pathway. Fas cross-linking results in the formation of a death-inducing signaling complex (15) composed of Fas, the adaptor molecule FADD/MORT-1 (16, 17), and the cysteine aspartic specific protease (caspase), FLICE/MACH/caspase-8 (18, 19). Caspase-8 is the initial caspase activated in response to Fas ligation (20) and potentially functions to activate downstream caspases such as ICE-LAP3/caspase-7 (21) and CPP32/caspase-3 (22), which have also been implicated in the Fas pathway. Fas cross-linking also leads to the rapid activation of acidic sphingomyelinase and the generation of intracellular ceramides (23, 24). The resulting ceramides have been implicated as second messengers in the Fas signal transduction pathway and can mediate apoptosis in the absence of Fas ligation (23, 24). Additionally,

134

REGULATION OF Fas FUNCTION IN MURINE THYMOCYTES

135

it has been demonstrated that Fas engagement results in the phosphorylation of tyrosine residues on a number of cellular proteins and that one or more of these events is required for the induction of apoptosis (25). Consistent with a role for modulation of tyrosine phosphorylation in Fas signal transduction is the observation that expression of the protein tyrosine kinase p59fyn (26) as well as the protein tyrosine phosphatases HCP (27) and FAP-1 (28) correlates with cellular susceptibility to Fas-mediated apoptosis. Given the potential value of modulating the outcome of Fas ligation on T cells for therapeutic purposes, it is of interest to determine the endogenous mechanisms by which T cell susceptibility to Fas-mediated apoptosis is regulated. To identify such mechanisms, we developed a system in which the susceptibility of wildtype murine thymocytes to Fas-mediated apoptosis is modulated. We found that thymocyte susceptibility to Fas could be decreased under in vitro culture conditions that promote at least some aspects of thymocyte maturation. The hyporesponsive state induced upon in vitro culture was specific for the Fas pathway, since cellular susceptibility to other apoptotic stimuli such as irradiation and glucocorticoids was not similarly reduced. Characterization of such in vitro cultured cells indicates that hyporesponsiveness to Fas-mediated apoptosis is not associated with alterations in the thymocyte subset distribution, decreased expression of full-length Fas protein, or alterations in the expression of FADD, Bcl-2, or Bcl-XL. Hyporesponsiveness could, however, be overcome by increasing the strength of the Fas cross-linking stimulus, leading us to propose that reduced susceptibility to Fas-mediated apoptosis results from altered Fas signal transduction. The block in Fas signaling within these cells resides proximal to ceramide generation, since Fas-hyporesponsive thymocytes are susceptible to ceramide-induced apoptosis. We anticipate that further characterization of the Fas signal transduction pathway in in vitro cultured thymocytes will allow us to identify factors regulating the susceptibility of wild-type lymphocytes to Fas. Given the reported lack of correlation between the quantitative level of Fas expression and cellular susceptibility to Fas-mediated apoptosis (2– 6), we expect that manipulation of these factors, rather than manipulation of Fas expression itself, may prove most effective in therapeutically modulating the outcome of Fas ligation.

Antibodies and chemicals. The anti-CD3 mAb, 2C11, was used as a tissue culture supernatant. The hamster anti-mouse Fas mAb, Jo2, hamster antimouse Bcl-2, PE-conjugated anti-CD4/RM4-5 (antiCD4-PE), FITC-conjugated anti-CD8/53-6.7 (anti-CD8FITC), PE-conjugated anti-CD16/CD32/2.4G2 (antiCD16-PE), and biotinylated anti-hamster IgG (clones G70-204 and G94-56) were purchased from PharMingen (San Diego, CA). Unconjugated anti-FcR mAb (2.4G2) was used as a tissue culture supernatant in apoptosis assays. Polyclonal rabbit anti-mouse Fas (C20) was purchased from Santa Cruz (Santa Cruz, CA), and monoclonal anti-rat Bcl-XL was from Transduction Laboratories (Lexington, KY). Polyclonal rabbit antimouse FADD was generously provided by Astar Winoto (University of California, Berkeley). SARED613 was purchased from Gibco BRL (Gaithersburg, MD). Protein A, C2-ceramide, and cycloheximide (CHX)4 were purchased from the Sigma Chemical Co. (St. Louis, MO). CHX was dissolved at a concentration of 10 mg/ml in ethanol and stored at 230°C. Protein A, ceramide, and CHX were diluted in tissue culture medium prior to use.

METHODS

DNA fragmentation assays. DNA fragmentation assays were performed as previously described (31).

Mice. Male and female C57BL/6J (B6) mice were purchased from either The Jackson Laboratory (Bar Harbor, ME) or the Charles Rivers Laboratory (Wilmington, MA). Mice were used between 7 and 13 weeks of age.

Thymocyte preparation and culture. Thymocytes were prepared by mechanical disruption of freshly isolated B6 thymi. Thymocyte suspensions were filtered through nylon mesh to remove cell aggregates and washed once. Washed cells were resuspended in fresh RPMI medium supplemented with 5% FCS, b-mercaptoethanol, glutamine, sodium pyruvate, nonessential amino acids, penicillin, and streptomycin and were precultured either at 4 or at 37°C in an atmosphere containing 7.5% CO2. Cells were precultured at densities of 2.5 3 106–5.0 3 106 viable cells per milliliter either in 24-well tissue culture plates (2 ml cell suspension per well) or, for experiments requiring large cell numbers, in 50-ml polypropylene OPTICUL tubes (Becton–Dickinson Co., Franklin Lakes, NJ). Following the preculture period, thymocytes were collected by centrifugation and resuspended in fresh tissue culture medium for use in DNA fragmentation assays. Control experiments demonstrated that the vessel in which the cells were precultured did not influence the outcome of the DNA fragmentation assay. Similar experimental results were obtained whether cells were precultured in the same tissue culture wells in which apoptosis was induced or were precultured in one vessel and then transferred to fresh tissue culture wells for the induction of apoptosis (Fig. 5).

4 Abbreviations used: Caspase, cysteine aspartic specific protease; CHX, cycloheximide; PVDF, poly(vinylidene difluoride); DN, double negative; DP, double positive; CD4SP, CD4 single positive; CD8SP, CD8 single positive; FcR, Fc receptor; TCR, T cell receptor.

136

HERSHBERGER, HE, AND MCCARTHY

Briefly, 5.0 3 106 thymocytes (freshly isolated or precultured at either 4 or 37°C) were incubated in 2 ml of tissue culture medium in the presence of the indicated stimuli for 12 h at 37°C, unless indicated otherwise. After treatment, the cells (1 3 107 per experimental group) were collected, washed three times in PBS, and lysed for 30 min at 4°C in a buffer containing 5 mM Tris, pH 8.0, 1 mM EDTA, and 0.5% Triton X-100. Fragmented and intact DNA were separated by centrifugation at 13,200 rpm at 4°C. Fragmented DNA (supernatant) and intact DNA (pellet) fractions were adjusted to the same final volume in the lysis buffer. Each sample was then sonicated, and the samples were plated in triplicate, 100-ml serial dilutions in Dynatech MicroFluor 96-well plates (Dynatech Labs, Alexandria, VA). One hundred microliters of a solution of 0.6 mg/ml of the fluorescent DNA dye, DAPI, was then added to each well. The relative amount of DNA in each sample was derived from the fluorescence emission at 465 nm, as measured on a Dynatech MicroFluor plate reader. The percentage of DNA fragmentation was calculated as % fragmentation 5 100 3 (DNA in supernatant/ (DNA in supernatant 1 DNA in pellet)). The percentage of specific DNA fragmentation was calculated by subtracting the percentage of DNA fragmentation observed for cells cultured in medium (spontaneous apoptosis) from the percentage of DNA fragmentation observed for cells cultured the same length of time with an apoptotic stimulus. Assays in which the Jo2 mAb was used to induce apoptosis were performed in the presence of 10 mg/ml CHX. The corresponding medium controls therefore also contained CHX. Thymocytes precultured at 4 and 37°C had comparable levels of spontaneous apoptosis. For Figs. 4A, 4B, and 5A, mean values for the percentage of specific DNA fragmentation from several independent experiments are shown, as indicated in the figure legends. In Figs. 4C, 5B, and 7 the absolute values for the percentage of specific DNA fragmentation varied substantially among independent experiments, although the patterns of apoptosis induction were consistent. In each of these figures, a representative experiment is therefore shown, as indicated in the figure legends. Immunofluorescence and flow cytometry. Thymocytes (1 3 106) were washed in FACS medium (13 HBSS, pH 7.2 (Gibco BRL) containing 0.1% BSA and 0.1% sodium azide) and collected by centrifugation at 1000 rpm for 10 min at 4°C. Washed cell pellets were resuspended in FACS medium and incubated for 30 min at 4°C with primary antibodies anti-CD4-PE, antiCD8-FITC, and either anti-Fas or anti-CD3. Unbound antibody was removed by washing the cells two times in FACS medium. To visualize Fas or CD3, the cells were subsequently incubated with biotinylated antihamster IgG for 30 min at 4°C. After washing to remove unbound secondary antibody, complexes contain-

ing biotinylated antibody were visualized with SARED613. Stained cells were then fixed in FACS medium containing 1% paraformaldehyde and stored at 4°C in the dark until analysis. For each sample 50,000 viable cells, as defined by forward- and side-scatter parameters, were analyzed on a Becton–Dickinson FACScan instrument. Data were analyzed using the LYSIS II software package. Net median fluorescence values were obtained by subtracting the median fluorescence values observed for cells stained with negative control antibodies (background fluorescence) from the median fluorescence values observed for cells stained in the presence of specific antibody. Immunoprecipitation and immunoblotting. Cell surface proteins were biotinylated essentially as described (32). Freshly isolated thymocytes, or thymocytes precultured at either 4 or 37°C, were washed in ice-cold PBS and then resuspended at a density of 1 3 107 cells/ml in biotinylation buffer (10 mM Na-borate, pH 8.8, and 150 mM NaCl). Cell suspensions were treated for 15 min at room temperature with D-biotinoyl-e-amidocaproic acid N-hydroxysuccinimide ester (Boehringer Mannheim, Indianapolis, IN) at a final concentration of 50 mg/ml. Biotinylation reactions were terminated by adding a one-tenth reaction volume of NH4Cl. Biotinylated cells were washed two times in ice-cold PBS and then lysed for 30 min on ice in NP-40 lysis buffer (0.2% NP-40, 50 mM Tris–Cl, pH 8.0, 150 mM KCl, 5 mM EDTA, 1 mM PMSF, 1 mg/ml aprotinin, and 1 mg/ml leupeptin). Lysates were clarified by centrifugation for 10 min at 13,200 rpm at 4°C. Fas was immunoprecipitated from biotinylated cell extracts by mixing 100 mg of total protein from each NP-40 lysate with 1 mg/ml Jo2 mAb for 1 h on ice. Antigen–antibody complexes were bound to protein A–Sepharose CL-4B beads (Sigma) overnight at 4°C. The following morning, the beads and associated proteins were collected and washed three times in the NP-40 lysis buffer. The washed beads were resuspended in 30 ml of 23 sample buffer (100 mM Tris–Cl, pH 6.8, 200 mM DTT, 4% SDS, 20% glycerol, 0.2% bromophenol blue) and boiled for 5 min. The proteins released by this treatment were resolved on 10% denaturing gels and then electrophoretically transferred to poly(vinylidene difluoride) (PVDF) membranes (Millipore, Bedford, MA) overnight at 4°C. To detect biotinylated proteins, the membranes were first blocked for several hours at room temperature in a 5% (w/v) solution of nonfat milk in TBST (18 mM Tris, pH 7.6, 122 mM NaCl, 0.1% Tween 20). The membranes were subsequently incubated for 1 h at room temperature in horseradish peroxidase conjugated to avidin D (Vector Laboratories, Burlingame, CA), diluted 1:20,000 in the blocking solution. After the blots were washed extensively in TBST, the biotinylated proteins were visual-

REGULATION OF Fas FUNCTION IN MURINE THYMOCYTES

137

FIG. 1. Schematic representation of thymocyte maturation. Thymocytes pass through several developmental stages characterized by their expression of the CD4 and CD8 differentiation antigens. The most immature thymocytes exhibit the CD42CD82 (double negative, DN) phenotype and express no cell surface T cell receptor complex (TCR/CD3). The majority of thymocytes are CD41CD81 (double positive, DP) and express undetectable cell surface TCR/CD3 or low levels of the complex. Within the DP population, the progression of surface TCR/CD3 expression, from undetectable to low to intermediate, represents a developmental progression from less mature to more mature cells. The most mature thymocytes are CD41CD82 (CD4 single positive, CD4SP) or CD42CD81 (CD8 single positive, CD8SP) and express high levels of surface TCR/CD3. The reported susceptibility of each thymocyte subset to apoptosis induced by Jo2 mAb is indicated (2, 3).

ized using Renaissance enhanced chemiluminescence reagents (Dupont NEN, Boston, MA). To analyze proteins directly from whole cell lysates, thymocytes were washed three times in PBS and then lysed on ice in a detergent buffer containing 1% Triton X-100, 0.1% SDS, 50 mM Tris–Cl, pH 8.0, 150 mM NaCl, 0.5 mM PMSF, and 5 mg/ml leupeptin. Lysates were clarified by centrifugation and their protein concentrations determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Thirty micrograms of protein from each cell lysate was electrophoresed and transferred to PVDF membranes as described above. Membranes were blocked and then incubated for 1 h at room temperature with primary antibody diluted 1:200 in blocking solution. The blots were then washed three times in TBST. Washed blots were subsequently incubated with an appropriate secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The blots were again washed extensively, and the proteins were detected by enhanced chemiluminescence. Statistical analysis. Statistical analyses were performed using the unpaired Student’s t test. RESULTS Thymocyte Susceptibility to Fas-Mediated Apoptosis Decreases upon in Vitro Culture Thymocyte susceptibility to Fas-mediated apoptosis is developmentally regulated such that immature DP thymocytes are susceptible to Fas-mediated apoptosis, whereas fully mature CD4SP and CD8SP thymocytes are refractory to Fas-mediated apoptosis, although they express levels of cell surface Fas comparable to the susceptible DP population (Fig. 1 and Refs. 2, 3).

We therefore hypothesized that thymocytes having an immature, TCR/CD3low phenotype and thymocytes having a somewhat more mature, TCR/CD3intermediate (TCR/CD3int) phenotype would display differential responses to Fas ligation and would thus provide us with paired populations of wild-type cells differing in the expression, or function, of endogenous regulators of cellular susceptibility to Fas. We chose to compare these two populations of cells, rather than comparing immature DP vs fully mature SP thymocytes, since our prior studies had demonstrated that immature DP thymocytes are susceptible to apoptosis induced by a variety of stimuli to which fully mature SP lymphocytes are refractory (31). Given that finding, we anticipated that the general decrease in the activity of apoptotic pathways in fully mature lymphocytes might interfere with our ability to identify candidate regulators of apoptosis specific for the Fas pathway. Freshly isolated thymocytes, or thymocytes maintained at 4°C following their isolation, have a predominantly immature TCR/CD3low phenotype (33). The conversion of DP thymocytes from a TCR/CD3low phenotype to a TCR/CD3int/hi phenotype can be achieved in vitro by culturing the cells in single-cell suspension at 37°C (33). This manipulation disrupts regulatory interactions between CD4 and MHC Class II molecules that normally retard thymocyte maturation in vivo (34). Compared to thymocytes precultured at 4°C, thymocytes precultured at 37°C display increased expression of the TCR/CD3 complex (Fig. 2A and Ref. 33) and increased TCR signaling capacity specifically within the DP subset (33). Preculture at 37°C does not alter the thymocyte subset distribution (Fig. 2 legend). Following an initial 10.5- to 12-h preculture period, we examined the susceptibility of thymocytes preincu-

138

HERSHBERGER, HE, AND MCCARTHY

FIG. 2. Preculture at 37°C results in increased TCR/CD3 expression within DP thymocytes and does not diminish Fas expression. Thymocytes were precultured for 18 h at either 4°C (4C) or 37°C (37C) and then analyzed for cell surface expression of CD4, CD8, and either CD3 or Fas. CD4 and CD8 were stained directly using antiCD4-PE and anti-CD8-FITC. CD3 and Fas were stained indirectly using the hamster-derived primary mAbs 2C11 and Jo2, respectively, followed by biotinylated anti-hamster-IgG secondary Ab plus SA-RED613. Single-color CD3 or Fas histograms for each subset were obtained by electronically gating on the appropriate regions of a CD4 3 CD8 contour plot. The percentages of cells in each thymocyte subset were as follows: 4°C preculture, 3.4% DN, 81.5% DP, 9.8% CD4SP, and 4.2% CD8SP; 37°C preculture, 2.9% DN, 83.9% DP, 8.9% CD4SP, and 3.5% CD8SP. Negative control antibodies comparably stained thymocytes precultured at 4 or 37°C, but were omitted here for clarity. The ratio of net median CD3 or Fas fluorescence at 37°C to net median fluorescence at 4°C is presented in the upper right corner of each panel. Similar results were obtained in three independent experiments.

bated at either 4°C (TCR/CD3low) or 37°C (TCR/CD3int) to Fas-mediated apoptosis by reculturing the cells with anti-Fas antibody (Jo2 mAb) and CHX for an additional 12 h at 37°C. CHX was added during the apoptosis induction culture to prevent the conversion of control cells from a TCR/CD3low phenotype to a TCR/ CD3int phenotype, which would otherwise occur at 37°C (data not shown). The extent of apoptosis induced in response to Fas ligation was then measured using a quantitative DNA fragmentation assay (31). The Jo2

mAb did not induce DNA fragmentation in thymocytes isolated from Fas-deficient lpr mice, verifying that the induction of apoptosis by Jo2 mAb was through the Fas pathway (data not shown). Approximately 20% of freshly isolated thymocytes (no preculture) undergo spontaneous apoptosis under our assay conditions. The addition of the Jo2 mAb induced an additional 33% of freshly isolated murine thymocytes to fragment their DNA (Table 1), resulting in a total of approximately 50 –55% of these cells undergoing apoptosis within 12 h. This is similar to the level of total thymocyte cell death induced in the presence of Jo2 mAb reported by others (2). Compared to fresh thymocytes, the level of spontaneous apoptosis observed for cells precultured at either 4 or 37°C was elevated (approximately 35% for both precultured cell populations). Despite their comparable levels of spontaneous apoptosis, control thymocytes precultured at 4°C and thymocytes precultured at 37°C differed significantly in their susceptibility to Fas-mediated apoptosis. Cells precultured at 37°C displayed approximately two-fifths as much DNA fragmentation in response to Fas ligation as did cells precultured at 4°C (Table 1, 12% versus 31%). This effect was Fasspecific, since the sensitivity of thymocytes precultured at 37°C to other apoptotic stimuli such as irradiation (Table 1) and ceramide (Fig. 7) was not reduced. Thymocytes precultured at 37°C also displayed no reduction in their susceptibility to dexamethasone-induced apoptosis when compared to freshly isolated thymocytes (data not shown). These observations are consisTABLE 1 Thymocytes Precultured at 37°C Have Reduced Susceptibility to Fas-Mediated Apoptosis % Specific DNA fragmentation Preculture

TCR/CD3 level

Anti-Fas

Irradiation

None 4°C 37°C

Low Low Intermediate

33 6 7 31 6 7 12 6 6

45 6 2 56 6 5 56 6 14

Note. B6 thymocytes were freshly isolated (no preculture) or precultured for 10.5–12 h at either 4 or 37°C. Cells were then either (a) recultured for an additional 12 h at 37°C in complete RPMI medium containing CHX and 50 ng/ml Jo2 mAb (9 to 12 independent experiments) or (b) irradiated (500 rads) and recultured for an additional 12 h in complete RPMI medium (2 to 3 independent experiments). The extent of apoptosis induced in each experimental group was determined using a quantitative DNA fragmentation assay as described under Materials and Methods. The percentage of specific DNA fragmentation was calculated by subtracting the percentage of DNA fragmentation observed for medium controls (spontaneous apoptosis) from the percentage of DNA fragmentation observed in the presence of an apoptotic stimulus. The reduction in susceptibility to Fas-mediated apoptosis observed for thymocytes precultured at 37°C is statistically significant (no preculture vs preculture at 37°C, P , 0.001; preculture at 4°C vs preculture at 37°C, P , 0.001).

REGULATION OF Fas FUNCTION IN MURINE THYMOCYTES

139

tent with the hypothesis that cellular susceptibility to Fas-mediated apoptosis specifically decreases in thymocytes precultured at 37°C. Thymocytes Precultured at 37°C Express Full-Length Fas Protein One hypothesis for the reduced susceptibility of thymocytes precultured at 37°C to Fas-mediated apoptosis is that these cells have decreased surface expression of Fas. To test this, we precultured thymocytes overnight at either 4 or 37°C and then used three-color immunofluorescence and flow cytometry to analyze Fas expression on each of the thymocyte subsets. These studies revealed that Fas is differentially expressed on the thymocyte subsets, with similar relative distributions observed for thymocytes precultured at 4 and 37°C (Fig. 2B). As reported for the distribution of Fas on freshly isolated thymocytes (2, 3), we find that the most immature DN thymocytes express little or no cell surface Fas. The progression of these cells to the DP stage is associated with a large increase in Fas expression. Relative to the DP population, Fas levels are slightly decreased on the most mature CD4SP and CD8SP thymocytes. In comparing the absolute level at which Fas is expressed, we find that Fas expression increases slightly in all thymocyte subsets upon overnight culture at 37°C (Fig. 2B), and each subset increases its Fas expression at 37°C relative to its expression at 4°C by approximately the same ratio. Thus, the reduced susceptibility to Fas-mediated apoptosis of thymocytes precultured at 37°C is not associated with reduced Fas expression. We next determined whether the Fas protein expressed at the cell surface was full length. Soluble (35) and membrane-associated (36) Fas variants arise by alternative splicing of the Fas transcript and have been shown to protect cells from Fas-mediated apoptosis. In light of these studies, we hypothesized that hyporesponsiveness to Fas-mediated apoptosis could result from reduced production of full-length Fas protein. To test this hypothesis, freshly isolated thymocytes (Fig. 3A, lanes 1 and 2) and thymocytes precultured at either 4°C (Fig. 3A, lanes 3 and 4) or 37°C (Fig. 3A, lanes 5 and 6) were surface biotinylated, and biotinylated cell surface proteins were immunoprecipitated using the Jo2 mAb. No biotinylated proteins were immunoprecipitated in the absence of the Jo2 mAb (Fig. 3A, lanes 1, 3, and 5). The Jo2 mAb, which recognizes an extracellular Fas epitope, specifically immunoprecipitated a protein of the size expected for full-length Fas (46 kDa) as well as a smaller protein of approximately 42 kDa from all three cell preparations (Fig. 3A, lanes 2, 4, and 6). Previous reports suggest that this smaller species may represent an incompletely glycosylated form of full-length Fas protein (37). The presence of full-length Fas protein in whole cell lysates prepared

FIG. 3. Cultured thymocytes express full-length Fas proteins. (A) Fas was immunoprecipitated from NP-40 lysates prepared from biotinylated thymocytes using 1 mg/ml Jo2 mAb. Immunoprecipitated proteins were resolved on 10% denaturing polyacrylamide gels and then were electrophoretically transferred to PVDF membranes overnight at 4°C. Biotinylated proteins were detected using horseradish peroxidase conjugated to avidin D followed by enhanced chemiluminescence. (B) Thirty micrograms of total cellular protein obtained from freshly isolated thymocytes, or thymocytes precultured at either 4 or 37°C, were analyzed by Western blot using polyclonal anti-Fas antisera (Santa Cruz). The blots shown are representative of two independent experiments.

from thymocytes precultured at 37°C was confirmed in additional Western blot experiments that utilized a polyclonal antiserum specific for the intracellular, carboxy-terminal sequences of Fas (Fig. 3B). The fact that we detect no unique Fas proteins in lysates prepared from thymocytes precultured at 37°C using two different Fas-specific antibodies suggests that production of alternative Fas species is not a likely explanation for the induction of hyporesponsiveness to Fas-mediated apoptosis. This finding was confirmed in RT-PCR experiments demonstrating that alternative Fas variants are not expressed in freshly isolated thymocytes or thymocytes precultured at 4 or 37°C (data not shown). Thymocytes Precultured at 37°C Display a Reduction in Fas Signal Transduction Data presented thus far indicate that thymocytes precultured at 37°C display reduced susceptibility to Fas-mediated apoptosis. Our studies suggest that reduced susceptibility does not result from an alteration in the thymocyte subset distribution (Fig. 2 legend), decreased Fas expression (Fig. 2), or production of alternative Fas species (Fig. 3 and data not shown). We therefore investigated the possibility that hyporesponsiveness to Fas is associated with an alteration in Fas signal transduction. To do so, we examined whether cellular responsiveness to Fas can be altered by changing the conditions under which Fas ligation occurs. We first examined whether the response of thymocytes precultured at 37°C to Fas can be increased by changing the concentration of antibody used to induce apoptosis. Thymocytes were precultured at either 4 or 37°C and then recultured for an additional 12 h at 37°C with increasing concentrations of Jo2 mAb. As shown

140

HERSHBERGER, HE, AND MCCARTHY

FIG. 4. Thymocytes precultured at 37°C are susceptible to apoptosis upon prolonged Fas engagement or extensive Fas cross-linking. Thymocytes were precultured at either 4°C (squares) or 37°C (circles) and then recultured at 37°C under the conditions indicated to induce apoptosis. DNA fragmentation was measured 12 h after the addition of apoptotic stimuli unless stated otherwise. (A) Cells were precultured at either 4 or 37°C for 10.5 h and then recultured in medium containing 10 mg/ml CHX and the indicated doses of Jo2 mAb. The values presented for the percentage of specific DNA fragmentation are the mean of three independent experiments. Statistically significant differences (P , 0.05) between thymocytes precultured at 4 and 37°C were obtained at concentrations of Jo2 mAb ranging from 50 to 100 ng/ml. (B) Cells were precultured at either 4 or 37°C for a fixed time and then recultured with 10 mg/ml CHX and 50 ng/ml Jo2 mAb for varying lengths of time. The values presented for the percentage specific DNA fragmentation at 12, 16, and 20 h are the mean of two independent experiments. DNA fragmentation at the 24-h timepoint was measured in a single experiment. (C) Cells were precultured at either 4 or 37°C and then recultured with varying concentrations of Jo2 mAb and 1 mg/ml protein A. The values presented are representative of three experiments.

in Fig. 4A, the extent of apoptosis induced in thymocytes precultured at 4°C was concentration-dependent, with optimal levels of apoptosis induced at 50 –75 ng/ml Jo2 mAb. The decrease in the apoptotic response of these cells at lower (25 ng/ml) and higher (200 ng/ml) Jo2 mAb concentrations is most likely related to suboptimal cross-linking of the Fas molecule under these conditions. A similar dose–response curve was obtained for freshly isolated thymocytes (data not shown). In contrast to these control cells, thymocytes precultured at 37°C showed no concentration dependence in their apoptotic response and remained hyporesponsive to Fas-mediated apoptosis over the entire range of Jo2 mAb concentrations tested. Hyporesponsiveness was not due to a lack of Jo2 mAb binding since comparable levels of antibody were bound to both precultured cell populations at each of the concentrations examined (data not shown). To determine whether thymocytes that had been precultured at 37°C could be induced to undergo Fasmediated apoptosis in response to more prolonged Fas engagement, we precultured cells at either 4 or 37°C for a fixed amount of time and then recultured these cells for increasing lengths of time at 37°C in the presence of Jo2 mAb. Whereas thymocytes precultured at 4°C displayed near-maximum levels of DNA fragmentation at the earliest time point examined following Jo2 addition, thymocytes precultured at 37°C displayed a time-dependent and delayed increase in DNA fragmentation (Fig. 4B). After 24 h of treatment with Jo2 mAb, the extent of DNA fragmentation induced by Fas ligation was comparable in cells precultured at 4 and 37°C. One possible explanation for this result is that prolonged exposure to Jo2 mAb is required to induce or allow the expression of molecules participating in Fas signal transduction in cells precultured at

37°C. We consider this explanation to be unlikely since CHX is added to the cultures at the same time as the Jo2 mAb and would be expected to inhibit new protein synthesis. An alternative possibility is that the cellular machinery necessary for the execution of Fas-mediated apoptosis is present in thymocytes precultured at 37°C, but that either it is harder to trigger or it takes longer to activate in these cells than in fresh cells or cells precultured at 4°C. This possibility led us to hypothesize that we could trigger Fas-mediated apoptosis in thymocytes that had been precultured at 37°C by providing a stronger signal through the Fas receptor. To test this hypothesis, we precultured thymocytes at either 4 or 37°C and then recultured the cells at 37°C with varying concentrations of Jo2 mAb and 1 mg/ml protein A. The Jo2 mAb is of an isotype that is effectively cross-linked by protein A. Whereas the addition of protein A alone did not induce thymocyte apoptosis, the addition of protein A in the presence of the Jo2 mAb resulted in strong apoptotic responses in both cell populations (Fig. 4C). Over the entire range of Jo2 mAb concentrations examined, thymocytes precultured at 4 and at 37°C displayed comparable levels of DNA fragmentation. We interpret these data as an indication that strong crosslinking of the Fas receptor is sufficient to circumvent or override the Fas-specific hyporesponsive state and efficiently induce apoptosis in thymocytes that have been precultured at 37°C. However, one alternative explanation for the observation that thymocytes precultured at 37°C are hyporesponsive to Jo2-mediated apoptosis in the absence (Table 1 and Fig. 4A) but not in the presence of protein A (Fig. 4C) is that preculture at 37°C results in a loss of FcR1 cells required for efficient cross-linking of Jo2 mAb, which is compensated for by protein A addition.

REGULATION OF Fas FUNCTION IN MURINE THYMOCYTES

141

FIG. 5. Preculture at 37°C does not result in the depletion or inactivation of FcR required for efficient cross-linking of the Jo2 mAb. (A) Thymocytes were precultured at 4 or 37°C for 10.5 h either in 50-ml Opticul tubes or in apoptosis assay wells. Following the preculture period, thymocytes that had been precultured in 50-ml tubes were collected by centrifugation and recultured in assay wells with 50 ng/ml Jo2 mAb. Jo2 mAb (50 ng/ml) was added directly to thymocytes that had been precultured in assay wells. DNA fragmentation was quantitated after 12 h at 37°C. The results are the means of three independent experiments. Statistically significant differences (P , 0.05) between thymocytes precultured at 4 and 37°C were obtained independently of the preculture method. (B) Thymocytes were precultured at 4 or 37°C for 10.5 h and then mixed with increasing amounts of freshly isolated B6 splenocytes. An aliquot of each mixture was analyzed for FcR expression by flow cytometry. Jo2 mAb (50 ng/ml) was added to a second aliquot of each mixture, and DNA fragmentation was quantitated after 12 h. The results shown are representative of two independent experiments.

To examine whether FcR1 cells were deleted as a consequence of our experimental protocol, thymocytes (a) were precultured at 4 or 37°C in tubes and then transferred to assay wells for reculture with Jo2 mAb (as per our standard protocol) or (b) were precultured in assay wells at 4 or 37°C to which Jo2 was added during the reculture period. As shown in Fig. 5A, thymocytes precultured at 37°C were hyporesponsive to Fas-mediated apoptosis using either of the preculture protocols, indicating that the transfer of thymocytes from preculture vessels to assay wells did not result in the depletion of a subpopulation of cells required for efficient cross-linking of the Jo2 mAb. To further investigate whether hyporesponsiveness to Fas-mediated apoptosis results from down-modulation or inhibition of FcR function upon preculture at 37°C, thymocytes were precultured at either 4 or 37°C, mixed with increasing numbers of FcR1 splenocytes, and then recultured at 37°C with Jo2 mAb. Flow cytometry was conducted on a separate aliquot of each mixture (thymocytes plus splenocytes) to determine the percentage of FcR1 cells present following the preculture period. The susceptibility of thymocytes precultured at 37°C to apoptosis induced by Jo2 mAb increased as the percentage of FcR1 cells in the culture increased (Fig. 5B), consistent with a role for FcR in modulating the strength of Jo2 cross-linking. However, at a fixed percentage of FcR1 cells, thymocytes precultured at 4°C were consistently more susceptible to Jo2-mediated apoptosis than were thymocytes precultured at 37°C. This indicates that hyporesponsiveness

to Fas-mediated apoptosis does not simply result from a decrease in FcR expression or function upon preculture at 37°C. Rather, these data support the conclusion that hyporesponsiveness to Fas-mediated apoptosis results from a decrease in Fas signaling within the thymocytes themselves. Expression of FADD, Bcl-2, and Bcl-XL Is Comparable in Freshly Isolated Thymocytes and Thymocytes Precultured at 4 or 37°C A decrease in Fas signaling within thymocytes precultured at 37°C could result from a decrease in the expression of FADD/MORT-1, an adaptor molecule required for the transmission of a death signal through Fas (16, 17). Alternatively, thymocyte susceptibility to Fas-mediated apoptosis could be decreased due to increased expression of the apoptotic suppressor proteins Bcl-2 and/or Bcl-XL, which have been reported to protect cells against Fas-mediated apoptosis (38, 39). We therefore used Western blot analysis to examine FADD, Bcl-2, and Bcl-XL expression in freshly isolated thymocytes (Fas sensitive) and thymocytes precultured at 4°C (Fas sensitive) or 37°C (Fas hyporesponsive). As shown in Fig. 6, comparable levels of FADD, Bcl-2, and Bcl-XL were detected in whole-cell lysates prepared from all three thymocyte populations, indicating that hyporesponsiveness to Fas-mediated apoptosis does not result from changes in the intracellular accumulation of these molecules.

142

HERSHBERGER, HE, AND MCCARTHY

in thymocytes that have been precultured at 37°C resides proximal to the generation or activity of ceramide. DISCUSSION

FIG. 6. FADD, Bcl-2, and Bcl-XL are expressed at comparable levels in freshly isolated thymocytes or thymocytes precultured at 4 or 37°C. Whole-cell lysates were prepared from either freshly isolated thymocytes or thymocytes that had been precultured at 4 or 37°C. Thirty micrograms of each whole-cell lysate was resolved by SDS–PAGE, transferred to PVDF membranes, and analyzed by Western blot using the antibodies indicated. The blots shown are representative of two independent experiments.

Thymocytes Precultured at 37°C Are Susceptible to Apoptosis Induced by Ceramides In Fas-sensitive cells, Fas ligation results in sphingomyelinase activation and the generation of intracellular ceramides (23, 24). This finding, combined with the observation that cell-permeant ceramides induce apoptosis (23, 24), has led to the hypothesis that ceramides are an important second messenger in the pathway leading from Fas ligation to cell death. Therefore, in a further effort to identify the molecular basis for reduced thymocyte susceptibility to Fas-mediated apoptosis, we examined whether thymocytes that were hyporesponsive to Fas-mediated apoptosis were susceptible to apoptosis induced by cell-permeant ceramides. Freshly isolated thymocytes, or thymocytes precultured at 4 or 37°C, were recultured at 37°C with increasing concentrations of cell-permeant C2-ceramide, and DNA fragmentation was quantitated after 12 h. In all three cell populations, an increase in the concentration of ceramide in the culture resulted in an increase in the level of DNA fragmentation (Fig. 7). Interestingly, thymocytes precultured at 37°C, although hyporesponsive to apoptosis induced by Fas ligation, were most susceptible to apoptosis induced by ceramide. Thus, the ‘‘block’’ in Fas signal transduction

Aberrations in lymphocyte susceptibility to Fas-mediated apoptosis are associated with several pathological conditions, including the depletion of T cells in HIV-infected individuals (40) and the accumulation of autoreactive lymphocytes in patients with autoimmune lymphoproliferative syndromes (9). Additionally, the induction of Fas-mediated apoptosis in tumor-infiltrating lymphocytes by FasL1 tumors has recently been shown to contribute to tumor evasion of immunemediated destruction (11–14). Thus, therapeutic strategies aimed at modulating lymphocyte susceptibility to Fas are expected to provide clinical benefit in the treatment of these diseases. To facilitate the identification of factors that regulate the apoptotic responses of wildtype lymphocytes to Fas ligation, we have developed a novel system in which the susceptibility of murine thymocytes to Fas-mediated apoptosis can be modulated. Association between Increased Thymocyte Maturation and Decreased Susceptibility to Fas: Implications for Thymic Selection By culturing thymocytes in single-cell suspension at 37°C, we reduce their susceptibility to Fas-mediated apoptosis (Table 1). Prior studies have documented that these culture conditions also result in at least partial maturation of DP thymocytes as evidenced by increased expression of the TCR/CD3 complex and increased TCR/CD3 signaling capacity specifically within this cell population (33). Based on these data, it is likely that the decrease in susceptibility to Fas-medi-

FIG. 7. Thymocytes precultured at 37°C are susceptible to apoptosis induced by cell-permeant ceramide. Freshly isolated thymocytes or thymocytes that were precultured at 4 or 37°C for 10.5 h were recultured at 37°C in the presence of the indicated concentrations of cell-permeant C2-ceramide. DNA fragmentation was quantitated after 12 h. Similar results were obtained in three experiments.

REGULATION OF Fas FUNCTION IN MURINE THYMOCYTES

ated apoptosis we observe in vitro occurs within DP thymocytes as they progress from a less mature TCR/ CD3low phenotype to a more mature TCR/CD3int/hi phenotype. A decrease in thymocyte susceptibility to Fasmediated apoptosis upon transition from the immature DP to the fully mature SP stage of development has previously been reported (2, 3). During their development, DP thymocytes bearing TCRs of autoreactive specificity are deleted by apoptosis (41). Since Fas-deficient mice develop autoimmune disease, it was initially proposed that Fas mediates the elimination of these cells (7). However, the majority of published data have not supported a role for Fas-mediated apoptosis in thymic negative selection (42, 43). Nevertheless, our system reveals active regulation of the Fas pathway in DP thymocytes, which are at a maturational stage appropriate for thymic selection. The existence of such regulation leads us to propose that Fas may participate in central tolerance induction. This hypothesis is supported further by the finding that FasL is expressed within the thymus (44) and by data demonstrating that in vivo administration of Fas-Fc (a reagent that blocks the interaction between Fas and Fas ligand) inhibits antigen-specific deletion of thymocytes (45). Interestingly, thymocytes precultured at 37°C display increased susceptibility to apoptosis induced by ceramides compared to control thymocytes (Fig. 7). This observation suggests that, although signaling from the Fas receptor itself is decreased in these cells, the activity of downstream components of the apoptosis pathway may actually be enhanced. If these components are common to other death receptor pathways, thymocytes precultured at 37°C would be expected to display enhanced apoptosis in response to ligation of such receptors. CD30 has been implicated in the negative selection of thymocytes (46) and may represent one receptor to which more mature thymocytes respond as they become hyporesponsive to Fas. What Is the Molecular Basis for the Reduced Susceptibility of Thymocytes Precultured at 37°C to Fas-Mediated Apoptosis? Protection from Fas-mediated apoptosis has been attributed to the expression of either soluble or membrane-associated Fas variants that arise by alternative splicing of the Fas transcript (35, 36). These variants, characterized most fully in the human system, are proposed to protect cells from Fas-mediated apoptosis by preventing the formation of functional Fas signaling complexes (35). It does not appear that production of Fas protein variants accounts for the induction of hyporesponsiveness to Fas in our studies since we did not detect any Fas proteins that were not full length using two different Fas-specific reagents (Figs. 3A and 3B) or using RT-PCR (data not

143

shown). Additionally, the only Fas variant identified in the mouse thymus (Fas b) has not been shown to be developmentally regulated (47). One trivial, alternative explanation for the induction of hyporesponsiveness is that by preculturing cells at 37°C we deplete the cultures of FcR1 cells required for efficient cross-linking of the Jo2 mAb during the subsequent reculturing step. We consider this to be an unlikely explanation for two reasons. First, in control experiments we precultured thymocytes at 37°C and then recultured them with Jo2 mAb in the same culture vessel, thereby eliminating the potential loss of FcR1 cells from our culture. In those experiments, the induction of hyporesponsiveness to Fas is not diminished (Fig. 5A). Second, at a fixed percentage of FcR1 cells in the culture (Fig. 5B), the susceptibility of thymocytes precultured at 37°C to Fas-mediated apoptosis is consistently lower than that of thymocytes precultured at 4°C. Rather, since hyporesponsiveness to Fasmediated apoptosis can be overcome either by protracted exposure to Jo2 mAb (Fig. 4B) or by a very strong cross-linking signal (Fig. 4C), we hypothesize that reduced responsiveness to Fas is associated with altered Fas signal transduction. Two candidate inhibitors of the Fas pathway that are expressed within the mouse thymus and could therefore influence the outcome of Fas ligation are the apoptotic suppressor proteins Bcl-XL (48) and Bcl-2 (49). Although enhanced Bcl-XL expression has been associated with protection of both cell lines and mature activated T cells from Fas-mediated apoptosis (38), it is unlikely to be the key regulator of thymocyte susceptibility to Fas since we found no difference in Bcl-XL expression between thymocytes precultured at 4°C and thymocytes precultured at 37°C, which display differential susceptibility to Fas-mediated apoptosis (Fig. 6). Furthermore, Bcl-XL is normally expressed at very high levels in immature DP thymocytes (which are susceptible to Fas-mediated apoptosis) and at much lower levels in the most mature, SP thymocytes (which are refractory to Fas-mediated apoptosis) (48). Thus, in thymocytes Bcl-XL expression correlates with susceptibility to, rather than protection from, Fas-mediated apoptosis. Thus, our findings support a model in which thymocytes precultured at 37°C are protected from Fas-mediated apoptosis by a Bcl-XL-independent mechanism. In contrast to Bcl-XL, Bcl-2 expression in murine thymocytes increases upon maturation from the DP to the SP stage (49). Like Bcl-XL, cellular expression of Bcl-2 has been associated with protection from Fas-mediated apoptosis in some circumstances (39). Taken together, these two observations prompted us to examine Bcl-2 levels in thymocytes precultured at 4 and 37°C. As shown in Fig. 6, we found that Bcl-2 is expressed at comparably low levels in both cell populations. These data suggest that Bcl-2 up-modulation is also not a likely reason for the establish-

144

HERSHBERGER, HE, AND MCCARTHY

ment of hyporesponsiveness in thymocytes precultured at 37°C. Although Bcl-XL (48) and Bcl-2 (49) expression levels have been shown to differ between immature doublepositive thymocytes and the most mature single-positive thymocytes, we see no changes in the expression of Bcl-XL or Bcl-2 in thymocytes that have converted from a less mature CD41CD81TCR/CD3low phenotype to a more mature CD41CD81TCR/CD3int/hi phenotype in vitro (Fig. 6). We interpret this to mean that preculture at 37°C does not promote the maturation of thymocytes to the developmental stage at which changes in the expression of these apoptosis regulatory molecules occurs. An alternative possibility that we cannot discount is that our in vitro cultures are deficient in a molecule or cell type required for developmental modulation of Bcl-2 and Bcl-XL expression in thymocytes. Several groups have recently reported the identification of a molecule (called FLIP, FLAME-1, or I-FLICE) (50 –52 and reviewed by 53) that can bind to FADD (50, 51) or FLICE/caspase-8 (50 –52) and suppress Fasmediated apoptosis presumably by inhibiting functional DISC assembly. Interestingly, this molecule is expressed at elevated levels in recently activated mature lymphocytes that are not susceptible to Fas-mediated apoptosis, and its expression is decreased at a later time when these cells become susceptible to Fasmediated apoptosis (50). Based on these studies, FLIP appears to be one of the key molecules regulating the susceptibility of normal, mature lymphocytes to Fasmediated apoptosis. It remains to be determined whether FLIP/FLAME-1/I-FLICE also participates in the developmental regulation of thymocyte susceptibility to Fas-mediated apoptosis. Rather than being actively inhibited, reduced susceptibility to Fas-mediated apoptosis may instead result from a maturation-associated decrease in the expression of an essential component of the Fas pathway. A naturally occurring, maturation-associated decrease in such a factor(s) upon transition from the DP to SP developmental stage would also explain why DP but not SP thymocytes are susceptible to Fas-mediated apoptosis (2, 3). Although we detected no difference in FADD expression among Fas-responsive and Fas-hyporesponsive thymocytes (Fig. 6), decreased expression of any one of the other molecules implicated in the Fas signal transduction pathway (including FLICE (18, 19), acidic sphingomyelinase (23, 24), p59fyn (26), or HCP (27)) may be involved. Future experiments will be designed to distinguish among these possibilities. Insight into the identity of the factor(s) that limits responsiveness to Fas ligation could have significant therapeutic value in the treatment of diseases resulting from either increased or decreased cellular susceptibility to Fas-mediated apoptosis.

ACKNOWLEDGMENTS We thank David Dougall, Esi Lamouse-Smith, Dr. Timothy Wright, and Dr. Lisa Kierstead for helpful suggestions and comments throughout the course of this work.

REFERENCES 1. Smith, C. A., Farrah, T., and Goodwin, R. G., Cell 76, 959, 1994. 2. Ogasawara, J., Suda, T., and Nagata, S., J. Exp. Med. 181, 485, 1995. 3. Nishimura, Y., Ishii, A., Kobayashi, Y., Yamasaki, Y., and Yonehara, S., J. Immunol. 154, 4395, 1995. 4. Owen-Schaub, L. B., Yonehara, S., Crump, W. L., III, and Grimm, E. A., Cell. Immunol. 140, 197, 1992. 5. Rothstein, T. L., Wang, J. K. M., Panka, D. J., Foote, L. C., Wang, Z., Stanger, B., Cui, H., Ju, S., and Marshak-Rothstein, A., Nature 374, 163, 1995. 6. Ashany, D., Song, X., Lacy, E., Nikolic-Zugic, J., Friedman, S. M., and Elkon, K. B., Proc. Natl. Acad. Sci. USA 92, 11225, 1995. 7. Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A., and Nagata, S., Nature 356, 314, 1992. 8. Adachi, M., Suematsu, S., Suda, T., Watanabe, D., Fukuyama, H., Ogasawara, J., Tanaka, T., Yoshida, N., and Nagata, S., Proc. Natl. Acad. Sci. USA 93, 2131, 1996. 9. Fisher, G. H., Rosenberg, F. J., Straus, S. E., Dale, J. K., Middelton, L. A., Lin, A. Y., Strober, W., Lenardo, M. J., and Puck, J. M., Cell 81, 935, 1995. 10. Nagata, S., and Suda, T., Immunol. Today 16, 39, 1995. 11. Hahne, M., Rimoldi, D., Schroter, M., Romero, P., Schreier, M., French, L. E., Schneider, P., Bornand, T., Fontana, A., Lienard, D., Cerottini, J.-C., and Tschopp, J., Science 274, 1363, 1996. 12. O’Connell, J., O’Sullivan, G. C., Collins, J. K., and Shanahan, F., J. Exp. Med. 184, 1075, 1996. 13. Walker, P. R., Saas, P., and Dietrich, P-Y., J. Immunol. 158, 4521, 1997. 14. Niehans, G. A., Brunner, T., Frizelle, S. P., Liston, J. C., Salerno, C. T., Knapp, D. J., Green, D. R., and Kratzke, R. A., Cancer Res. 57, 1007, 1997. 15. Kischkel, F. C., Hellbardt, S., Behrmann, I., Germer, M., Pawlita, M., Krammer, P. H., and Peter, M. E., EMBO J. 14, 5579, 1995. 16. Chinnaiyan, A. M., O’Rourke, K., Tewari, M., and Dixit, V. M., Cell 81, 505, 1995. 17. Boldin, M. P., Varfolomeev, E. E., Pancer, Z., Mett, I. L., Camonis, J. H., and Wallach, D., J. Biol. Chem. 270, 7795, 1995. 18. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M., Cell 85, 817, 1996. 19. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D., Cell 85, 803, 1996. 20. Medema, J. P., Scaffidi, C., Kischkel, F. C., Shevchenko, A., Mann, M., Krammer, P. H., and Peter, M. E., EMBO J. 16, 2794, 1997. 21. Duan, H., Chinnaiyan, A. M., Hudson, P. L., Wing, J. P., He, W., and Dixit, V. M., J. Biol. Chem. 271, 1621, 1996. 22. Schlegel, J., Peters, I., Orrenius, S., Miller, D. K., Thornberry, N. A., Yamin, T., and Nicholson, D. W., J. Biol. Chem. 271, 1841, 1996. 23. Cifone, M. G., De Maria, R., Roncaioli, P., Rippo, M. R., Azuma, M., Lanier, L. L., Santoni, A., and Testi, R., J. Exp. Med. 177, 1547, 1993.

REGULATION OF Fas FUNCTION IN MURINE THYMOCYTES 24.

25. 26.

27. 28. 29. 30. 31. 32. 33.

34. 35. 36. 37. 38. 39.

Tepper, C. G., Jayadev, S., Liu, B., Bielawska, A., Wolff, R., Yonehara, S., Hannun, Y. A., and Seldin, M. F., Proc. Natl. Acad. Sci. USA 92, 8443, 1995. Eischen, C. M., Dick, C. J., and Leibson, P. J., J. Immunol. 153, 1947, 1994. Atkinson, E. A., Ostergaard, H., Kane, K., Pinkoski, M. J., Caputo, A., Olszowy, M. W., and Bleackley, R. C., J. Biol. Chem. 271, 5968, 1996. Su, X., Zhou, T., Wang, Z., Yang, P. A., Jope, R. S., and Mountz, J. D., Immunity 2, 353, 1995. Sato, T., Irie, S., Kitada, S., and Reed, J. C., Science 268, 411, 1995. Gamen, S., Marzo, I., Anel, A., Pin˜eiro, A., and Naval, J., FEBS Lett. 390, 233, 1996. Pronk, G. J., Ramer, K., Amiri, P., and Williams, L. T., Science 271, 808, 1996. Cairns, J. S., Mainwaring, M. S., Cacchione, R. N., Walker, J. A., and McCarthy, S. A., Thymus 21, 177, 1993. Meier, T., Arni, S., Malarkannan, S., Poincelet, M., and Hoessli, D., Anal. Biochem. 204, 220, 1992. Nakayama, T., June, C. H., Munitz, T. I., Sheard, M., McCarthy, S. A., Sharrow, S. O., Samelson, L. E., and Singer, A., Science 249, 1558, 1990. McCarthy, S. A., Kruisbeek, A. M., Uppenkamp, I. K., Sharrow, S. O., and Singer, A., Nature 336, 76, 1988. Papoff, G., Cascino, I., Eramo, A., Starace, G., Lynch, D. H., and Ruberti, G., J. Immunol. 156, 4622, 1996. Cascino, I., Papoff, G., De Maria, R., Testi, R., and Ruberti, G., J. Immunol. 156, 13, 1996. Mariani, S. M., Matiba, B., Armandola, E. A., and Krammer, P. H., Eur. J. Immunol. 24, 3119, 1994. Boise, L. H., Minn, A. J., Noel, P. J., June, C. H., Accavitti, M. A., Lindsten, T., and Thompson, C. B., Immunity 3, 87, 1995. Armstrong, R. C., Aja, T., Xiang, J., Gaur, S., Krebs, J. F., Hoang, K., Bai, X., Korsmeyer, S. J., Karanewsky, D. S., Fritz, L. C., and Tomaselli, K. J., J. Biol. Chem. 271, 16850, 1996.

40. 41. 42. 43.

44. 45.

46.

47. 48.

49. 50.

51.

52. 53.

145

Katsikis, P. D., Wunderlich, E. S., Smith, C. A., Herzenberg, L. A., and Herzenberg, L. A., J. Exp. Med. 181, 2029, 1995. Surh, C. D., and Sprent, J., Nature 372, 100, 1994. Singer, G. G., and Abbas, A. K., Immunity 1, 365, 1994. Adachi, M., Suematsu, S., Suda, T., Watanabe, D., Fukuyama, H., Ogasawara, J., Tanaka, T., Yoshida, N., and Nagata, S., Proc. Natl. Acad. Sci. USA 93, 2131, 1996. French, L. E., Wilson, A., Hahne, M., Viard, I., Tschopp, J., and Robson MacDonald, H., J. Immunol. 159, 2196, 1997. Castro, J. E., Listman, J. A., Jacobson, B. A., Wang, Y., Lopez, P. A., Ju, S., Finn, P. W., and Perkins, D. L., Immunity 5, 617, 1996. Amakawa, R., Hakem, A., Kundig, T. M., Matsuyama, T., Simard, J. J. L., Timms, E., Wakeham, A., Mittruecker, H-W., Griesser, H., Takimoto, H., Schmits, R., Shahinian, A., Ohashi, P. S., Penninger, J. M., and Mak, T. W., Cell 84, 551, 1996. Hughes, D. P. M., and Crispe, I. N., J. Exp. Med. 182, 1395, 1995. Ma, A., Pena, J. C., Chang, B., Margosian, E., Davidson, L., Alt, F. W., and Thompson, C. B., Proc. Natl. Acad. Sci. USA 92, 4763, 1995. Veis, D. J., Sentman, C. L., Bach, E. A., and Korsmeyer, S. J., J. Immunol. 151, 2546, 1993. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J-L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E., and Tschopp, J., Nature 388, 190, 1997. Srinivasula, S. M., Ahmad, M., Ottilie, S., Bullrich, F., Banks, S., Wang, Y., Fernandes-Alnemri, T., Croce, C. M., Litwack, G., Tomaselli, K. J., Armstrong, R. C., and Alnemri, E. S., J. Biol. Chem. 272, 18542, 1997. Hu, S., Vincenz, C., Ni, J., Gentz, R., and Dixit, V. M., J. Biol. Chem. 272, 17255, 1997. Wallach, D., Nature 388, 123, 1997.