Comparison of Apoptosis Signaling through T Cell Receptor, Fas, and Calcium Ionophore

EXPERIMENTAL CELL RESEARCH

222, 95–102 (1996)

Article No. 0012

Comparison of Apoptosis Signaling through T Cell Receptor, Fas, and Calcium Ionophore HOLDEN T. MAECKER,*,1 SASCHA HEDJBELI,† MORTIMER ALZONA,†

AND

PHONG T. LE‡

*Department of Medicine/Oncology, Stanford University Medical Center, Stanford, California 94305; †Department of Biology, Loyola University Chicago, Chicago, Illinois 60626; and ‡Department of Cell Biology, Neurobiology, and Anatomy, Loyola University Medical Center, Maywood, Illinois 60153

active thymocytes, as well as positive selection of thymocytes with low affinity for thymic MHC, can both be accomplished via signals presumably generated through the TCR/CD3 complex (reviewed in [4–5]). Bcl-2, an oncogene known to protect many types of cells from various apoptosis-inducing agents, is expressed preferentially in the thymic medulla [6–8], in cells which presumably have been spared negative selection and are undergoing positive selection. However, transgenic mice which constitutively express bcl-2 still show evidence of negative selection (clonal deletion) in their thymuses [9]. Recently, in situ staining experiments have demonstrated the presence of apoptotic cells in the thymic cortex, a minority of which are bcl-2positive [10]. Thus, the existence of a bcl-2-independent negative selection pathway appears inevitable. However, the general correlation of bcl-2 expression with positive selection makes the role of bcl-2 in the thymus still unclear. SUP-T13 is a clonal T leukemia cell line which appears to represent the developmental stage of immature thymocytes. These cells are CD4/CD8/ and can be induced to undergo apoptosis by ligation of their TCR with a mAb [11]. Interestingly, cotreatment with a mAb to CD3 rescues SUP-T13 cells from apoptosis, a phenomenon which may be analogous to positive selection in the thymus, since it involves the generation of additional signals through the TCR/CD3 complex. SUP-T13 cells are also susceptible to apoptosis by a variety of other agents, making them amenable to studies comparing the signaling pathways leading to apoptosis. In this study, we attempted to dissect some of the signaling pathways which lead to apoptosis of SUPT13 cells by different stimuli. We were particularly interested in the role of bcl-2 in regulating the cytotoxic effect of anti-TCR mAb and/or the rescuing effect of anti-CD3 mAb. Our results argue that both of these pathways occur independent of changes in bcl-2 expression.

The SUP-T13 cell line, a human T leukemia, is susceptible to apoptosis by various inducers, including anti-TCR mAb, calcium ionophores, and anti-fas mAb. Induction of apoptosis by these three agents was investigated, and several differences were found. All three agents induced DNA fragmentation with a similar time course, but the kinetics of cell death were different for the three agents. Anti-TCR mAb-induced apoptosis, but not A23187- or anti-fas-induced apoptosis, was rescued by anti-CD3 mAb treatment. In contrast, only anti-fas mAb-mediated apoptosis was rescued by PKC activators such as PMA. These differences suggest that each of these three agents mediate apoptosis by unique signaling pathways. Nevertheless, two variant subclones of SUP-T13 were found to be resistant to all three apoptosis-inducing agents, suggesting a point(s) of common regulation between the different pathways. To determine whether this regulation occurred through bcl-2, p53, or c-myc, their expression in the parental and variant cells was determined. The three clones expressed approximately equal amounts of these proteins, and their levels did not change significantly upon treatment with anti-TCR or anti-TCR plus anti-CD3 mAb. Thus, although the proximal signaling by various apoptosis inducers was quite different, a common mediator(s) (as yet unknown) may still regulate apoptosis induced by these multiple agents. q 1996 Academic Press, Inc.

INTRODUCTION

The vast majority of developing thymocytes in mammals are known to die by a process of apoptosis, as characterized by chromatin condensation, surface blebbing, cell shrinkage, and the characteristic cleavage of DNA into oligonucleosomal fragments (reviewed in [1– 3]). The signaling pathways which control apoptosis of thymocytes are not fully understood. In particular, it remains paradoxical how negative selection of self-re-

MATERIALS AND METHODS 1

To whom correspondence and reprint requests should be addressed.

Cell lines. SUP-T13, a gift of Steven Smith (University of Chicago, Chicago, IL), is a CD3/CD4/CD8/ human T acute lymphoblas95

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0014-4827/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tic leukemia cell line. HPB-ALL, another CD3/CD4/CD8/ human T acute lymphoblastic leukemia cell line, was a gift of Brad McIntyre (University of Texas, Houston, TX). All cell lines were grown in RPMI 1640 media supplemented with 10% fetal or newborn calf serum (Irvine Scientific, Santa Ana, CA) and 10 mg/ml gentamicin (Gibco, Grand Island, NY), hereafter referred to as ‘‘media.’’ Variants of SUP-T13, termed SUP-T13.A3 and SUP-T13.C8, were obtained by limiting dilution of parental SUP-T13 cells in microtiter plates. The media was supplemented with 50% culture supernatant from human thymic epithelial cells grown as described previously [12, 13]. Growing SUP-T13 clones were screened by addition of 10 mg/ml LC4 anti-TCR mAb and assessed for cell viability after 2 days. MAb. Clonotypic anti-TCR mAb to SUP-T13, named LC4 and LC11, were made by Shuji Takahashi (Sapporo Medical University, Sapporo, Japan); LC4 is described elsewhere [11]. Clonotypic antiTCR mAb to HPB-ALL, named 4-9E and 5-10C, are described elsewhere [14]. OKT3, a mAb to human CD3, was purchased from Ortho Diagnostic Systems (Raritan, NJ). JE6, another mAb to human CD3, is described elsewhere [11]. 2D1, a mAb to the human fas antigen, was a gift of Shuji Takahashi and is described elsewhere [15]. DNA fragmentation assays. SUP-T13 (5 1 106) cells were incubated in 5 ml media in the presence or absence of apoptosis inducing agents: 500 nM A23187, 500 ng/ml anti-fas mAb, or 500 ng/ml LC4. After incubation of 2–8 h at 377C, cells were washed in phosphatebuffered saline (PBS) and then lysed in 500 ml of TTE (0.2% Triton X-100, 10 mM Tris, pH 7.6, 15 mM EDTA). After 10 min at room temperature, samples were spun at 10,000g for 20 min at 47C in a microfuge. Supernatants were removed to new tubes and 25 ml of 10 mg/ml proteinase K was added. The samples were left at 507C overnight and then DNA was precipitated by addition of 100 ml 5 M NaCl and 0.7 ml isopropanol. Tubes were chilled on dry ice, then spun again as above, the pellets washed once with 70% ethanol, dried, and resuspended in 15 ml TE (10 mM Tris, pH 8, 15 mM EDTA) containing 10 mg/ml RNase A. After 5 min at 377C, samples were electrophoresed in 2% agarose gels and stained with ethidium bromide by a standard method [16]. Calcium flux assays. Ca2/ flux experiments were performed using a Ca2/-sensitive dye, FLUO-3/AM (Molecular Probes, Inc., Eugene, OR) and flow cytometry. Cells (106) were resuspended in 1.0 ml Hanks’ balanced salt solution containing 5% bovine calf serum (HBSS-BCS). Fifty micrograms of FLUO-3/AM was dissolved in 43.8 ml dimethyl sulfoxide. Twenty microliters of this solution and 5.0 ml of Pluronic F127 were added to the cells. Cells were rotated at 257C for 1 h and then washed three times and resuspended in 0.5 ml of HBSS-BCS. Two hundred microliters of cell suspension was added to 200 ml of mAb (8 mg/ml) and immediately analyzed by flow cytometry. Shifts in fluorescence were monitored for 180 s using the Chronys program (Becton–Dickinson, Mountain View, CA). IL-2 receptor (IL-2R) expression assays. Cells were incubated at 5 1 105 cells/ml in media in the presence of 1 mg/ml mAb for 72 h. Direct staining of 106 cells per sample was performed using 100 ml of FITC-conjugated anti-CD25 (1 mg/106 cells) (T Cell Sciences, Cambridge, MA). Samples were fixed using 2% paraformaldehyde in PBS prior to flow cytometry analysis. Cytokine secretion assays. Cells were incubated with 1 mg/ml mAb as above for 72 h and then supernatants were collected and used for enzyme-linked immunosorbent assays (ELISA). Interferon-g (IFNg) and tumor necrosis factor-a (TNF-a) ELISA reagents were a gift of Dr. Anita Chong (Rush Medical College, Chicago, IL). Microtiter plates were coated with 50 ml of either anti-IFNg mAb (15 mg/ml, Olympus, Lake Success, NY) or anti-TNF-a mAb (Endogen, Boston, MA). Plates were washed with PBS containing 0.05% Tween 20 (Sigma Chemical Co., St. Louis, MO) and blotted dry. Cytokine standards were made by diluting stock IFN-g or TNF-a to achieve a standard curve from 0 to 20 ng/ml. Fifty microliters of each standard or sample was incubated on the plates at 257C for 90 min, followed by 50 ml of either rabbit anti-human IFN-g (anti-serum prepared by Phil Scuderi, Miles Laboratories, Berkeley, CA) or goat anti-human

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TNF-a (polyclonal anti-serum, Endogen), and finally 50 ml of goat anti-rabbit or swine anti-goat Ig peroxidase (Boehringer-Mannheim, Indianapolis, IN). Optimum dilutions of each of these antisera were determined empirically and ranged from 1:1000 to 1:10,000. Plates were washed twice between each step and developed with 100 ml of a standard substrate (Sigma). Absorbances were read on an ELISA plate reader at 405 nm. Intracellular staining. Cells (106) were incubated in 1 ml media in the presence or absence of 50 mg/ml LC4 and/or 50 mg/ml JE6 for 1–8 h at 377C. The cells were washed once in cold PBS and then incubated for 10 min on ice in 2% paraformaldehyde in PBS. Triton X-100 was then added to 0.25% for an additional 10 min on ice. Next, cells were washed once with cold PBS and once with PBS containing 2% bovine serum albumin (BSA). The pellets were then resuspended in 10 ml of anti-bcl-2-FITC mAb (Dako, Carpenteria, CA), or 100 ml of anti-myc mAb culture supernatant (clone 9E10, American Type Culture Collection, Rockville, MD), or 100 ml of anti-p53 culture supernatant (clone DO-1, M. Oren, Weisman Institute, Rehovat, Israel) and incubated for 30 min on ice. For indirect staining, the cells were washed once in PBS / 2% BSA, resuspended in 100 ml of a 1:100 dilution of Fab2 goat anti-mouse IgG-FITC (Caltag, South San Francisco, CA), and incubated for 30 min on ice. Finally, the cells were washed with PBS / 2% BSA and resuspended in PBS / 2% paraformaldehyde for later analysis by flow cytometry as above.

RESULTS

Various agents induce DNA fragmentation in SUPT13 cells. SUP-T13 cells were incubated for 6 h with various concentrations of mAb against the TCR or fas antigen, or with A23187 (a calcium ionophore). Each of these agents induced DNA fragmentation characteristic of apoptosis (Fig. 1A), with similar dose–responses. A23187, however, induced slightly less DNA fragmentation than the two mAb. The kinetics of DNA fragmentation (Fig. 1B) were also similar with all three agents, although A23187 did not reach peak fragmentation until about 12 h (not shown). These kinetics of DNA fragmentation were not correlated with the kinetics of cell death, however. As seen in Fig. 1C, cell death induced by anti-fas mAb was much more rapid and complete than that induced by the other agents, and cell death induced by A23187 was slightly more rapid and complete than that induced by anti-TCR mAb. Thus, although anti-fas and anti-TCR mAb induced equally rapid and strong DNA fragmentation, they resulted in vastly different kinetics of cell death. Cotreatment with anti-CD3 mAb rescues apoptosis induced by anti-TCR mAb, but not other agents. The existence of different pathways to apoptosis in SUPT13 cells was further demonstrated by comparative killing and rescue experiments. Previously, cotreatment of SUP-T13 cells with anti-CD3 and anti-TCR mAb had been shown to rescue or prevent apoptosis [11]. We have now shown that this effect is specific to apoptosis by anti-TCR mAb (Fig. 2A). While treatment with anti-CD3 mAb rescued SUP-T13 cells from antiTCR mAb-induced apoptosis, anti-CD3 mAb failed to rescue A23187- or anti-fas mAb-induced apoptosis. In

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CD3 mAb must therefore trigger an additional intracellular signaling event. The timing of this signaling was investigated as shown in Fig. 2B. Cells were treated with anti-TCR mAb alone or with addition of anti-CD3 mAb at times from 0 to 20 h after anti-TCR mAb addition. It can be seen that the protective effect of antiCD3 mAb declines in as little as 1 h, but is still measurable 3 h (but not 20 h) after addition of anti-TCR mAb. Three of three anti-CD3 mAb tested were found to rescue anti-TCR mAb killing, and two of two anti-TCR mAb to SUP-T13 cells induced killing (data not shown). Thus, these phenomena appear to be target-specific, rather than peculiarities of one particular mAb. Early signaling events and cytokine induction patterns do not distinguish apoptosis-inducing anti-TCR mAb from apoptosis-inhibiting anti-CD3 mAb. Some signaling mechanisms which might explain the differential effects of anti-TCR and anti-CD3 mAb on SUPT13 cells include calcium flux, phosphorylation events, and cytokine/cytokine receptor induction. Because of the ability of anti-CD3 mAb to act late after addition of anti-TCR mAb, distal events such as cytokine/cytokine receptor induction seemed to be the most likely candidates for differences in signaling between the two types

FIG. 1. (A) Dose–response of DNA fragmentation of SUP-T13 by various agents. Anti-TCR mAb (LC4), A23187 (calcium ionophore), and anti-fas mAb (2D1) were used at the indicated concentrations and cells treated for 6 h as described under Materials and Methods. (B) Kinetics of DNA fragmentation by various agents. SUP-T13 cells were treated for 2–8 h, as indicated, with 500 ng/ml LC4, 500 nM A23187, or 500 ng/ml 2D1. (C) Kinetics of cell death by various agents. 106 SUP-T13 cells in 1 ml of media were treated with the above concentrations of LC4, A23187, or 2D1, and viability was determined by trypan blue exclusion at 1, 2, and 3 days. Although the kinetics of DNA fragmentation were similar for all three agents, the kinetics of cell death were quite different.

fact, it may even have enhanced killing by these two agents. The protective effect of anti-CD3 mAb was not caused by cell-surface blocking, since the two mAb do not inhibit each other’s binding [11]. Treatment with anti-

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FIG. 2. (A) Rescue of anti-TCR mAb-mediated apoptosis by antiCD3 mAb treatment. 106 SUP-T13 cells in 1 ml of media were incubated for 3 days in the presence of 10 mg/ml anti-TCR mAb LC4 or 2 mM A23187, or for 1 day in the presence of 1 mg/ml anti-fas mAb 2D1, with or without 10 mg/ml anti-CD3 mAb JE6, as indicated. Cell viability was determined by trypan blue exclusion. (B) Time course of anti-CD3 mAb rescue. Cells were treated with anti-TCR mAb, with anti-CD3 mAb added at the indicated times after anti-TCR mAb addition. Viability was determined after 2 days of culture.

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TABLE 1 Early Signaling Events and Cytokine Induction Profiles of Anti-TCR and Anti-CD3 mAb Cell line

mAb

Ca2/ fluxa (%)

IL2R exp.b (%)

IFN-g ng/ml

TNF-a ng/ml

SUP-T13

Anti-CD3 (OKT3) Anti-CD3 (JE6) Anti-TCR (LC4) Anti-TCR (LC11) Anti-CD3 (OKT3) Anti-CD3 (JE6) Anti-TCR (4-9E) Anti-TCR (5-10C)

192 152 155 155 155 133 158 142

536 165 1327 1246 134 132 166 155

0.2 17.0 ú20 ú20 0.2 0.2 0.3 0.3

ú20 ú20 ú20 ú20 4.5 4.2 3.8 3.8

HPB-ALL

a b

Maximum percent of baseline mean channel fluorescence during 3-min treatment. Percent of control expression after 72-h treatment.

of mAb. Nevertheless, calcium flux as well as induction of IL-2R, and secretion of IFN-g and TNF-a were chosen for study (Table 1). SUP-T13 cells were compared to HPB-ALL cells, a phenotypically similar T leukemia cell line for which anti-TCR mAb were available, but for which such mAb did not cause cell death. No significant differences in calcium fluxes were seen between two anti-CD3 and two anti-TCR mAb in SUPT13 cells. Also, levels of secreted TNF-a were similarly high in response to all anti-TCR and anti-CD3 mAb in SUP-T13. However, anti-TCR mAb in SUP-T13 cells (but not in HPB-ALL cells) induced high levels of IL-2R expression after 72 h incubation. To investigate whether IL-2 had any effect on apoptosis of SUP-T13, exogenous IL-2 was added in the presence or absence of anti-TCR and/or anti-CD3 mAb. IL-2 was not found to influence the kinetics or degree of cell killing or rescue (data not shown). Also, anti-TCR mAb induced a higher level of IFN-g secretion in SUP-T13 (but not HPB-ALL) cells. The importance of IFN-g in apoptosis was therefore tested by addition of exogenous IFN-g in the presence or absence of anti-TCR and/or anti-CD3 mAb. It also did not influence the kinetics or degree of cell killing or rescue (data not shown). Furthermore, addition of neutralizing antibodies to IFN-g had no effect (data not shown). From these experiments, we conclude that any differences seen in early signaling events or cytokine induction between anti-CD3 and anti-TCR mAb are not sufficient to explain their differential properties with regard to apoptosis induction. PMA rescues anti-fas-induced apoptosis, but not apoptosis induced by other agents. To further study differences in the signaling pathways leading to apoptosis, the ability of protein kinase A (PKA) and protein kinase C (PKC) activators to rescue or enhance apoptosis of SUP-T13 cells was investigated. Activators of PKA (IBMX and forskolin) showed little or no reproducible effects on apoptosis induced by various agents

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(data not shown). In addition, a PKA-specific peptide phosphorylation assay (Pierce Chemical Co., Rockford, IL) showed no difference in PKA activity between untreated SUP-T13 cells and cells treated for 1 h with anti-TCR mAb, anti-fas mAb, or A23187 (data not shown). By contrast, PKC activators were found to have differential effects upon apoptosis induced by these three agents. As shown in Figs. 3A–3C, PMA enhanced the killing of SUP-T13 cells treated with anti-TCR mAb or A23187, when used at levels (1–5 nM) that were not cytotoxic to SUP-T13 cells on their own. However, similar concentrations of PMA rescued the killing of SUP-T13 cells by anti-fas mAb (Fig. 3C). This rescuing ability was specific to the active (PKCbinding) isoform of PMA (Fig. 3D); a non-PKC-stimulating isomer, 4aPDD, did not rescue anti-fas killing. Furthermore, other activators of PKC (OAG and 1,2DiC8) showed similar, but less dramatic, effects on anti-fas killing (Fig. 3E). Similar rescue experiments with anti-fas mAb / PMA, OAG, and DiC8 were also carried out using normal, ConA-activated human T cells. They showed a similar pattern of rescue as in Fig. 3E, although there was even less killing by the anti-fas mAb (data not shown). These data suggest that the anti-fas-mediated pathway is differently coupled to PKC as compared to the anti-TCR and A23187 pathways. SUP-T13 variants are partially resistant to multiple apoptosis-inducing agents. In an attempt to further distinguish differences in signaling pathways leading to SUP-T13 apoptosis, apoptosis-resistant variants of SUP-T13 were isolated. SUP-T13 cells were plated at limiting dilution and screened for clones that were not killed by anti-TCR mAb. Two clones, termed A3 and C8, were selected in this manner. Surprisingly, these clones were found to be also resistant to killing by A23187 and anti-fas mAb, as well as anti-TCR mAb (Fig. 4). Anti-fas mAb led to the swiftest and most complete killing of parental SUP-T13 cells (Fig. 4C); anti-TCR

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resistant to all three apoptosis-inducing agents than were the parental cells. Apoptosis induced through the TCR/CD3 complex in normal T cells has been shown to proceed through induction of fas expression and secretion of fas ligand, a phenomenon termed activation-induced cell death. Thus, a possible explanation for the resistance of A3 and C8 cells to both fas and anti-TCR mAb would be decreased expression of cell-surface fas. However, we checked fas expression by flow cytometry in both untreated and A23187-activated SUP-T13, A3, and C8 cells. No difference in the level of fas expression was seen between different cells or between resting and activated cells (data not shown). Endogenous bcl-2 levels are not higher in SUP-T13 variants than in parental SUP-T13 cells. Overexpression of the bcl-2 oncoprotein has been found to protect various cells from apoptosis induced by multiple agents (reviewed in [17]). Thus, differences in bcl-2 lev-

FIG. 3. Effect of PMA treatment on apoptosis induced by various agents. (A–C) PMA enhances killing by anti-TCR mAb and A23187, but partially rescues killing by anti-fas mAb. (D) The rescue effect is specific to the PKC-binding isomer of PMA and is not seen with 4aPDD, an inactive isomer. (E) The rescue effect could be demonstrated to a lesser degree with other, less potent, PKC activators such as OAG and DiC8. H7, aPKC inhibitor, negates these effects. Each panel is representative of at least two similar experiments. Concentrations used were: PMA and 4aPDD, 1 nM; anti-TCR mAb, 10 mg/ml; A23187, 4 mM; anti-fas mAb, 100 ng/ml; OAG, 25 mM; DiC8, 20 mM; H7, 20 mM. 106 SUP-T13 cells in 1 ml were treated for 2–3 h, washed twice, and resuspended in fresh media. Cell viability was determined by trypan blue exclusion after 2 days (C) or 3 days (A, B, D, and E).

mAb, by contrast, was the slowest and weakest killing agent (Fig. 4A). The variants A3 and C8 appeared to have a general but incomplete resistance to apoptosis induction, as they demonstrated the greatest resistance to anti-TCR mAb, and the least resistance to antifas mAb. There were slight differences between the two variants. A3 cells were slightly more resistant to A23187 than C8 cells, and C8 cells were slightly more resistant to anti-fas mAb than A3 cells. Although these differences were reproducible in multiple experiments (not shown), they were overshadowed by the larger differences between either of the variants and the parental SUP-T13 cells. A3 and C8 cells were always more

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FIG. 4. Resistance of SUP-T13 variants to multiple agents. 106 parental SUP-T13 cells, SUP-T13.A3, or SUP-T13.C8 variants were treated in 1 ml of media with 10 mg/ml LC4 anti-TCR mAb (A), or 2 mM A23187 (B), or 100 ng/ml 2D1 anti-fas mAb (C). Cell viability was assessed by trypan blue exclusion after 1, 2, and 3 days.

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FIG. 5. (A) Levels of constitutive expression of bcl-2 in SUP-T13 parental and variant cells. Intracellular bcl-2 staining was done as described under Materials and Methods. Normal human PBL were used as a negative control, and the EBV-transformed B cell line EBNA was used as a positive control. All samples were stained in triplicate, and error bars represent {SD of the mean. Mean channel fluorescences were normalized to parental SUP-T13 cells, which were arbitrarily assigned a value of 100%. (B) Time course of bcl-2 expression in SUP-T13 parental and variant cells. Intracellular bcl-2 staining was done as described under Materials and Methods. All samples were stained in triplicate, and the means were expressed as a percent of control (untreated) cells for each time point.

els might explain the observed resistance of A3 and C8 cells to various apoptotic stimuli. To test this hypothesis, cells were permeabilized and stained with a mAb to bcl-2 (Fig. 5). As controls, an EBV-transformed B cell line and normal human PBL were used, respectively, to show the upper and lower ranges of detection of the assay. Parental SUP-T13 cells were found to express an intermediate level of bcl-2 protein (Fig. 5A). This was also confirmed by reverse transcription and PCR assays (not shown). Neither variant, A3 or C8, expressed elevated levels of bcl-2 compared to parental SUP-T13; A3 cells, in fact, reproducibly stained slightly lower with the anti-bcl-2 mAb, though this difference was not statistically significant. Thus, differences in endogenous levels of bcl-2 could not account for the apoptosis resistance of variants A3 and C8. Parental and variant SUP-T13 cells show no change in bcl-2 expression in response to anti-TCR or anti-TCR plus anti-CD3 mAb. It remained possible that treatment with anti-TCR mAb might induce changes in bcl2 expression, which were different in A3 and C8 cells compared to parental SUP-T13 cells. To test this, time courses of anti-TCR mAb treatment were performed,

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and cells stained for bcl-2 (Fig. 5B). Although slight fluctuations in bcl-2 levels were observed in parental cells, no significant differences in response patterns could be seen that might explain the resistance of the variants. In fact, bcl-2 levels in both parent and variant cells never fluctuated by more than 20% from control (untreated) levels during 8 h of treatment. This result was confirmed for the 4-h treatment point by using the same anti-bcl-2 mAb (in unconjugated form) in a Western blot (not shown). No difference in staining intensity could be seen between control and anti-TCRtreated cells in that experiment. Finally, the ability of anti-CD3 mAb to rescue antiTCR mAb-mediated apoptosis might have been explained by an induction of bcl-2 expression. However, Fig. 5B shows that parental cells treated with antiTCR / anti-CD3 mAb expressed similar levels of bcl2 as did cells treated with anti-TCR mAb alone. Thus, induction or repression of bcl-2 expression cannot explain the protective effect of anti-CD3 mAb versus antiTCR mAb. Parental and variant SUP-T13 cells also show little difference in p53 or c-myc levels. Two gene products which have been shown to have apoptosis-inducing capabilities in various systems are p53 and c-myc. Since bcl-2 levels did not explain the resistance of A3 and C8 cells to apoptosis, we decided to measure levels of these two proteins as well. Using a similar permeabilization and fluorescent staining assay, we saw only very slight differences in endogenous p53 levels between parental and variant cells (Fig. 6A). These differences were probably not biologically significant, as the variants expressed only about 15% less p53 protein than the parental cells. These differences also disappeared after 8 h of treatment with anti-TCR mAb. No difference was seen between anti-TCR versus anti-TCR / anti-CD3 mAb-treated parental cells. It should be noted that the mAb used, DO-1, did not distinguish between wild-type and mutant forms of p53, but rather measured total p53 protein. There may have been a difference in the ratio of mutant to wild-type p53 in variant cells compared to parental cells that was not reflected in the overall level of p53 expression. Similar results were obtained with a mAb to c-myc (Fig. 6B). No differences in endogenous levels were seen between A3, C8, and parental cells, and only slight differences between the variants and parental cells were seen after 1–8 h of anti-TCR mAb treatment. The direction and magnitude of these differences make them unlikely to account for the apoptosis resistance of the variant cells. Instead, we postulate that there may be other unknown mediators that impact upon the sensitivity of these cells to multiple apoptosis-inducing agents. DISCUSSION

Multiple lines of evidence now point to the conclusion that TCR-mediated apoptosis, such as during thymic

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FIG. 6. Time course of p53 and myc expression in SUP-T13 parental and variant cells. Intracellular staining for p53 (A) or c-myc (B) was carried out as described under Materials and Methods. The mean channel fluorescence of 10,000 cells for each time point is shown.

selection, is at least partially bcl-2-independent. Our results using a potential model of thymic selection, SUP-T13 cells and anti-TCR/CD3 mAb, are in agreement with this notion. We found that variant SUP-T13 cells which were not killed by anti-TCR mAb did not express higher levels of bcl-2, nor did they regulate bcl-2 levels differently during treatment with antiTCR mAb. While it is still possible that bcl-2 overexpression might rescue SUP-T13 cells from anti-TCR-mediated apoptosis, such a result is unlikely based on the results of other groups working in different systems. For example, Sentman et al. [9] found that negative selection of Vbeta5, Vbeta11, and Vbeta17a/ thymocytes still occurred in bcl-2 transgenic mice. However, thymocytes from such mice did show resistance to anti-CD3 mAb-induced apoptosis in vitro. Cuende et al. [18] found that overexpression of bcl2 did not confer resistance to anti-IgM mAb-induced apoptosis on WEHI-231 cells, an immature B cell line. Thus, bcl-2-independent pathways may exist for antigen-receptor stimulation in both B and T cells. On the other hand, stimulation of germinal center B cells via CD40 results in a bcl-2-dependent rescue from apoptosis [19]. In addition, overexpression of a bcl-2 transgene under control of an immunoglobulin enhancer element leads to an increase in memory B cells, increased B cell lifespan, and increased B cell neoplasia

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[20, 21]. Thus, bcl-2 clearly is important for B cell development and differentiation. Other apoptosis pathways in T cells may be bcl-2dependent as well. For example, Itoh et al. [22] found that expression of a bcl-2 transgene in the T lymphoma cell line WR19L partially rescued the cells from fasmediated killing. In B-CLL cells, Mapara et al. [23] found a correlation between bcl-2 downregulation and susceptibility to fas-mediated killing. In fact, we also found a partial loss of bcl-2 staining intensity in SUPT13 cells treated with anti-fas mAb, prior to cell death (data not shown). Multiple other genes have been shown to impact upon the susceptibility of various cell types to apoptosis. These include homologues of bcl-2 (e.g., bclx/bax) as well as genes involved in transformation and cell cycle control (e.g., c-fos, c-myc and p53). While we examined p53 and c-myc levels in SUP-T13 cells and their variants, levels of all of these protein products have not been examined in sufficient detail to rule out their involvement in the resistance of A3 and C8 cells to apoptosis. Signals for apoptosis have also been shown to be generated through the action of cytokines, notably IL-2 [24], TNF-a [25], and IFN-g [26, 27] in T cells. In our cells, correlations were found between IL-2R induction, IFN-g production, and apoptosis by anti-TCR mAb. However, addition of exogenous IL-2 or IFN-g, or blocking antibody to IFN-g, did not affect the outcome of anti-TCR or anti-TCR / CD3 mAb treatment. Thus, we do not postulate a role for these cytokines in TCRmediated apoptosis. Activation of protein kinases, particularly PKA and PKC, has also been reported to influence apoptosis of T cells [28, 29] as well as other cell types. We directly assayed PKA activation by a peptide phosphorylation assay in SUP-T13 cells and variants, with or without stimulation by anti-TCR mAb, anti-fas mAb, or A23187. While forskolin was found to upregulate PKA activity in all cells, no differences in PKA activity were found between parent and variant cells or between any of the apoptosis-inducing treatments and control cells. Although PKC activity was not measured directly in our cells, we found that PKC activators such as PMA could rescue SUP-T13 cells from fas-mediated death, while enhancing the killing by anti-TCR mAb and A23187. Thus, PKC appears to be differently coupled to these three apoptotic pathways. Inhibitors of PKC could not be productively examined in this system, since they were too toxic to use at physiologically active concentrations (data not shown). Another signaling difference between apoptosis inducers in SUP-T13 was the ability of anti-CD3 mAb to preferentially rescue killing by anti-TCR mAb. Even though CD3 and TCR are part of the same cell-surface complex, the rescue effect was not due to cell-surface blocking. This implies the existence of signals gener-

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ated through CD3 that uniquely block those generated through TCR. Similarly, in thymic selection, signals generated through the TCR/CD3 complex can presumably lead to either positive or negative selection, based upon the affinity of the interaction. Thus, there is precedence for qualitatively different signals being generated through the same surface receptor complex. Finally, a role for oxidative metabolism as a final common pathway for apoptosis has been postulated (reviewed in [30]). In particular, alteration of redox potential by treatment of cells with 2-mercaptoethanol can prevent some forms of growth inhibition in B cells [31]. Although 2-mercaptoethanol provided a growth advantage to SUP-T13 cells, it provided only slight inhibition of killing by anti-TCR mAb (data not shown). Thus, the role of intracellular thiols in TCR-mediated apoptosis of SUP-T13 cells is unclear. In conclusion, we have described a model system for apoptosis, SUP-T13, which may have relevance to thymic selection. Three lines of evidence serve to distinguish the different apoptosis-inducing agents examined in this system. First, different kinetics of cell death (which are not correlated with the kinetics of DNA fragmentation) could be seen with the three apoptosis mediators studied. Second, susceptibility to anti-CD3 mAb rescue could discriminate apoptosis induced by anti-TCR mAb from that induced by other agents. Third, susceptibility to PKC activators could be used to differentiate the fas-mediated pathway from those mediated by anti-TCR mAb or A23187. Despite these differences, we isolated variants of SUP-T13 with partial resistance to multiple inducers of apoptosis, suggesting the action of a common mediator on the various apoptosis pathways. This common mediator does not appear to be bcl-2 and is unlikely to be p53 or c-myc, based on our data. Thus, we postulate that other unknown factors can regulate the multiple apoptosis pathways in these cells. The authors thank Patricia Simms and Debra Czerwinski for technical assistance with flow cytometry experiments; Danish Haque and Mary Ibrahim for technical assistance with cell viability experiments; and Shoshana and Ronald Levy for providing reagents and helpful discussions. This work was supported by a grant from the American Cancer Society, Illinois Division, to H.T.M, and by PHS Grant AI38865 to P.T.L.

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Received July 10, 1995 Revised version received September 15, 1995

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