Cell Receptor-induced Apoptosis Research Articles

STAYING ALIVE! SIGNAL PROCESSING BY NF-κB PROTECTS NKT CELLS FROM TNFR1-TRIGGERED DEATH

Abstract A unique ontogenetic program controls the thymic development of type I nature killer T (NKT) cells. For example, signal integration by NF-κB plays a critical role in NKT cell development such that RelA and RelB play cell intrinsic and cell extrinsic roles, respectively. The key cell extrinsic role played by NF-κB seems to be the induction of IL-15 production that is required for the development and homeostasis of NKT cells. On the other hand, cell intrinsic NF-κB activity is essential for the development of both NK1.1neg and NK1.1pos NKT cells. We have demonstrated that NKT cells in mice expressing a dominant negative mutant IκBDNtg that prevents NF-κB activation were arrested at a very early stage of their development. Importantly, introgression of Bcl-xL-coding Bcl2l1 transgene into IκBDNtg mice lead to the development of NK1.1pos NKT cells. Further investigations revealed that NKT cell intrinsic NF-κB activation was required to protect developing NKT cells from tumor necrosis factor (TNF) receptor 1-induced cell death and not from Fas/FasL- or NKT T cell receptor-induced apoptosis. Consistent with this finding, we found that TNF-α induces the activation of caspase 8- and 9-mediated apoptosis in NKT cells in which NF-κB activation was blocked. Thus, akin to hepatocytes and some cancer cells, TNF-α induces both cell intrinsic (caspase 8-mediated) and cell extrinsic (caspase 9-mediated) apototic pathways in developing NKT cells that are offset by the activation of NF-κB and the expression of one of its target gene, Bcl2l1.

Inhibition of p300 impairs Foxp3+ T regulatory cell function and promotes antitumor immunity

Foxp3+ T-regulatory (Treg) cells maintain immune homeostasis and limit autoimmunity, but can also curtail host immune responses to various types of tumors1,2. Foxp3+ Tregs are therefore considered promising targets to enhance anti-tumor immunity, and efforts are underway to develop approaches for their therapeutic modulation. However, while studies showing that Foxp3+ Treg depletion experimentally can enhance anti-tumor responses provide proof-of-principle, they lack clear translational potential and have various shortcomings. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and non-histone proteins3,4. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (Ep300, KAT3B), in Foxp3+ Tregs, increased TCR-induced apoptosis in Tregs, impaired Treg suppressive function and peripheral Treg induction, and limited tumor growth in immunocompetent, but not in immunodeficient, hosts. Our data thereby demonstrate that p300 is important for Foxp3+ Treg function and homeostasis in vivo and in vitro, and identify novel mechanisms by which appropriate small molecule inhibitors can diminish Treg function without overtly impairing T-effector (Teff) cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.

Kaposi's Sarcoma-Associated Herpesvirus Oncoprotein K13 Protects against B Cell Receptor-Induced Growth Arrest and Apoptosis through NF-κB Activation

Kaposi's sarcoma-associated herpesvirus (KSHV) has been linked to the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (MCD). We have characterized the role of KSHV-encoded viral FLICE inhibitory protein (vFLIP) K13 in the modulation of anti-IgM-induced growth arrest and apoptosis in B cells. We demonstrate that K13 protects WEHI 231, an immature B-cell line, against anti-IgM-induced growth arrest and apoptosis. The protective effect of K13 was associated with the activation of the NF-κB pathway and was deficient in a mutant K13 with three alanine substitutions at positions 58 to 60 (K13-58AAA) and a structural homolog, vFLIP E8, both of which lack NF-κB activity. K13 upregulated the expression of NF-κB subunit RelB and blocked the anti-IgM-induced decline in c-Myc and rise in p27(Kip1) that have been associated with growth arrest and apoptosis. K13 also upregulated the expression of Mcl-1, an antiapoptotic member of the Bcl2 family. Finally, K13 protected the mature B-cell line Ramos against anti-IgM-induced apoptosis through NF-κB activation. Inhibition of anti-IgM-induced apoptosis by K13 may contribute to the development of KSHV-associated lymphoproliferative disorders.

C-Cbl Promotes T Cell Receptor-induced Thymocyte Apoptosis by Activating the Phosphatidylinositol 3-Kinase/Akt Pathway

The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.

B cell receptor-induced growth arrest and apoptosis in WEHI-231 immature B lymphoma cells involve cyclic AMP and Epac proteins

Signaling by the B cell antigen receptor (BCR) is essential for B lymphocyte homeostasis and immune function. In immature B cells, ligation of the BCR promotes growth arrest and apoptosis, and BCR-driven balancing between pro-apoptotic extracellular signal-regulated kinase 1 and 2 (ERK1/2) and anti-apoptotic phosphoinositide 3-kinase-dependent Akt seems to define the final cellular apoptotic response. Dysfunction of these late BCR signaling events can lead to the development of immunological diseases. Here we report on novel cyclic AMP-dependent mechanisms of BCR-induced growth arrest and apoptosis in the immature B lymphoma cell line WEHI-231. BCR signaling to ERK1/2 and Akt requires cyclic AMP-regulated Epac, the latter acting as a guanine nucleotide exchange factor for Rap1 and H-Ras independent of protein kinase A. Importantly, activation of endogenously expressed Epac by a specific cyclic AMP analog enhanced the induction of growth arrest (reduced DNA synthesis) and apoptosis (nuclear condensation, annexin V binding, caspase-3 cleavage and poly-ADP-ribose polymerase processing) by the BCR. Our data indicate that cyclic AMP-dependent Epac signals to ERK1/2 and Akt upon activation of Rap1 and H-Ras, and is involved in BCR-induced growth arrest and apoptosis in WEHI-231 cells.

RasGRP1 Sensitizes an Immature B Cell Line to Antigen Receptor-induced Apoptosis

RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that confer survival and proliferation, but RasGRP1 also promotes T cell receptor-mediated deletion of mature T cells. We used the WEHI-231 cell line as an experimental system to determine whether RasGRP1 can serve as a quantitative modifier of B cell receptor-induced deletion of immature B cells. A 2-fold elevation in RasGRP1 expression markedly increased apoptosis of WEHI-231 cells following B cell receptor ligation, whereas a dominant negative mutant of RasGRP1 suppressed B cell receptor-induced apoptosis. Activation of ERK1 or ERK2 kinases was not required for RasGRP1-mediated apoptosis. Instead, elevated RasGRP1 expression caused down-regulation of NF-kappaB and Bcl-x(L), which provide survival signals counter-acting apoptosis induction by B cell receptor. Inhibition of NF-kappaB was sufficient to enhance B cell receptor-induced apoptosis of WEHI-231 cells, and ligation of co-stimulatory receptors that activate NF-kappaB suppressed the ability of RasGRP1 to promote B cell receptor-induced apoptosis. These experiments define a novel apoptosis-promoting pathway leading from B cell receptor to the inhibition of NF-kappaB and demonstrate that differential expression of RasGRP1 has the potential to modulate the sensitivities of B cells to negative selection following antigen encounter.

NFκB Controls the Balance between Fas and Tumor Necrosis Factor Cell Death Pathways during T Cell Receptor-induced Apoptosis Via the Expression of Its Target Gene A20

Activation-induced cell death (AICD), a term originally coined for the anti-CD3-induced apoptosis of T cell hybridomas and thymocytes, is predominantly driven by death receptors and has been involved in the control of autoreactive T cells in the periphery. In the Do-11.10 T cell hybridoma model of AICD, activation of the T cell receptor (TCR) results in Fas-dependent apoptosis. Here, we show that inhibition of the transcription factor nuclear factor kappa B (NF kappa B) in Do-11.10 cells resulted in increased sensitivity to TCR-mediated apoptosis, correlating with defective induction of the anti-apoptotic NF kappa B target gene A20. Stable expression of the zinc finger protein A20 in NF kappa B-negative Do-11.10 cells rescued the phenotype. TCR activation in NF kappa B-deficient Do-11.10 cells resulted predominantly in tumor necrosis factor (TNF) receptor 2 (TNFR2)-dependent bystander cell death rather than classical Fas-dependent AICD. Strikingly, A20 blocked TNF-mediated apoptosis and simultaneously restored TCR-induced Fas-dependent AICD. In addition, NF kappa B downstream of TNFR was required for up-regulation of Fas expression by endogenous TNF secreted in response to TCR stimulation. Together, these results suggest that NF kappa B can play both pro- and anti-apoptotic roles during AICD. We propose that NF kappa B controls the balance between Fas and TNF cell death pathways during AICD via the expression of the zinc finger protein A20.

Src homology region 2 domain-containing phosphatase 1 positively regulates B cell receptor-induced apoptosis by modulating association of B cell linker protein with Nck and activation of c-Jun NH2-terminal kinase.

Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a key mediator in lymphocyte differentiation, proliferation, and activation. We previously showed that B cell linker protein (BLNK) is a physiological substrate of SHP-1 and that B cell receptor (BCR)-induced activation of c-Jun NH(2)-terminal kinase (JNK) is significantly enhanced in cells expressing a form of SHP-1 lacking phosphatase activity (SHP-1-C/S). In this study, we confirmed that SHP-1 also exerts negative regulatory effects on JNK activation in splenic B cells. To further clarify the role of SHP-1 in B cells, we examined how dephosphorylation of BLNK by SHP-1 affects downstream signaling events. When a BLNK mutant (BLNK Delta N) lacking the NH(2)-terminal region, which contains four tyrosine residues, was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK activation was inhibited. Among candidate proteins likely to regulate JNK activation through BLNK, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of dominant-negative forms of Nck inhibited BCR-induced JNK activation. Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing cells and coexpression of Nck SH2 mutants or a dominant-negative form of SEK1 reversed this phenotype. Collectively, these results suggest that SHP-1 acts on BLNK, modulating its association with Nck, which in turn negatively regulates JNK activation but exerts a positive effect on apoptosis.

An absolute requirement for Fyn in T cell receptor-induced caspase activation and apoptosis.

SPECIFIC AIMWe investigated the possible implication of the tyrosine kinase p59Fyn in TCR-mediated caspase activation and apoptosis in a T cell hybridoma (T8.1 cells) overexpressing different Fyn constructs and in mouse embryo fibroblasts (MEFs) and thymocytes from control or Fyn knockout mice.PRINCIPAL FINDINGS1. Cleavage of Fyn upon TCR triggering in T lymphocytesWe first analyzed the cleavage of different molecular forms of Fyn stably expressed in a T cell hybridoma, T8.1. Soluble anti-CD3 mAb failed to induce the cleavage of WT Fyn, but immobilized anti-CD3 was found to generate the cleaved p57 form of the kinase (Fig. 1A⤻ ). Cleavage of Fyn was clearly detectable within 4 h of anti-CD3 treatment and was found to be maximal at 8 h (Fig. 1B⤻ ). Conversely, a kinase-dead mutant (Fyn KD) and a soluble form of Fyn (Fyn ΔN-ter) were not cleaved after CD3 triggering. Cleavage of Fyn upon TCR engagement, which is reminiscent of our previous observation that Fas induces Fyn cleavage in T lymphocytes, prompted...

Moving in for a Kill

The TR3 protein (also named nur77 or NGFI-B) is a member of the nuclear hormone receptor family and is required for T cell receptor-induced apoptosis. These receptors are thought to act primarily as ligand-dependent transcription factors. However, Li et al . found that TR3 function does not require the DNA binding domain of the protein. Instead, TR3 promotes apoptosis by translocating to the mitochodria, where it causes release of cytochrome c. Li, H., Kolluri, S.K., Gu, J., Dawson, M.I., Cao, X., Hobbs, P.D., Lin, B., Chen, G., Lu, J., Lin, F., Xie, Z., Fontana, J.A., Reed, J.C., and Zhang X. (2000) Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science 289 : 1159-1164. [Abstract] [Full Text]

T cell receptor-induced activation and apoptosis in cycling human T cells occur throughout the cell cycle.

Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0-G1, S, and G2-M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca(2+) flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle.

Mitochondria Connects the Antigen Receptor to Effector Caspases During B Cell Receptor-Induced Apoptosis in Normal Human B Cells

We have previously reported that CD40 stimulation sensitizes human memory B cells to undergo apoptosis upon subsequent B cell receptor (BCR) ligation. We have proposed that activation stimuli connect the BCR to an apoptotic pathway in mature B cells and that BCR-induced apoptosis of activated B cells could serve a similar function as activation-induced cell death in the mature T cell compartment. Although it has been reported that caspases are activated during this process, the early molecular events that link the Ag receptor to these apoptosis effectors are largely unknown. In this study, we report that acquisition of susceptibility to BCR-induced apoptosis requires entry of memory B cells into the S phase of the cell cycle. We also show that transduction of the death signal via the BCR sequentially proceeds through a caspase-independent and a caspase-dependent phase, which take place upstream and downstream of the mitochondria, respectively. Furthermore, our data indicate that the BCR-induced alterations of the mitochondrial functions are involved in activation of the caspase cascade. We have found both caspases-3 and -9, but not caspase-8, to be involved in the BCR apoptotic pathway, thus supporting the notion that initiation of the caspase cascade could be under the control of the caspase-9/Apaf-1/cytochrome c multimolecular complex. Altogether, our findings establish the mitochondria as the connection point through which the Ag receptor can trigger the executioners of apoptotic cell death in mature B lymphocytes.

A role for neutral sphingomyelinase-mediated ceramide production in T cell receptor-induced apoptosis and mitogen-activated protein kinase-mediated signal transduction.

Studying apoptosis induced by T cell receptor (TCR) cross-linking in the T cell hybridoma, 3DO, we found both neutral sphingomyelinase activation and production of ceramide upon receptor engagement. Pharmacological inhibition of ceramide production by the fungal toxin, fumonisin B1, impaired TCR-induced interleukin (IL)-2 production and programmed cell death. Addition of either exogenous ceramide or bacterial sphingomyelinase reconstituted both responses. Moreover, specific inactivation of neutral sphingomyelinase by antisense RNA inhibited IL-2 production and mitogen-activated protein kinase activation after TCR triggering. These results suggest that ceramide production by activation of neutral sphingomyelinase is an essential component of the TCR signaling machinery.

B cell receptor-induced apoptosis in primary transitional murine B cells: signaling requirements and modulation by T cell help.

Self-reactive immature B cells may be eliminated in the bone marrow (BM) after B cell receptor (BCR) engagement in a process known as negative selection. Immature B cells emigrating from the BM, the so-called transitional cells, remain sensitive to negative selection and are likely to be important targets of tolerance towards peripheral antigens. Transitional cells are deleted through apoptosis after BCR cross-linking in vitro. Using anti-Ig as a surrogate antigen, we determined the signaling requirements for the induction of apoptosis in transitional cells. Treatment with anti-Ig for only 20 min causes most cells to be apoptotic 16 h later. Furthermore, apoptosis of transitional cells is induced with low doses of anti-Ig while mature cell proliferation requires extended culture at 30-fold higher concentrations. For both populations of B cells, total surface Ig expression is equivalent, therefore indicating that the threshold of BCR signaling required to elicit these responses is different. T cell help can modulate B cell tolerance. However, specific help may not be available when apoptosis is triggered by a peripheral antigen. The opportunity to reverse apoptosis of transitional cells is surprisingly long. Even 8 h after anti-Ig treatment, IL-4 or anti-CD40 antibody can block apoptosis. The upper time limit of protection is concurrent with irreversibility of apoptosis as measured by DNA fragmentation. These findings indicate that B cell negative selection is more easily triggered than activation, and that the induction of apoptosis and its reversal by T cell help can be events that occur in distinct microenvironments.

Molecular and Cellular Mechanisms of T Lymphocyte Apoptosis

This chapter focuses on the genetic and molecular mechanisms of T lymphocyte apoptosis and also deals with the various phases of apoptosis initiation, execution, and degradation of lymphocytes. Apoptosis is a regulated (programmed) device for the removal of superfluous, aged, or damaged cells within the immune system, as well as in all other organ systems. It is fundamental for development, throughout embryogenesis, organ metamorphosis and organogenesis, including synaptic interactions of neurons, and repertoire selection of T lymphocytes. The activation of T cells generates lymphocytes that are prone to apoptosis, unless these cells are rescued by IL-2 or reside in a specialized environment. Thus, activated lymphocytes undergo programmed cell death by default in the absence of rescue signals mediated by cytokines or cell-cell contacts. Although in determined circumstances T cell death can be induced by starvation from IL-2, this lymphokine can also facilitate T cell apoptosis. Thus, pretreatment with IL-2 renders T cells susceptible to T cell receptor-induced apoptosis, perhaps by dysregulated activation of the cell cycle or by influencing Fas/Fas-L signaling.

Role of Egr-1 Gene Expression in B Cell Receptor-induced Apoptosis in an Immature B Cell Lymphoma

Ligation of B cell receptor (BCR) on BKS-2, an immature B cell lymphoma by anti-IgM antibodies (Ab) caused apoptosis. Here we report that signaling through B cell receptor in wild type BKS-2 cells down-regulated the expression of Egr-1, a zinc finger-containing transcription factor. A reduction in the level of Egr-1 mRNA could be demonstrated as early as 30 min after the ligation of BCR on BKS-2 cells. Immunocytochemical and Western blot analysis revealed that the expression of EGR-1 protein was also inhibited by anti-IgM treatment. Antisense oligonucleotides to Egr-1 caused growth inhibition and apoptosis in BKS-2 cells, suggesting that expression of Egr-1 is important for the survival of these B lymphoma cells. In contrast to wild type BKS-2 cells, the mutant 1. B5 cell line, which is refractory to B cell receptor-mediated growth-inhibitory signals, showed an increased expression of Egr-1 upon treatment with anti-IgM. These results implicate a role for Egr-1 in blocking B cell receptor-mediated apoptosis in immature B cells.

A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis.

By comparing untreated and dexamethasone-treated murine T cell hybridoma (3DO) cells by the differential display technique, we have cloned a new gene, GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) encoding a new member of the tumor necrosis factor/nerve growth factor receptor family. GITR is a 228-amino acids type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain and similar to CD27 and 4-1BB in the intracellular domain. GITR resulted to be expressed in normal T lymphocytes from thymus, spleen, and lymph nodes, although no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. Furthermore, GITR expression was induced in T lymphocytes upon activation by anti-CD3 mAb, Con A, or phorbol 12-myristate 13-acetate plus Ca-ionophore treatment. The constitutive expression of a transfected GITR gene induced resistance to anti-CD3 mAb-induced apoptosis, whereas antisense GITR mRNA expression lead to increased sensitivity. The protection toward T cell receptor-induced apoptosis was specific, because other apoptotic signals (Fas triggering, dexamethasone treatment, or UV irradiation) were not modulated by GITR transfection. Thus, GITR is a new member of tumor necrosis factor/nerve growth factor receptor family involved in the regulation of T cell receptor-mediated cell death.

Immune responses in mice deficient in Ly-GDI, a lymphoid-specific regulator of Rho GTPases

Ly-GDI (lymphoid-specific guanosine diphosphate (GDP) dissociation inhibitor), also called D4-GDI, is preferentially expressed in hematopoietic tissues including bone marrow, thymus, spleen and lymph nodes. It binds to the small guanosine triphosphate (GTP)-binding protein Rho and inhibits GDP dissociation from Rho proteins. To explore the function of Ly-GDI in lymphocytes, we have generated Ly-GDI-deficient mice by gene targeting. These mice showed no striking abnormalities of lymphoid development or thymocyte selection. The mice also exhibited, for the most part, normal immune responses including lymphocyte proliferation, IL-2 production, cytotoxic T lymphocyte activity, antibody production, antigen processing and presentation, immune cell aggregation and migration, and protection against an intracellular protozoan. However, Ly-GDI-deficient mice exhibited deregulated T and B cell interactions after in vitro cultivation of mixed lymphocyte populations in concanavalin A (Con A) leading to overexpansion of B lymphocytes. Further studies revealed that Ly-GDI deficiency decreased IL-2 withdrawal apoptosis of lymph node cells while dexamethasone- and T cell receptor-induced apoptosis remained intact. These data implicate the regulation of the Rho GTPase by Ly-GDI in lymphocyte survival and responsiveness, but suggest that these functions may be partially complemented by other Rho regulatory proteins when the LyGDI protein is deficient.

Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided by CD28.

CD4+CD8+ thymocytes expressing self-reactive T cell antigen receptors (TCR) are deleted in the thymus as a consequence of TCR/self-antigen/major histocompatibility complex interactions. However, the signals that are necessary to initiate clonal deletion have not yet been clarified. Here we demonstrate that TCR engagement does not efficiently induce apoptosis of CD4+CD8+ thymocytes, although it generates signals that increase expression of CD5, a thymocyte differentiation marker. In fact, TCR signals fail to induce thymocyte apoptosis even when augmented by simultaneous engagement with CD4 or lymphocyte function 1-associated molecules. In marked contrast, signals generated by engagement of both TCR and the costimulatory molecule CD28 potently induce apoptosis of CD4+CD8+ thymocytes. Thus, the present results define a requirement for both TCR and costimulatory signals for thymocyte apoptosis and identify CD28 as one molecule that is capable of providing the necessary costimulus. These results provide a molecular basis for differences among cell types in their ability to mediate negative selection of developing thymocytes.

Propriocidal apoptosis of mature T lymphocytes occurs at S phase of the cell cycle

We found that mature nontransformed CD4+ and CD8+ T lymphocytes could be made susceptible to T cell receptor(TcR)-mediated apoptosis by pretreatment with interleukin-4 (IL-4) or interleukin-2 (IL-2). The degree of susceptibility to death could be correlated with the level of cell cycling as measured by thymidine incorporation, cell doubling times, or the number of cells incorporating bromodeoxyuridine during S phase. However, using pharmacologic cell cycle blocking agents, we found that progression through the cell cycle was not required for cell death. Rather, we found that cells must be in a certain phase of the cell cycle to be susceptible to TcR-mediated death. Cells blocked in G1 phase were resistant to T cell receptor-induced apoptosis, whereas cells blocked in S phase were susceptible. These observations suggest that an important feature of growth lymphokines is their ability to drive T cells into portions of the cell cycle where they are sensitive to antigen receptor-induced apoptosis. Furthermore, these results provide additional evidence that the T cell growth lymphokines IL-2 and IL-4 may participate in the down-regulation of T cell responses by apoptosis-a pathway we have termed "propriocidal regulation".