Deficiency of Trp53 rescues the male fertility defects of Kit(W-v) mice but has no effect on the survival of melanocytes and mast cells.

Mutations of the receptor tyrosine kinase, Kit, or its ligand, mast growth factor (Mgf), affect three unrelated cell populations: melanocytes, germ cells, and mast cells. Kit signaling is required initially to prevent cell death in these lineages both in vitro and in vivo. Mgf appears to play a role in the survival of some hematopoietic cells in vitro by modulating the activity of p53. Signaling by Mgf inhibits p53-induced apoptosis of erythroleukemia cell lines and suppresses p53-dependent radiation-induced apoptosis of bone marrow cells. We tested the hypothesis that cell survival in Kit mutant mice would be enhanced by p53 deficiency in vivo. Double-mutant mice, which have greatly reduced Kit receptor tyrosine kinase activity and also lack Trp53, were generated and the affected cell lineages examined. Mast cell, melanoblast, and melanocyte survival in the double Kit(W-v/W-v):Trp53(-/-) mutants was not increased compared to the single Kit(W-v/W-v):Trp53(+/+) mutants. However, double-mutant males showed an increase in sperm viability and could father litters, in contrast to their homozygous Kit mutant, wild-type p53 littermates. This germ cell rescue appears to be male specific, as female ovaries were similar in mice homozygous for the Kit mutant allele with or without p53. We conclude that defective Kit signaling in vivo results in apoptosis by a p53-independent pathway in melanocyte and mast cell lineages but that in male germ cells apoptosis in the absence of Kit is p53-dependent.     

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITCconjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germcells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flowcytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes ofepithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphologywas normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.     

Apoptosis,onset and maintenance of spermatogenesis: evidence for the involvement of Kit in Kit-haplodeficient mice

Kit/stem cell factor (SCF ) has been reported to be involved in survival and proliferation of male differentiating spermatogonial cells. This kinetics study was designed to assess the role of Kit/SCF during spermatogenesis in mice, and the extent of male programmed germ cell death was measured between 8 and 150 days of age. For this purpose, 129/Sv inbred mice in which one Kit allele was inactivated by an nlslacZ sequence insertion (Kit(W-lacZ/+)) were compared with 129/Sv inbred mice with wild-type alleles at the Kit locus. Four different approaches were used: 1) morphometric study to assess spermatogenesis, 2) flow cytometry to study testicular cell ploidy, 3) in situ end labeling to detect apoptosis, and 4) follow-up of reporter gene expression. Spermatogenesis was lower in Kit(W-lacZ/+) heterozygous mice both at the onset of spermatogenesis and during adulthood. Indeed, greater apoptosis occurred at the onset of spermatogenesis. This was followed in the adult by a smaller seminiferous tubule diameter and a lower ratio between type B spermatogonia and type A stem spermatogonia in Kit(W-lacZ/+) mice compared with Kit(+/+) mice. These differences are probably related to the Kit haplodeficiency, which was the only difference between the two genotypes. Germ cell counts and testicular cell ploidy revealed delayed meiosis in Kit(W-lacZ/+) mice. Reporter gene expression confirmed expression of the Kit gene at the spermatogonial stage and also revealed Kit expression during the late pachytene/diplotene transition. These results suggest involvement of Kit/SCF at different stages of spermatogenesis.     

Induction of allograft tolerance in the absence of Fas-mediated apoptosis.

Using certain immunosuppressive regimens, IL-2 knockout (KO) mice, in contrast to wild-type (wt) controls, are resistant to the induction of allograft tolerance. The mechanism by which IL-2 regulates allograft tolerance is uncertain. As IL-2 KO mice have a profound defect in Fas-mediated apoptosis, we hypothesized that Fas-mediated apoptosis of alloreactive T cells may be critical in the acquisition of allograft tolerance. To definitively study the role of Fas in the induction of transplantation tolerance, we used Fas mutant B6.MRL-lpr mice as allograft recipients of islet and vascularized cardiac transplants. Alloantigen-stimulated proliferation and apoptosis of Fas-deficient cells were also studied in vivo. Fas mutant B6.MRL-lpr (H-2b) mice rapidly rejected fully MHC-mismatched DBA/2 (H-2d) islet allografts and vascularized cardiac allografts with a tempo that is comparable to wt control mice. Both wt and B6.MRL-lpr mice transplanted with fully MHC-mismatched islet allografts or cardiac allografts can be readily tolerized by either rapamycin or combined costimulation blockade (CTLA-4Ig plus anti-CD40L mAb). Despite the profound defect of Fas-mediated apoptosis, Fas-deficient T cells can still undergo apoptotic cell death in vivo in response to alloantigen stimulation. Our study suggests that: 1) Fas is not necessarily essential for allograft tolerance, and 2) Fas-mediated apoptosis is not central to the IL-2-dependent mechanism governing the acquisition of allograft tolerance.     

Apoptose et spermatogenèse

Onset of spermatogenesis is associated with a wave of apoptosis, which limits its efficacy during the first cycles in most mammals. After the first cycles, the actual efficacy of spermatogenesis always remains below the theoretical yield. Among the germinal cells, spermatogonia are the main targets of physiological apoptosis. This physiological apoptosis partly depends on the relationships between germ cells and Sertoli cells. The impact of the Sertoli cell/germ cell number ratio on the efficacy of spermatogenesis is well accepted, the concept of density-dependent regulation in the seminiferous tubule was proposed in the early eighties. Since the steps of spermatogenesis require a continuous progression of the cell cycle rather than an arrest, germ cells might therefore be more sensitive to apoptosis. This may also lead to severe disturbances between proliferation and cell death. The first experiments designed to elucidate the mechanisms of germ cell apoptosis were based on hormonal deprivation or cryptorchidism. However, the link between hormonal or cellular action and cell survival remained to be established. Analysis of signal transduction pathways involved in germ cell apoptosis and their regulation were the next steps. The involvement of bcl-2 family genes has been confirmed, although the expression of some of its members remains more controversial. Data derived from overexpression of some genes (Bcl-2, Bcl-xl) or resulting from gene inactivation (Bax) at the testicular level have highlighted the role of these genes in the control of germ cell apoptosis and have also provided some evidence for the strict requirement for density-dependent regulation of spermatogenesis. More recently, variations in the pattern of expression of these genes or proteins helped to explain some of the discrepancies in the literature. The place of the Fas/Fas ligand system during the first cycle of spermatogenesis remains a matter of debate, with controversies concerning the precise site of expression of this oncogene and its receptor. Conversely, its role in the testis after chemotoxic or radiotoxic treatments is well established. However, the normal fertility of animals with a spontaneous inactivation of Fas or Fas L genes does not support a physiological role of these factors during spermatogenesis. While factors involved in TNF/TNF R1 (Tumor Necrosis Factor) are under study, some data have been reported concerning the role of TRAIL (TNFalpha Related Apoptosis Inducing Ligand) and its active or decoy receptors in the testis. Among the oncogenes which may modulate the apoptotic process, Kit/Stem Cell Factor is particularly interesting, as Kit is expressed in some germ cells and Leydig cells, whereas SCF is expressed by Sertoli cells. Its impact during gonadal development and in the survival and proliferation of differentiated spermatogonia has been clearly established. Using a transgenic mice model, in which the Kit gene was inactivated by the insertion of a nls-lacZ sequence in its first exon, we showed that one single copy of the gene was unable to sustain physiological spermatogenesis and fertility in male mice. Our results also suggest that the Kit gene might be expressed at different steps of spermatogenesis, with different signal transduction pathways and biological actions. Finally, analysis of the signal transduction pathways involved in testicular apoptosis and their mechanisms of control is one of the key steps to a better understanding of both impairment of spermatogenesis and the pathogenesis of certain germ cell tumours.     

Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.     

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

Role of acidic sphingomyelinase in Fas/CD95-mediated cell death

Engagement of the Fas receptor has been reported to induce ceramide generation via activation of acidic sphingomyelinase (aSMase). However, the role of aSMase in Fas-mediated cell death is controversial. Using genetically engineered mice deficient in the aSMase gene (aSMase(-/-)), we found that thymocytes, concanavalin A-activated T cells, and lipopolysaccharide-activated B cells derived from both aSMase(-/-) and aSMase(+/+) mice were equally sensitive to Fas-mediated cell death, triggered by either anti-Fas antibody or Fas ligand in vitro. Similarly, activation-induced apoptosis of T lymphocytes was unaffected by the status of aSMase, and aSMase(-/-) mice failed to show immunological symptoms seen in animals with defects in Fas function. In vivo, intravenous injection of 3 microg/25 g mouse body weight of anti-Fas Jo2 antibody into aSMase(-/-) mice failed to affect hepatocyte apoptosis or mortality, whereas massive hepatocyte apoptosis and animal death occurred in wild type littermates. Animals heterozygous for aSMase deficiency were also significantly protected. Susceptibility of aSMase(-/-) mice to anti-Fas antibody was demonstrated with higher antibody doses (>/=4 microg/25 g mouse). These data indicate a role for aSMase in Fas-mediated cell death in some but not all tissues.     

The role of mast cells in atrial natriuretic peptide-induced cutaneous inflammation

Atrial natriuretic peptide (ANP) is widely distributed throughout the heart, skin, gastrointestinal and genital tracts, and nervous and immune systems. ANP acts to mediate vasodilation and induces mast cell activation in both human and rats in vitro. However, the mechanisms of ANP-induced mast cell activation, the extent to which ANP can induce tissue swelling, mast cell degranulation, and granulocyte infiltration in mouse skin are not fully understood. This issue was investigated by treatment with ANP in rat peritoneal mast cells (RPMCs) and mouse peritoneal mast cells (MPMCs) in vitro and by injection of ANP into the skin of congenic normal WBB6F1/J-Kit+/Kit+ +/+, genetically mast cell-deficient WBB6F1/J-Kit(W)/Kit(W-v) (W/W(v)) and mast cell-engrafted W/W(v) (BMCMC→W/W(v)) mice in vivo. ANP induced the release of histamine and TNF-α from RPMCs and enhanced serotonin release from MPMCs, in a dose-dependent fashion, as well as reduced cAMP level of RPMCs in vitro. In +/+ mice, ANP induced significant tissue swelling, mast cell degranulation, and granulocyte infiltration in a dose-dependent manner, whereas not in genetically mast cell-deficient W/W(v) mice. However, ANP-induced cutaneous inflammation has been restored in BMCMC→W/W(v) mice. These data indicate that mast cells play a key role in the ANP-induced cutaneous inflammation.     

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.     

Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells

Apoptosis plays an important role in controlling germ cell numbers and restricting abnormal cell proliferation during spermatogenesis. The tumor suppressor protein, p53, is highly expressed in the testis, and is known to be involved in apoptosis, which suggests that it is one of the major causes of germ cell loss in the testis. Mice that are c-kit/SCF mutant (Sl/Sld) and cryptorchid show similar testicular phenotypes; they carry undifferentiated spermatogonia and Sertoli cells in their seminiferous tubules. To investigate the role of p53-dependent apoptosis in infertile testes, we transplanted p53-deficient spermatogonia that were labeled with enhanced green fluorescence protein into cryptorchid and Sl/Sld testes. In cryptorchid testes, transplanted p53-deficient spermatogonia differentiated into spermatocytes, but not into haploid spermatids. In contrast, no differentiated germ cells were observed in Sl/Sld mutant testes. These results indicate that the mechanism of germ cell loss in the c-kit/SCF mutant is not dependent on p53, whereas the apoptotic mechanism in the cryptorchid testis is quite different (i.e., although the early stage of differentiation of spermatogonia and the meiotic prophase is dependent on p53-mediated apoptosis, the later stage of spermatids is not).     

Over-expressed Fas improves the apoptosis of malignant T-cells in vitro and vivo

Fas play a critical role in T-cell apoptosis by functioning as a major cell-surface death receptor. To explore a potential method that can improve the sensitivity to Fas-mediated apoptosis in malignant precursor T-cells. Fas gene was stable transfected into Jurkat cells to establish a new cell line named Jurkat-Fas with over-expressed Fas. RT-PCR, real-time RT-PCR, flow cytometry, and confocal microscopy assay were performed to detect the Fas level of mRNA and protein in the two cell lines. The sensitivities to Fas-mediated apoptosis of the two cell lines were evaluated by flow cytometry with Alexa Fluor 488 annexin V/PI staining in vitro. Tumor xenograft models were prepared with Jurkat and Jurkat-Fas cells for in vivo study. Fas mRNA and protein levels in Jurkat-Fas cell line were higher than that in Jurkat cell line. Compared to Jurkat cells, apoptosis rates of Jurkat-Fas cells were remarkably higher in vitro, and the tumor growth of Jurkat-Fas cells in nude mice was significantly inhibited in vivo. Stable over-expression of extrinsic Fas gene can significantly ameliorate the sensitivity to Fas-mediated apoptosis in human malignant T-cell, which indicates a novel strategy to improve therapeutic effects on precursor T-cell malignancy.     

Evidence for altered circular smooth muscle cell function in lower esophageal sphincter of W/Wv mutant mice

Nitrergic neurotransmission to gut smooth muscle is impaired in W/W(v) mutant mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). In addition, these mice have been reported to have smaller amplitude unitary potentials (UPs) and a more negative resting membrane potential (RMP) than control mice. These abnormalities have been attributed to absence of ICC-IM, but it remains possible that they are due to alterations at the level of the smooth muscle itself. Amphotericin-B-perforated patch-clamp recordings and Ca(2+) imaging (fura 2) were compared between freshly isolated single circular smooth muscle cells (CSM) from W/W(v) mutant and control mice lower esophageal sphincter (LES). There was no significant difference in seal resistance, capacitance, or input resistance in response to applied electrotonic current pulses between CSM cells from W/W(v) mutants and controls. Compared with control mice, RMP was more negative and UPs significantly smaller in CSM cells from mutant mice LES. Administration of caffeine induced an inward current in cells from both mutant and control mice, but the current density was significantly larger in cells from W/W(v) mutants. Membrane potential hyperpolarization induced by sodium nitroprusside was larger in cells from control mice vs. W/W(v) mutants. In addition, intracellular Ca(2+) transients induced by caffeine were significantly increased in cells from mutants. These findings indicate that LES CSM is abnormal in W/W(v) mutant mice. Thus some physiological functions attributed to ICC-IM based on experiments in smooth muscle of ICC deficient mice may need to be reconsidered.     

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.     

The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor.

The W/c-kit and Steel loci respectively encode a receptor tyrosine kinase (Kit) and its extracellular ligand, Steel factor, which are essential for the development of hematopoietic, melanocyte, and germ cell lineages in the mouse. To determine the biochemical basis of the Steel/W developmental pathway, we have investigated the response of the Kit tyrosine kinase and several potential cytoplasmic targets to stimulation with Steel in mast cells derived from normal and mutant W mice. In normal mast cells, Steel induces Kit to autophosphorylate on tyrosine and bind to phosphatidylinositol 3'-kinase (PI3K) and phospholipase C-gamma 1 but not detectably to Ras GTPase-activating protein. Additionally, we present evidence that Kit tyrosine phosphorylation acts as a switch to promote complex formation with PI3K. In mast cells from mice homozygous for the W42 mutant allele, Kit is not tyrosine phosphorylated and fails to bind PI3K following Steel stimulation. In contrast, in the transformed mast cell line P815, Kit is constitutively phosphorylated and binds to PI3K in the absence of ligand. These results suggest that Kit autophosphorylation and its physical association with a unique subset of cytoplasmic signaling proteins are critical for mammalian development.     

Superoxide anion is a natural inhibitor of FAS-mediated cell death.

The cell surface receptor Fas is a major trigger of apoptosis. However, expression of the Fas receptor in many tumor cell types does not correlate with sensitivity to Fas-mediated cell death. Because a prooxidant state is a common feature of tumor cells, we examined the role of intracellular reactive oxygen intermediates in the regulation of Fas-mediated cytotoxicity. Our results show that an oxidative stress induced by increasing the intracellular superoxide anion (O2-) concentration can abrogate Fas-mediated apoptosis in cells which are constitutively sensitive to Fas. Conversely, an O2- concentration decrease is observed to sensitize cells which are naturally resistant to Fas signals. These observations suggest that intracellular O2- may play a key role in regulating cell sensitivity to a potentially lethal signal and provide tumor cells with a natural, inducible mechanism of resistance to Fas-mediated apoptosis.     

Primed T cells are more resistant to Fas-mediated activation-induced cell death than naive T cells.

Memory T cells respond in several functionally different ways from naive T cells and thus function as efficient effector cells. In this study we showed that primed T cells were more resistant to Fas-mediated activation-induced cell death (AICD) than naive T cells using OVA-specific TCR transgenic DO10 mice and Fas-deficient DO10 lpr/lpr mice. We found that apoptosis was efficiently induced in activated naive T cells at 48 and 72 h after Ag restimulation (OVA peptide; 0.3 and 3 microM), whereas apoptosis was not significantly increased in activated primed T cells at 24-72 h after Ag restimulation. We further showed that the resistance to AICD in primed T cells was due to the decreased sensitivity to apoptosis induced by Fas-mediated signals, but TCR-mediated signaling equally activated both naive and primed T cells to induce Fas and Fas ligand expressions. Furthermore, we demonstrated that primed T cells expressed higher levels of Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP), an inhibitor of Fas-mediated apoptosis, at 24-48 h after Ag restimulation than naive T cells. In addition, Bcl-2 expression was equally observed between activated naive and primed T cells after Ag restimulation. Thus, these results indicate that naive T cells are sensitive to Fas-mediated AICD and are easily deleted by Ag restimulation, while primed/memory T cells express higher levels of FLIP after Ag restimulation, are resistant to Fas-mediated AICD, and thus function as efficient effector cells for a longer period.