IL-7 sensitizes human pre-B cells but not pro-B cells to Fas/APO-1 (CD95)-mediated apoptosis

Homeostasis of human B cell development is maintained by a complex network of cytoplasmic and surface expressed molecules. Abnormalities in this process may result in the expansion of malignant B cell precursors in B lineage acute lymphoblastic leukaemia (ALL). ALL cells share surface antigens with normal early precursor B cells. We have studied here the role of Fas/APO-1 (CD95) antigen on leukaemic precursor B cell line growth and survival, and the modulation of its effects by signals involved in normal early B cell development. Four ALL cell lines representative of the early steps of B cell differentiation are shown to express surface Fas/APO-1 (CD95) antigen and to undergo apoptosis in the presence of anti-Fas cross-linking antibodies. This effect is strongly enhanced when pre-B, but not pro-B cells, are pretreated with IL-7 but not with IL-2, IL-3, IL-4 or IL-10. Furthermore, pre-B cell death induced by anti-Fas antibodies in combination with IL-7 is increased upon pre-B receptor but not CD19 cross-linking. Bcl-2 and Bax protein expression is not influenced by IL-7 or pre-BR stimulation in either pro-B or pre-B cell lines. These results indicate that signals involved in normal early B cell development can modulate the Fas (CD95)-mediated apoptosis of leukaemic precursor B cells.     

TGF-beta1 modulates Fas (APO-1/CD95)-mediated apoptosis of human pre-B cell lines

We have previously shown that Fas-induced apoptosis is markedly enhanced by IL-7 in human pre-B but not pro-B cell lines. In addition, pre-B cell receptor (pre-BCR) ligation significantly potentiates the IL-7 effects on Fas-triggered pre-B cell death. We show herein that transforming growth factor (TGF)-beta 1 sharply reduces Fas-induced death rate of pre-B but not pro-B cells. TGF-beta 1 causes inhibition of Fas-mediated disruption of mitochondrial transmembrane potential and cleavage of caspase 8, Bid and caspase 3. Bcl2 expression is markedly increased in TGF-beta 1-treated pre-B cells, whereas cellular FLICE-like inhibitory protein long (c-FLIPL), Bcl-XL, Bax, and Bad expression remains unchanged. TGF-beta 1 causes a selective growth arrest of pre-B cells in G0/G1 phase of the cell cycle and induces a partial down-modulation of both Fas and pre-BCR expression. All TGF-beta 1-mediated effects, but Bcl2 up-regulation, can be reproduced by the LY294002 phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor but not by inhibitors of the MAPK/ERK (MEK) and Janus kinase (Jak)/STAT pathways, which promote cell death. Akt phosphorylation is strongly inhibited by TGF-beta1 in pre-B but not pro-B cells and is not modified by Fas engagement. Altogether, our findings suggest that TGF-beta1 prevents Fas-induced apoptosis of pre-B lines by inhibiting PI3K pathway and by enhancing expression of Bcl2. They also suggest that the PI3K/Akt pathway is involved in the control of Fas and pre-BCR expression, a checkpoint in B cell development.     

Rapid B cell apoptosis induced by antigen receptor ligation does not require Fas (CD95/APO-1), the adaptor protein FADD/MORT1 or CrmA-sensitive caspases but is defective in both MRL-+/+ and MRL-lpr/lpr mice

Antigen receptor ligation-induced apoptosis is thought to play a role in self-tolerance by deleting autoreactive lymphocytes. Antigen receptor ligation-induced apoptosis of mature T cells and T cell lines requires autocrine or paracrine activation of Fas (CD95/APO-1). Whether B cell antigen receptor (BCR)-mediated apoptosis requires Fas or related molecules is unclear. Here we demonstrate that expression of either CrmA, the cowpox virus serpin, or an inhibitor of the adapter protein FADD/MORT1 blocks Fas-mediated apoptosis but has no effect on BCR ligation-induced apoptosis of the B cell line WEHI-231. In contrast, expression of Bcl-2 blocks BCR-mediated but not Fas-induced apoptosis in WEHI-231 cells. These results indicate that BCR ligation activates an apoptotic signaling pathway distinct from Fas-mediated apoptosis in WEHI-231 cells, and that BCR-mediated apoptosis of WEHI-231 cells does not require Fas or related molecules such as DR3, DR4 and DR5, as all of these death receptors require FADD/MORT1 and/or CrmA-sensitive caspases for induction of apoptosis. Moreover, extensive BCR ligation induces death of mature B cells from C57BL/6-lpr/lpr mice as efficiently as those from C57BL/6 mice, indicating that Fas is not essential for BCR-mediated apoptosis of mature B cells. In contrast, BCR ligation-induced apoptosis is reduced in mature B cells from MRL mice and this is not affected by the lpr mutation. Since MRL-lpr/lpr mice but not C57BL/6-lpr/lpr mice develop severe autoimmune disease, defects in BCR-mediated apoptosis in the MRL background, together with lpr mutation, may contribute to the development of severe autoimmune disease in MRL-lpr/lpr mice by allowing survival of self-reactive B cells.     

Cord Blood–Derived and Peripheral Blood–Derived Cytokine-Induced Killer Cells Are Sensitive to Fas-Mediated Apoptosis

Fas-mediated apoptosis is one of the mechanisms used by tumor cells to escape the cytotoxicity of tumor-infiltrating lymphocytes. It has been suggested that cytokine-induced killer (CIK) cells are resistant to Fas-mediated apoptosis, thereby rendering them more attractive for use in cellular immunotherapy. Unlike what was observed by others, here we show that CIK cells are sensitive to Fas-mediated apoptosis. We have observed an increase in Fas expression in the different CIK cell subpopulations (CD3⁺CD56⁻, CD3⁺CD56⁺, and CD3⁻CD56⁺) isolated from both cord blood (CB) and peripheral blood (PB). We also show that the bulk, as well as the CD3⁺CD56⁻ and CD56⁺ CB- and PB-CIK cell subpopulations were sensitive to Fas-mediated apoptosis induced by both CH11 and APO-1 antibodies, albeit with a weaker effect for the CH11 antibody on CB-CIK cells. In addition, in the presence of the APO-1 and CH11 inducers, Fas engagement inhibited the cytotoxic activity of CB- and PB-CIK cells. This new contradictory result may help explain the variable efficacy observed with CIK cells in the clinic.     

Immunofluorescent studies of the development of pre-B cells, B lymphocytes and immunoglobulin isotype diversity in humans.

Immunofluorescent techniques were used to examine several aspects of B cell ontogeny in humans. Large lymphoid cells containing intracytoplasmic IgM (pre-B cells) were present in fetal liver as early as 7 weeks of gestation, approximately 2 weeks prior to the appearance of surface IgM positive (sIgM⁺) B lymphocytes. Pre-B cells outnumbered sIgM⁺ B lymphocytes in fetal liver up until the 13th week of gestation. In fetuses older than 13 weeks, pre-B cells and sIgM⁺ B lymphocytes were present in approximately equal proportions in liver and bone marrow. Pre-B cells in fetal liver, and fetal and adult marrow, were large and small (indicating a heterogeneous population of cytoplasmic IgM⁺. SIg^- cells in these sites), while only the small pre-B cells were present in fetal spleen, blood and lymph node. Lymphocytes bearing sIgG were detected earlier than those bearing sIgD or sIgA, which were present by the 12th gestational week. Using double-staining techniques, we determined that during fetal life, (a) the proportion of B lymphocytes bearing only sIgM, as opposed to those bearing both sIgM and sIgD, was much higher in liver and bone marrow than in spleen, blood and lymph node, and (b) sIgG, sIgA and sIgD appear independently on lymphocytes bearing sIgM. Studies of the frequency of double-stained cells for each combination of the four sIg isotypes indicated that B lymphocytes from neonatal humans may simultaneously bear three or more sIg isotypes, whereas sIgG⁺ and sIgA⁺ B lymphocytes in adult blood usually express only the single isotype. Lower concentrations of anti-y antibodies were required for modulation of sIgM from B lymphocytes of fetal liver and adult bone marrow than for equivalent removal of sIgM from circulating B cells of mature individuals. In conjunction with data obtained in mice, our observations indicate that (a) the presence of large and small pre-B cells, (b) a high ratio of sIgM single to sIgM.sIgD double B lymphocytes, and (c) increased sensitivity to modulation of B cell sIgM by divalent anti-μ antibodies define the fetal liver and adult bone marrow as bursa-equivalent sites in humans. Our results are consistent with a model of isotype diversification in which immature sIgM⁺ cells give rise to B cell sublines devoted to synthesis of each of the Ig classes, and sIgD is secondarily expressed on unstimulated B cells of all of these sublines.     

Apoptosis in labial salivary glands from Sjögren's syndrome (SS) patients: comparison with human T lymphotropic virus-I (HTLV-I)-seronegative and -seropositive SS patients

Apoptosis is a type of cell death that occurs during morphogenesis and development of the immune system. One of the mechanisms is mediated through the Fas and Fas ligand (FasL) pathway. To determine the possible involvement of Fas and its ligand in salivary gland destruction, we analysed the appearance of nuclei with DNA fragmentation by using nick end labelling (TUNEL) and the expression of Fas and FasL by immunohistochemistry in labial salivary glands. Furthermore, we compared the features of apoptosis in labial salivary glands between HTLV-I^? and HTLV-I⁺ SS. When the frozen sections of 10 primary SS patients in the absence of anti-HTLV-I antibody were examined, several apoptotic cells were found in the acinar and ductal epithelial cells as well as infiltrated mononuclear cells. Both Fas and FasL were detected in the infiltrated mononuclear cells. Acinar epithelial cells, which are surrounded by FasL⁺ mononuclear cells, were also double-positive with Fas and FasL, although the expression of FasL was localized at their apical border, suggesting that apoptosis of mononuclear cells was achieved by activation-induced mechanisms through Fas/FasL pathways, and that of acinar epithelial cells was mediated by FasL derived from either acinar epithelial cells themselves or infiltrated mononuclear cells. Interestingly, Fas expression in ductal epithelial cells was localized around the lumen side of the ducts, indicating that FasL secreted from acinar epithelial cells may induce Fas-mediated apoptosis of ductal epithelial cells. We also studied the labial salivary glands from nine SS patients with anti-HTLV-I antibodies. There was no significant difference in the occurrence of apoptotic cells or in the expression of Fas and FasL between HTLV-I⁺ and HTLV-I^? SS patients. It was of note that neither the expression of Fas and FasL nor the presence of apoptotic cells were determined in labial salivary glands from subjects without SS. These findings indicate that Fas-mediated apoptosis in salivary glands could be involved in the pathological manifestations of SS, irrespective of HTLV-I seropositivity.     

Increased lymphocyte Fas expression and high incidence of common variable immunodeficiency disorder in childhood Evans' syndrome

Evans' syndrome (ES) is characterized by autoimmune hemolytic anemia and thrombocytopenia and has been associated with immune deficiency and lymphoproliferation in some cases. Abnormalities of Fas-mediated apoptosis have been reported in various immune dysregulation disorders associated with autoimmunity and lymphoproliferation. We measured lymphocyte Fas expression and Fas-mediated T lymphocyte apoptosis in 7 children with ES, 7 with acute idiopathic thrombocytopenic purpura (ITP) and 9 with non-immune-mediated disorders. Patients with ES had higher Fas expression on peripheral blood T and B lymphocytes (P<0.001 and P=0.046, respectively) and increased Fas-mediated elimination of activated T lymphocytes compared with the control groups. While two ES patients had panhypogammaglobulinemia at testing, three more developed it later, reaching a frequency of 83%. Some children with ES have increased lymphocyte Fas expression and Fas-mediated T lymphocyte apoptosis and these may be early signs of common variable immunodeficiency disorder in ES.     

The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4

In the bone marrow, progenitor (pro-) and precursor (pre-) B cells depend on close contact with stromal cells for growth and maturation. Stromal cell-derived factor 1 (SDF-1), also known as pre-B cell growth-stimulating factor, is produced by bone marrow stromal cells and was reported to act together with interleukin-7 as co-mitogen for pre-B cells. SDF-1 was recently shown to be a chemokine which is chemotactic for different types of leukocytes and acts via the chemokine receptor CXCR4. Using sorted B220⁺ bone marrow cells and several B cell lines characteristic for different stages of B lymphopoiesis, we now show that SDF-1 is a potent attractant for pro- and pre-B cells, but is inactive on B cells at later stages of development. In early B cell precursors, SDF-1 induced intracellular Ca²⁺ mobilization and in vitro migration with a potency and efficacy similar to that observed for chemokines acting on blood leukocytes. These responses were mediated via CXCR4 as they could be inhibited by an anti-receptor antibody. SDF-1 is the first chemokine shown to act on early-stage B cell precursors. Mice lacking SDF-1 die perinatally and show a severe deficiency in B lymphopoiesis. We propose that SDF-1 released from the stromal cells exerts its critical hematopoietic function by selectively attracting and confining early B cell precursors within the bone marrow microenvironment that provides the necessary factors for growth and differentiation.     

Expression and Function of Fas during Differentiation and Activation of B Cells

Fas (Apo-1, CD95) cell surface antigen belongs to the tumor necrosis factor receptor family and mediates apoptosis of a variety of cell types, including lymphocytes, after ligation with Fas ligand (FasL). Recent studies on the role of Fas/FasL interaction in the immune responses strongly suggest the relevance of dysregulation in Fas-mediated apoptosis as a cause of autoimmune disorders. While Fas is not an essential molecule in the elimination or functional inactivation (anergy) of autoreactive B cells, it is indispensable to the maintenance of peripheral tolerance and prevention of autoimmunity. Studies in the past few years have begun to reveal the mechanism by which susceptibility to Fas-mediated apoptosis in B cells is regulated to allow antigen-specific B cells survive and differentiate and to eliminate nonspecifically activated, potentially selfreactive B cells.     

Expression and function of Fas during differentiation and activation of B cells

Fas (Apo-1, CD95) cell surface antigen belongs to the tumor necrosis factor receptor family and mediates apoptosis of a variety of cell types, including lymphocytes, after ligation with Fas ligand (FasL). Recent studies on the role of Fas/FasL interaction in the immune responses strongly suggest the relevance of dysregulation in Fas-mediated apoptosis as a cause of autoimmune disorders. While Fas is not an essential molecule in the elimination or functional inactivation (anergy) of autoreactive B cells, it is indispensable to the maintenance of peripheral tolerance and prevention of autoimmunity. Studies in the past few years have begun to reveal the mechanism by which susceptibility to Fas-mediated apoptosis in B cells is regulated to allow antigen-specific B cells survive and differentiate and to eliminate nonspecifically activated, potentially selfreactive B cells.     

Rapid up-regulation of c-FLIP expression by BCR signaling through the PI3K/Akt pathway inhibits simultaneously induced Fas-mediated apoptosis in murine B lymphocytes

Cross-linking of BCR rapidly induces protection of B cells from Fas-mediated apoptosis, which has been assumed one of the important survival mechanisms of B cells during antigen stimulation. In the mouse B cell line A20, which is sensitive to Fas-mediated apoptosis, stimulation of BCR inhibited apoptosis induced via Fas upstream of caspase-8 activation with an associated rapid increase in the expression of both short and long forms of cellular caspase-8/FLICE-inhibitory protein (c-FLIP). The c-FLIP competitively inhibited the recruitment of caspase-8 to the death-inducing signaling complex (DISC), which took as long as 3h to form after the stimulation of Fas in A20 cells. Knockdown of c-FLIP by a short hairpin RNA-expressing method rendered BCR-stimulated A20 cells sensitive to Fas-mediated apoptosis. The BCR-induced rapid expression of c-FLIP was not affected by inactivation of NF-kappaB, but was inhibited by either treatment with a PI3K inhibitor, LY294002, or expression of a dominant negative PI3K p85 subunit, both of which suppressed phosphorylation of Akt and sensitized BCR-stimulated A20 cells to Fas-mediated apoptosis. Overexpression of constitutively active Akt was shown not only to up-regulate c-FLIP expression but also to render A20 cells resistant to Fas-mediated apoptosis. Moreover, treatment with LY294002 also suppressed BCR-induced up-regulation of c-FLIP expression in spleen B cells. Taken together, BCR-stimulation was shown to rapidly trigger a survival signal against simultaneously or ongoingly stimulated Fas-mediated apoptosis by promoting a PI3K/Akt signaling pathway-mediated up-regulation of c-FLIP expression.     

Expression and function of Fas antigen on activated murine B cells

We have studied the expression and function of Fas antigen on murine B lymphocytes. While Fas was present on only a few B cells in the bone marrow, spleen, lymph node or peripheral blood, its expression could be strongly up-regulated by stimulation with soluble CD40 ligand (CD40L). Treatment with anti-IgM and interleukin-4 (IL-4) alone did not induce significant Fas expression but enhanced CD40L-mediated up-regulation of Fas expression. The T cell-derived signal via CD40 is therefore a potent inducer of Fas expression by B lymphocytes. The sensitivity to Fas-mediated apoptosis was found to depend on the duration of B cell activation. B cells activated for 1 day were resistant to Fas-mediated cell death, whereas B cells activated for 3 days were relatively sensitive. Interestingly, different sensitivity to Fas-mediated death signal was observed in 2-day activated B cells. It was found that B cells stimulated with CD40 L alone were more sensitive to Fas-mediated apoptosis than were cells stimulated with CD40L plus anti-IgM or IL-4, and in particular, the combination of the two. The greater sensitivity exhibited by B cells stimulated with CD40L alone seems to be related to limited activation of these cells in the absence of additional stimulation. Co-stimulation of B cells in the presence of CD40L and anti-Fas antibody resulted initially in activation of B lymphocytes, as reflected by the expression of activation markers and cell growth, but this was followed by growth inhibition and cell death. The data demonstrate that the B cell response can be regulated positively and negatively by signaling through CD40 and Fas antigens, respectively.     

Significance of Fas receptor protein expression in epithelial ovarian cancer

Fas receptor (FasR) is a cell surface receptor that, when activated, triggers apoptosis. It has been postulated that this receptor may be involved in the clearance of benign ovarian epithelial inclusion cysts (IC). In this study, we test the hypothesis that the expression of FasR changes among IC, cystadenoma (AD), tumors of low malignant potential (LMP), and invasive cancer (cystadenocarcinoma, CA). Formalin-fixed paraffin-embedded sections from 53 oophorectomy specimens representing 26 IC, 17 AD, 17 LMP, and 24 CA were stained using the immunohistochemical avidin-biotin-peroxidase method. We used a mouse antihuman monoclonal antibody Apo-1/Fas (Dako), at 1:5 dilution, after antigen retrieval. The stain was semiquantitatively scored by 3 observers evaluating the intensity of the stain and percentage of positive tumor cells. Statistical analysis was performed using the Wilcoxon rank sum test. Strong (score 6+/7+) and diffuse (>75%) luminal FasR stain was identified in 22 (85%) of 26 IC and 16 (94%) of 17 AD, but only in 6 (35%) of 17 LMP and 1 (4%) of 24 CA. Conversely, weak (score 2+/3+) and focal (<25%) FasR staining was observed in 7 (29%) of 24 CA, but in none of the IC, AD, or LMP. These differences were statistically significant (P < .05). The decreased expression of FasR in malignant ovarian epithelial neoplasms as compared with benign ovarian epithelial lesions suggests that a decreased sensitivity to Fas-mediated apoptosis may be involved in ovarian epithelial carcinogenesis.     

Fas and FasL in the homeostatic regulation of immune responses

Studies of the biological effects of Fas signaling, using transformed cell lines as targets, indicate that ligation of the Fas receptor induces an apoptotic death signal. Chronically activated normal human T cells are also susceptible to Fas-mediated apoptosis. However, interactions between Fas and Fas ligand can also yield a costimulatory signal. Here, David Lynch, Fred Ramsdell and Mark Alderson present a model for the role of Fas and FasL in the homeostatic regulation of normal immune responses. They discuss how dysregulation of the Fas apoptotic pathway may contribute to certain disease states, including autoimmune disease and human immunodeficiency virus (HIV)-induced depletion of CD4⁺ T cells.     

Involvement of the Fas (CD95) system in peripheral cell death and lymphoid organ development

Fas-mediated apoptosis is a form of cell death that operates through a Fas-Fas ligand (FasL) interaction. In this study we investigated the role of the Fas system during development of normal and Fas-mutated lymphocytes. Irradiated RAG2^–/– recipients were reconstituted with bone marrow cells from B6 and lpr mice (Fas defective) or from B6 and gld mice (FasL defective), and analyzed for long-term development. The results showed a primary role of the Fas system in peripheral cell death and thymic colonization. In the periphery, the interaction in vivo between Fas⁺ and Fas^– T cell populations indicated that cellular homeostasis was defective. Indeed, we observed a FasL-mediated cytotoxic effect on normal-derived T cells, explaining the dominance of lpr T cells in the mixed chimeras. The Fas mutation affected neither cell activation nor cell proliferation, as the effector (Fas^–) and target (Fas⁺) cells behaved similarly with regard to activation marker expression and cell cycle status. However, Fas^– T cells failed to seed the periphery and the thymus in the long term. We suggest that this could be due to the fact that FasL is involved in the structural organization of the lymphoid compartment.     

Resistance to Fas-mediated apoptosis of human T-cell lines expressing human T-lymphotropic virus type-2 (HTLV-2) Tax protein

The susceptibility to Fas-mediated apoptosis was evaluated in seven T-cell lines (two infected with HTLV-2, one with HTLV-1, and four HTLV-free) as well as in Jurkat cells transfected with a Tax-2 expressing vector. Fas-mediated apoptosis was significantly reduced in the HTLV-1- and HTLV-2-infected lines in comparison with the HTLV-free lines regardless of the surface expression of Fas antigen (which was no different in the infected and uninfected cells). Fas-mediated apoptosis was also significantly inhibited in Jurkat cells transfected with the Tax-2 expressing vector without any modification in Fas expression. There was significantly more antiapoptotic Bcl-x(L) mRNA and protein in the transfected than in the untransfected Jurkat T cells. In conclusion, our results suggest that HTLV-2 is capable of inhibiting Fas-mediated apoptosis by means of a mechanism involving the tax-2 gene and probably the expression of bcl-x(L) messenger and protein.     

Human B-lymphocyte precursors do not express the N-myc gene.

N-myc expression has been reported in neuroblastoma, retinoblastoma and small cell lung carcinoma. Increased expression associated with gene amplification in neuroblastoma correlates with disease stage and prognosis. N-myc expression has been observed in diverse murine tissues during early stages of development with loss of expression in later stages. Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells express N-myc, whereas mature B cells do not. To determine whether human B-lymphocyte precursors also have increased N-myc expression, we extracted DNA and RNA from representative cell lines, prepared Southern and Northern blots and examined them with the N-myc probe, pNB-1. RNA from the following B-cell developmental stages were examined. One null, 1 pre-pre-B, 3 pre-B (including pre-B-lymphoblastic leukemia, a poor prognostic category) and 5 mature B. Neuroblastoma cells and tissues served as positive controls; negative controls included human muscle, placenta, epithelial cell lines, monocytic, promyelocytic, and T-cell lines. N-myc expression was detected in neuroblastoma cells, but in none of the mature human B or B-lymphocyte precursor cells. Additional immunocytochemical studies performed for N-myc nuclear protein likewise failed to detect this gene product. We conclude that human pre-B cells, unlike murine B-cell precursors, do not express increased levels of N-myc RNA. Expression of this oncogene in human neoplastic B cells does not appear to correlate with developmental stage or prognostic group.     

Reduced susceptibility to Fas-mediated apoptosis in B-1 cells

Elimination of activated T and B cells by Fas-dependent apoptosis may contribute to the maintenance of peripheral tolerance. CD40 ligation was recently shown to up-regulate Fas expression and enhance susceptibility to Fas-mediated apoptosis in mouse splenic B cells. In the present study, we have investigated the regulation of Fas expression and Fas-triggered apoptotis in mouse peritoneal B-1 cells. B-1 cells expressed a similar level of CD40 as that on B-2 cells, and proliferated in response to a soluble CD40 ligand (CD40L)-CD8α chimeric protein, suggesting that CD40 on B-1 cells is functional. In contrast to B-2 cells, B-1 cells expressed Fas at only low levels in response to CD40L-CD8α alone or CD40L-CD8α+interleukin-4, and were resistant to Fas-mediated apoptosis following these treatments. While Fas expression could be induced in B-1 cells to a comparable level as that in B-2 cells by cross-linking CD40L-CD8α with an anti-CD8α antibody, the sensitivity to Fas-mediated apoptosis in B-1 cells was significantly reduced compared with B2 cells. These results suggest that peritoneal B-1 cells from normal mice have a lower susceptibility to Fas-mediated apoptosis and may distinguish B-1 from B-2 cells. Similarly, B-1 cells from the peritoneal cavity and spleen of autoimmune-prone NZB mice exhibited reduced susceptibility to Fas-mediated apoptosis relative to their B-2 counterparts. NZB splenic B-1 cells, however, were more susceptible to Fas-mediated apoptosis than NZB peritoneal B-1 cells. The results presented here raise the possibility that the reduced susceptibility to Fas-triggered apoptosis in B-1 cells might be an accelerating factor for the autoantibody production in NZB mice.