IL-4 counteracts anti-mu-induced human B cell proliferation: involvement of a cAMP-dependent inhibitory pathway.

In this report we show that IL-4 inhibits DNA synthesis induced by stimulation of human B cells with mitogenic doses of either soluble anti-mu mAb DA44 or phorbol ester. In contrast, earlier steps of anti-mu-induced B cell stimulation, such as RNA synthesis, CD23 expression and IL-6 production, were not inhibited but rather increased in the presence of IL-4. From these results, IL-4 appears therefore to exert two opposite effects on DA44 anti-mu mAb-induced human B cell activation: early steps are stimulated, and later steps inhibited. The results of kinetic analysis were consistent with this model. The inhibitory activity of IL-4 required an active cAMP-dependent pathway since IL-4-mediated inhibition of anti-mu-induced B cell proliferation was abolished in the presence of two specific inhibitors of the cAMP pathway (H8 and 2',5'-dideoxyadenosine which are specific for cAMP-dependent protein kinase and adenylate cyclase respectively). Furthermore, IL-4 induced a delayed and prolonged increase in intracellular cAMP concentrations (observed between 4 and 48 hours of culture), and this strongly suggests that the late inhibitory effects of IL-4 is cAMP-dependent. Moreover, this delayed IL-4-mediated cAMP production is probably sufficient to prevent anti-mu induced DNA synthesis since addition of the cAMP agonist forskolin on day 1 or 2 of culture also suppresses the anti-mu-mediated B cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequential effect of a high molecular weight B cell growth factor and of interleukin 2 on activated human B cells

A high m.w. B cell growth factor (50,000 BCGF) prepared from lectin-activated human peripheral blood lymphocyte culture supernatants acts only on B cells preactivated by a first signal. This first signal can be delivered in vitro (with anti-mu antibody (Ab)) or in vivo. Upon costimulation with anti-mu Ab the 50,000 BCGF induces an early and transient proliferative response, whereas the response to interleukin 2 (IL-2) develops more progressively. To determine the respective targets of the 50,000 BCGF and of IL-2, B cells were activated with anti-mu Ab and separated according to the expression of the IL-2 receptor (cluster designation (CD)25 antigen). CD25+ B cells do not respond to the 50,000 BCGF and do not acquire this responsiveness after an additional culture with IL-2. CD25- B cells respond to the 50,000 BCGF and not to IL-2. However, when CD25- B cells are cultured for 3 days with the 50,000 BCGF they become responsive to IL-2. These results demonstrate a pathway of B cell activation based on the ordered and sequential action of anti-mu Ab, the 50,000 BCGF, and IL-2.     

Effects of diacetyl diamines on in vitro activation and proliferation of human B lymphocytes

N,N'-Diacetylputrescine (tetramethylenebisacetamide [TMBA]) and its six carbon analog, hexamethylenebisacetamide (HMBA), inhibited the proliferative response of human B lymphocytes to anti-mu and formalinized Cowan I strain Staphylococcal aureus (SAC) stimulation. In contrast, B cell growth factor-stimulated proliferation of human B cells was minimally inhibited by TMBA or HMBA. The antiproliferative effect of these diamine derivatives was specific for anti-mu (or SAC) activation of normal B cells, because the proliferation of PHA-stimulated human T cells and transformed human B cells was not affected by the presence of TMBA or HMBA. The inhibitory effect of diacetyl diamines on anti-mu (or SAC)-induced B cell activation was dose dependent and persisted after removal of the diamine derivatives from the culture media. These studies show that diacetylated derivatives of polyamines modulate human B cell activation in vitro by specific abrogation of anti-mu or SAC activation.     

Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis

IL-21 costimulates B cell proliferation and cooperatively with IL-4 promotes T cell-dependent Ab responses. Somewhat paradoxically, IL-21 also induces apoptosis of B cells. The present study was undertaken to more precisely define the expression of the IL-21R, using a novel mAb, and the circumstances by which IL-21 promotes B cell growth vs death. The IL-21R was first detected during T and B cell development, such that this receptor is expressed by all mature lymphocytes. The IL-21R was further up-regulated after B and T activation, with the highest expression by activated B cells. Functional studies demonstrated that IL-21 substantially inhibited proliferation and induced Bim-dependent apoptosis for LPS or CpG DNA-activated B cells. In contrast, IL-21 induced both costimulation and apoptosis for anti-CD40-stimulated B cells, whereas IL-21 primarily costimulated B cells activated by anti-IgM or anti-IgM plus anti-CD40. Upon blocking apoptosis using C57BL/6 Bim-deficient or Bcl-2 transgenic B cells, IL-21 readily costimulated responses to anti-CD40 while proliferation to LPS was still inhibited. Engagement of CD40 or the BCR plus CD40 prevented the inhibitory effect by IL-21 for LPS-activated B cells. Collectively, these data indicate that there are three separable outcomes for IL-21-stimulated B cells: apoptosis, growth arrest, or costimulation. We favor a model in which IL-21 promotes B cell maturation during a productive T cell-dependent B cell response, while favoring growth arrest and apoptosis for nonspecifically or inappropriately activated B cells.     

The Ba fragment of complement factor B inhibits human B lymphocyte proliferation

Normal human B lymphocyte function is finely regulated by both positive and negative signals at each stage of activation, proliferation, and differentiation. Activation signals include antigen and surface Ig cross-linking agents such as anti-mu or anti-delta. Signals inducing proliferation include IL-2, high m.w.-B cell growth factor (BCGF), and low m.w.-BCGF. IL-2 as well as IL-6 and other partially characterized B cell differentiation factors can induce terminal differentiation of proliferating B cells into Ig-secreting plasma cells. Various C components have been described to regulate B cell function including Bb that enhances proliferation, C5a that enhances Ig production, and C3a that inhibits Ig production. In our study, we examined the ability of the factor B cleavage fragment Ba to influence human B cell function. Ba did not affect the activation of resting B cells but inhibited the proliferation of activated B cells stimulated with either high m.w.-BCGF or low m.w.-BCGF. The inhibition occurred with doses of Ba as low as 1 microgram/ml (29 nM). Ba was found to bind to activated human B lymphocytes in a saturable manner with an apparent K of approximately 25 nM and an apparent Bmax of 56,000 sites/cell. A peptide made of the carboxy terminal 10 amino acids of Ba (GHGPGEQQKR), was also found to inhibit growth factor induced proliferation of activated B cells but at an ID50 of approximately 5 microM. Finally, Ba was found to inhibit the terminal differentiation of Staphylococcus aweus Cowan-activated B cells stimulated with B cell differentiation factors but not Ig secretion by the partially differentiated EBV-transformed cell line SKW.6. Thus, concentrations of Ba achievable in vivo at sites of active inflammation were found to act on human B lymphocytes by inhibiting their proliferation. This may act to limit the immune response to a specific antigenic challenge.     

Heterogeneity of B-cell growth factor receptor reactivity in healthy donors and in patients with chronic lymphatic leukemia: relationship to B-cell-derived lymphokines

Twenty-five long-term B-cell lines were studied for B-BCGF activity. The cell lines were cultured in the presence or absence of the new tumor promoter teleocidin, and control and teleocidin-treated derived supernatants were cocultured with purified B cells obtained from healthy donors and patients with B chronic lymphatic leukemia (B-CLL), in the presence of anti-mu. In attempt to delineate the role of other B-cell lymphokines in promoting proliferation of activated B cells, the supernatants were also studied for interleukin 1 (IL-1), interleukin 2 (IL-2), and interferon gamma (IFN-gamma). The effect of B-cell-derived lymphokines on the proliferation of activated B cells obtained from the 14 donors was heterogeneous, and three types of response were observed: In four healthy donors there was induction of B-cell proliferation by B-cell lymphokines derived from both control cells and teleocidin-treated cells. In cells obtained from the other five healthy donors there was induction of B-cell proliferation by B-cell lymphokines derived from teleocidin-treated cells. In the five B-CLL patients, B-cell proliferative response to B-cell lymphokines derived from both control cells and teleocidin-activated cells was absent. Comparison of B-BCGF reactivity to T-BCGF reactivity demonstrated that B-CLL B lymphocytes did not respond to either B-BCGF or T-BCGF, whereas normal B cells responded to T-BCGF and may proliferate upon stimulation with B-cell-derived IL-2 and/or B-BCGF. These results suggest heterogeneity of B-BCGF receptor reactivity in B lymphocytes derived from healthy donors, and lack of both B-BCGF and T-BCGF receptor reactivities in B lymphocytes derived from B-CLL patients; B-cell-derived lymphokines influence normal B-cell response but not leukemic B cells; B-BCGF optimal effect is in large part due to other B-cell lymphokines, especially B-cell-derived IL-2; the possible existence of various B-BCGFs.     

Differential effects of the stimulation of complement receptors CR1 (CD35) and CR2 (CD21) on cell proliferation and intracellular Ca2+ mobilization of chronic lymphocytic leukemia B cells.

The regulatory role of CR1 and CR2 on B cell activation and proliferation has been investigated by using B cells from patients with chronic lymphocytic leukemia. The chronic lymphocytic leukemia B cells are clonal expansions of B lymphocytes frozen at specific stages of activation. They displayed two patterns of response upon surface Ig (sIg) cross-linking in terms of in vitro proliferation and intracellular free Ca2+ mobilization: cells from patient F (first pattern) proliferated in the presence of mitogenic anti-mu antibodies, whereas cells from patient A (second pattern) did not respond to sIg cross-linking but proliferated in the presence of low m.w. B cell growth factor and IL-2. Coculture of A or F cells with C3b-bearing SRBC led to a two- to four-fold increase in thymidine incorporation in cultures containing low m.w. B cell growth factor but not in cultures containing rIL-2. This enhanced proliferation was inhibited by F(ab')2 polyclonal rabbit antihuman CR1 antibodies. Only cells which proliferated in the presence of anti-mu (cells F) responded to cross-linking of sIg with a rise in intracellular Ca2+. No increase in calcium mobilization was observed after co-cross-linking of CR1 and sIg on A and F cells with mAb or polyclonal anti-CR1 antibodies. Co-cross-linking of CR2 with sIg only led to an enhanced intracellular Ca2+ rise in F cells but not in A cells. The lack of CR2-mediated synergy in Ca2+ rise in A cells indicates that the synergy occurs only if there is a proper coupling of sIg to phospholipase C. CR1-induced proliferation of B cells does not involve the signaling pathways of sIg. These results provide additional evidences for the role of C3 fragments in modulation of human B cell activation.     

Modulation of IL-2-induced human B cell proliferation in the presence of human 50-kDa B cell growth factor and IL-4

Several human T cell derived factors capable of stimulating human B cells to synthesize DNA have been previously described. One such factor exhibits an apparent m.w. of 50,000 Da and has been termed 50-kDa-B cell growth factor (BCGF). In this report, we show that a human B cell proliferation pathway based on the sequential action of anti-mu antibody, 50-kDa-BCGF and IL-2 is inhibited in the presence of human rIL-4. Although IL-4 itself is capable of triggering B cell DNA synthesis as measured at 72 h, this lymphokine inhibits, in a dose-related manner, the 50-kDa-BCGF driven response of B cells to IL-2 when such proliferation is determined after 144 h. This inhibition takes place at an early step of the B cell activation and does not require the presence of IL-4 during the whole culture period. Such inhibitory activity of IL-4 is specific to the IL-2-induced B cell proliferation because DNA synthesis measured in the presence of semi-purified human 12-kDa-BCGF is not affected by the presence of IL-4. Our results suggest that a particular pathway of human B cell activation leading to the proliferation of these cells in the presence of IL-2 could be either up- or down-modulated by 50-kDa-BCGF and IL-4, respectively.     

Functional analysis of B and T lymphocyte attenuator engagement on CD4+ and CD8+ T cells

T cell activation can be profoundly altered by coinhibitory and costimulatory molecules. B and T lymphocyte attenuator (BTLA) is a recently identified inhibitory Ig superfamily cell surface protein found on lymphocytes and APC. In this study we analyze the effects of an agonistic anti-BTLA mAb, PK18, on TCR-mediated T cell activation. Unlike many other allele-specific anti-BTLA mAb we have generated, PK18 inhibits anti-CD3-mediated CD4+ T cell proliferation. This inhibition is not dependent on regulatory T cells, nor does the Ab induce apoptosis. Inhibition of T cell proliferation correlates with a profound reduction in IL-2 secretion, although this is not the sole cause of the block of cell proliferation. In contrast, PK18 has no effect on induction of the early activation marker CD69. PK18 also significantly inhibits, but does not ablate, IL-2 secretion in the presence of costimulation as well as reduces T cell proliferation under limiting conditions of activation in the presence of costimulation. Similarly, PK18 inhibits Ag-specific T cell responses in culture. Interestingly, PK18 is capable of delivering an inhibitory signal as late as 16 h after the initiation of T cell activation. CD8+ T cells are significantly less sensitive to the inhibitory effects of PK18. Overall, BTLA adds to the growing list of cell surface proteins that are potential targets to down-modulate T cell function.     

Proliferation of peripheral lymphocytes to interleukin-2 and interleukin-4 after marrow transplantation.

Defects in the interleukin-2 (IL-2)-mediated T-lymphocyte activation/proliferation pathway have been implicated as contributing to the compromised immune function observed in patients following bone marrow transplantation (BMT). Since interleukin-4 (IL-4) is also involved in T-lymphocyte function, we have examined whether phytohemagglutinin (PHA)- or anti-CD3 (OKT3)-activated lymphocytes obtained from patients after allogeneic or autologous BMT are capable of proliferating in response to human recombinant IL-4, and compared these results to those obtained using human recombinant IL-2. Peripheral blood lymphocytes from marrow graft recipients were initially cultured for 3 days in the presence of PHA or OKT3. Such mitogen-activated lymphocytes exhibited little or no proliferation (as assessed by incorporation of [3H]-thymidine) following culture for an additional 3 days in the presence of IL-4 or IL-2. Results were similar for lymphocytes obtained from patients early (less than 4 months) after marrow grafting and those obtained from long-term marrow graft recipients with chronic graft-vs-host disease at the time of testing. In contrast, lymphocytes obtained from healthy individuals proliferated in response to IL-4, as well as to IL-2, following initial activation with PHA or OKT3. Immunofluorescence analysis showed that in normals equal numbers of CD4 and CD8 cells proliferated after stimulation with anti-CD3 antibody and IL-2. However, in BMT patients there was a predominant proliferation of CD8 cells using the same stimulator. These results indicate that defects in the IL-4-mediated T-lymphocyte activation/proliferation pathway may also contribute to the immunodeficiency observed following BMT.     

Selective suppressive effects of glucocorticoids on the early events in the human B cell activation process

The effect of hydrocortisone on the induction of human B cell activation and proliferation has been described. Hydrocortisone prevents the anti-mu-induced cell enlargement of small tonsillar B cells, blocks expression of the activation markers 4F2 and 5E9 induced by anti-mu, inhibits RNA synthesis of small B cells stimulated by anti-mu with or without BCGF, and suppresses B cell proliferation in response to anti-mu and BCGF or to Staphylococcus aureus Cowan I. In contrast, hydrocortisone does not affect the proliferative response of in vitro or in vivo preactivated B cells. Therefore, hydrocortisone has a selective inhibitory effect on early events in the human B cell cycle that subsequently leads to inhibition of total RNA and DNA synthesis. Possible mechanisms of this action are discussed. These studies further define the nature of glucocorticoid-induced modulation of human B cell activation and proliferation.     

Synergistic effect of recombinant IL 2 and interferon-gamma on the proliferation of human monoclonal lymphocytes

We studied the effect of interferon-gamma (IFN-gamma) on the proliferation of lymphocytes from 10 B-type chronic lymphocytic leukemia (B-CLL) patients. In no instance did IFN-gamma induce a proliferative response whether used alone or in combination with anti-mu antibody (Ab). This was observed regardless of the responsiveness of a given patient's cells to interleukin 2 (IL 2) and to B cell growth factor (BCGF). In contrast IFN-gamma strongly and reproducibly synergized with IL 2 (but not with BCGF) to support B-CLL proliferation in five of the 10 patients. The effect of IFN-gamma was dose related and could be inhibited by an anti-IFN-gamma monoclonal Ab. A monoclonal Ab toward the IL 2 receptor molecule was also inhibitory. Preincubation with IFN-gamma potentiated the responsiveness of B-CLL to IL 2 in secondary cultures, showing that IFN-gamma exerts its effect before that of IL 2.     

Interactions between B lymphocyte subpopulations. Augmentation of the responses of resting B lymphocytes by activated B lymphocytes

Mouse B lymphocytes were fractionated from normal T lymphocyte-depleted spleen cell populations using discontinuous percoll gradients and were stimulated with rabbit F(ab')2 anti-mouse mu-specific antibodies (anti-mu) plus the supernatant of Con A-stimulated rat spleen cells (SN) as a source of lymphokines. The responses of small (mean volume 120 mu 3), dense (greater than 1.087 specific gravity), resting (least spontaneous thymidine incorporation) B lymphocytes were augmented by irradiated (4000 rad), larger (mean volume greater than 170 mu 3), less dense (less than 1.081 specific gravity), activated (greater spontaneous thymidine incorporation) B lymphocytes. Proliferation was augmented 2- to 4-fold and polyclonal antibody-forming cell responses three- to sixfold. Maximal augmentation of the responses of 5 X 10(4) resting B cells was obtained with 10(4) activated B cells. Augmenting activity was specific for activated B lymphocytes in that responses were not augmented by irradiated thymocytes, T lymphoblasts, macrophages, or additional supernatant. B lymphocytes activated in vitro by LPS or anti-mu also had augmenting activity. Augmentation of responses was maximal only when activated B lymphocytes were added simultaneously with anti-mu. The interaction between activated and resting B lymphocytes did not appear to be genetically restricted. Interestingly, the augmenting activity of activated B cells could be reconstituted by a combination of supernatant and cell membranes from these cells but not by either alone, suggesting that two components are required, one soluble and the other membrane-bound. Thus, a functional interaction has been demonstrated between B lymphocyte subpopulations which differ in their state of activation, and this interaction appears to involve a novel mechanism of action.     

IL-4 inhibits apoptosis and prevents mitochondrial damage without inducing the switch to necrosis observed with caspase inhibitors.

We previously demonstrated that the broad-spectrum caspase inhibitor, zVAD-fmk, totally deviated apoptosis to necrosis in B lymphocytes. We report here that, in contrast with zVAD-fmk, IL-4 protected B cells from spontaneous and from dexamethasone-induced apoptosis and actually maintained cell viability. This was assessed by morphological and biochemical criteria and accompanied by the maintenance of mitochondrial transmembrane potential (DeltaPsiCm) and elevated glutathione (GSH) levels. Under these conditions, zVAD-fmk also totally inhibited apoptosis in thymocytes, but it partly preserved cell viability with a parallel increase in the percentage of cells exhibiting high DeltaPsiCm and elevated GSH levels. Nevertheless, non-rescued cells were deviated to necrosis. Therefore, the pathway leading to either apoptosis or necrosis appears to involve common mitochondrial dysfunctions which could not be reversed by caspase inhibition, suggesting that the pharmacological inhibition of cell death should occur at an earlier stage.     

The modulation of membrane Ia on human B lymphocytes

Using flow cytometry techniques, changes in surface Ia (DR and DS) expression on human B lymphocytes were correlated with changes in the cell cycle following stimulation with anti-mu. The effect of interleukin (IL)-1, IL-2, B-cell growth factor (BCGF), and interferons on Ia expression on resting B cells was also examined. A population of resting B lymphocytes was cultured in vitro with 100 micrograms/ml of anti-mu and immunofluorescently stained for DR and DS at various times following stimulation. Detectable increases in DR and DS expression were found within 8 hr, and the major increases (twofold and fourfold) in DR and DS expression occurred over the next 48 hr. Using cell cycle inhibitors and propidium iodide staining, it was demonstrated that the enhanced DR and DS expression following anti-mu stimulation began during G0 to G1 transition and increased as the cells progressed through G1 phase. During S and G2/M phases, there were minimal further increases in surface Ia. Although prolonged exposure of B cells to anti-mu was required for cellular activation, cell size enlargement, and progression into S phase, a brief exposure to anti-mu, insufficient for cellular activation, markedly enhanced Ia expression. Thus anti-mu-stimulated resting human B lymphocytes rapidly increase their surface Ia expression. This increase occurs predominantly prior to entrance into S phase and can occur in the absence of significant cellular activation. Interferons have been reported to modulate surface Ia expression on a human lymphoid cell line and on monocytes and supernatants with BCGF activity to enhance surface Ia expression on murine B cells; however, neither alpha-interferon, gamma-interferon, IL-1, IL-2, nor BCGF modified surface DR expression on normal resting human B cells.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

The effects of interleukin 1 on human B cell activation and proliferation

The precise role of B cell surface immunoglobulin (slg) in the activation of B cells is unclear at present. In particular, it is uncertain whether ligands interacting with the B cell slg suffice to induce proliferation, or simply induce a state of activation in which the B cell becomes responsive to growth factors made by accessory cells. We have examined the effects of two ligands, Staphylococcus aureus Cowan strain I (SAC) and antihuman mu chain (anti-mu), which interact with B cell slg on highly purified human peripheral blood and tonsillar B cells cultured at low cell concentrations. The effects on B cell proliferation of these ligands alone or in combination with highly purified interleukin 1 (IL 1) or a supernatant of a human T-T hybridoma containing a B cell growth factor (BCGF) were studied. SAC with its high cell wall content of protein A triggered maximal B cell proliferation which was not increased further by IL 1 or BCGF. High concentrations of soluble F(ab')2 fragments of goat anti-mu chain also induced significant B cell proliferation. Lower concentrations of anti-mu resulted in little or no B cell proliferation but activated the B cell to a state of responsiveness to both IL 1 and BCGF. IL 1 by itself had no effect on the proliferation of unstimulated B cells or on the proliferation of in vivo-activated B cells which responded to BCGF in vitro, but demonstrated clear synergy with low concentrations of anti-mu antibody. BCGF alone augmented the proliferation of unstimulated B cells, presumably by acting on B cells which had undergone some degree of activation in vivo. In addition, it showed marked synergy with anti-mu antibody, which resulted in proliferation similar in magnitude to that induced by SAC. This synergy was far greater than that seen between anti-mu antibody and IL 1, and the resulting proliferative response was only slightly increased by the presence of IL 1. We conclude that the importance of accessory cell factors for the initial rounds of B cell proliferation depends on the strength of the initial slg-mediated activation signal. When this is strong, the response is maximal and independent of accessory cells or accessory cell factors. When it is suboptimal, a moderate synergy is seen with IL 1 and a dramatic synergy with BCGF.     

IL-21 induces the apoptosis of resting and activated primary B cells

Cytokines play an important role in regulating the development and homeostasis of B cells by controlling their viability. In this study, we show that the recently described T cell-derived cytokine IL-21 induces the apoptosis of resting primary murine B cells. In addition, the activation of primary B cells with IL-4, LPS, or anti-CD40 Ab does not prevent IL-21-mediated apoptosis. The induction of apoptosis by IL-21 correlates with a down-regulation in the expression of Bcl-2 and Bcl-x(L), two antiapoptotic members of the Bcl-2 family. Furthermore, the reconstitution of Bcl-x(L) or Bcl-2 expression protects primary B cells from IL-21-induced apoptosis. In addition, a short-term preactivation of B cells with anti-CD40 Ab confers protection from IL-21-mediated apoptosis through the up-regulation of Bcl-x(L). These studies reveal a novel pathway that mediates B cell apoptosis via the IL-21R and suggest that IL-21 may play a role in regulating B cell homeostasis.