Kinetics of human B cell behavior and amplification of proliferative responses following stimulation with IL-21

Although recent studies indicated that IL-21 is an important regulator of human B cell activation, detailed comparison of the effects of IL-21 on distinct B cell subsets have not been performed. Our studies revealed that IL-21R is expressed by naive and germinal center B cells, but not memory or plasma cells. IL-21R was increased on naive and memory B cells following in vitro activation. Investigation into the kinetics and magnitude of responses of human B cells to IL-21 revealed that IL-21 potently augmented proliferation of CD40L-stimulated neonatal, splenic naive, and memory and tonsil germinal center B cells. This response exceeded that induced by IL-4, IL-10, and IL-13, cytokines that also induce B cell proliferation. Remarkably, CD40L/IL-21-stimulated naive B cells underwent the same number of divisions as memory cells and exhibited a greater enhancement in their response compared with CD40L alone than memory B cells. Therefore, IL-21 is a powerful growth factor for naive B cells. This may result from the higher expression of IL-21R on naive, compared with memory, B cells. Stimulation of human B cells with CD40L/IL-21 also induced IL-10 production and activation of STAT3. We propose that IL-21 may have therapeutic application in conditions of immunodeficiency where it could expand naive B cells, the predominant B cell subset in such patients. Conversely, because IL-21 is increased in murine models of lupus, dysregulated IL-21 production may contribute to perturbed B cell homeostasis observed in systemic lupus erythematosus. Thus, antagonizing IL-21 may be a novel strategy for treating Ab-mediated autoimmune diseases.     

An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+ T cells

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-gamma production. It has been recently reported that IL-27 induces T-bet and IL-12Rbeta2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rbeta2 expression, and synergistic IFN-gamma production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rbeta2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation.     

Signaling events involved in interleukin 27 (IL-27)-induced proliferation of human naive CD4+ T cells and B cells

IL-27 induces stronger proliferation of naive than memory human B cells and CD4(+) T cells. In B cells, this differential response is associated with similar levels of IL-27 receptor chains, IL-27Rα and gp130, in both subsets and stronger STAT1 and STAT3 activation by IL-27 in naive B cells. Here, we show that the stronger proliferative response of CD3-stimulated naive CD4(+) T cells to IL-27 is associated with lower levels of IL-27Rα but higher levels of gp130 compared with memory CD4(+) T cells. IL-27 signaling differs between naive and memory CD4(+) T cells, as shown by more sustained STAT1, -3, and -5 activation and weaker activation of SHP-2 in naive CD4(+) T cells. In the latter, IL-27 increases G0/G1 to S phase transition, cell division and, in some cases, cell survival. IL-27 proliferative effect on naive CD4(+) T cells is independent of MAPK, but is dependent on c-Myc and Pim-1 induction by IL-27 and is associated with induction of cyclin D2, cyclin D3, and CDK4 by IL-27 in a c-Myc and Pim-1-dependent manner. In BCR-stimulated naive B cells, IL-27 only increases entry in the S phase and induces the expression of Pim-1 and of cyclins A, D2, and D3. In these cells, inhibition of Pim-1 inhibits IL-27 effect on proliferation and cyclin induction. Altogether, these data indicate that IL-27 mediates proliferation of naive CD4(+) T cells and B cells through induction of both common and distinct sets of cell cycle regulators.     

Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rbeta2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-gamma production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of T-bet, IL-12Rbeta2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of IL-12Rbeta2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression.     

Novel diversity in IL-4-mediated responses in resting human naive B cells versus germinal center/memory B cells

Recent studies have defined several phenotypic and molecular changes associated with the maturation of naive human B cells within the milieu of germinal centers. Although naive B cells serve as natural precursors to germinal center (GC)/memory (M) subpopulations, little is known about the physiological requirements for the survival of the naive B cell pool in the absence of cell-cell contact or Ag-mediated activation. Because IL-4 induces expression of several membrane receptors such as CD23 which are uniquely present on resting human naive B lymphocytes, we hypothesized that these cells might be intrinsically programmed to respond to IL-4 in the absence of cell division. Using buoyant density-dependent isolation and further enrichment by negative/positive selection of human naive and GC/M subpopulations, we characterized cytokine receptor moieties on these cells and analyzed their survival and growth in the presence of IL-4 or IL-10. Resting naive B cells expressed significantly higher IL-4 receptor alpha-chain on their cell surface than the combined GC/M subpopulation. The IL-10 receptor and the IL-2 receptor gammac chain were almost equally expressed on both subpopulations. When cultured in vitro, the addition of IL-4, but not IL-10, protected naive B cells from apoptosis in the absence of activation and growth. However, IL-4 exerted no such effect on resting GC/M B cells. These data support the hypothesis that IL-4 plays a pivotal role in the survival and maintenance of resting human naive B cells.     

STAT3 is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production

IL-27, a member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in augmentation of Th1 differentiation and suppression of proinflammatory cytokine production. In the present study, we investigated the role of STAT3 in the IL-27-mediated immune functions. IL-27 induced phosphorylation of STAT1, -2, -3 and -5 in wild-type naive CD4+ T cells, but failed to induce that of STAT3 and STAT5 in STAT3-deficient cohorts. IL-27 induced not only proinflammatory responses including up-regulation of ICAM-1, T-box expressed in T cells, and IL-12Rbeta2 and Th1 differentiation, but also anti-inflammatory responses including suppression of proinflammatory cytokine production such as IL-2, IL-4, and IL-13 even in STAT3-deficient naive CD4+ T cells. In contrast, IL-27 augmented c-Myc and Pim-1 expression and induced cell proliferation in wild-type naive CD4+ T cells but not in STAT3-deficient cohorts. Moreover, IL-27 failed to activate STAT3, augment c-Myc and Pim-1 expression, and induce cell proliferation in pro-B BaF/3 transfectants expressing mutant gp130, in which the putative STAT3-binding four Tyr residues in the YXXQ motif of the cytoplasmic region was replaced by Phe. These results suggest that STAT3 is activated through gp130 by IL-27 and is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production. Thus, IL-27 may be a cytokine, which activates both STAT1 and STAT3 through distinct receptor subunits, WSX-1 and gp130, respectively, to mediate its individual immune functions.     

IL-27 suppresses CD28-mediated [correction of medicated] IL-2 production through suppressor of cytokine signaling 3

IL-27 is a novel IL-6/IL-12 family cytokine that not only plays a role in the early regulation of Th1 differentiation, but also exerts an inhibitory effect on immune responses, including the suppression of proinflammatory cytokine production. However, the molecular mechanism by which IL-27 exerts the inhibitory effect remains unclear. In this study we demonstrate that IL-27 inhibits CD28-mediated IL-2 production and that suppressor of cytokine signaling 3 (SOCS3) plays a critical role in the inhibitory effect. Although IL-27 enhanced IFN-gamma production from naive CD4+ T cells stimulated with plate-coated anti-CD3 and anti-CD28 in the presence of IL-12, IL-27 simultaneously inhibited CD28-mediated IL-2 production. Correlated with the inhibition, IL-27 was shown to augment SOCS3 expression. Analyses using various mice lacking a signaling molecule revealed that the inhibition of IL-2 production was dependent on STAT1, but not on STAT3, STAT4, and T-bet, and was highly correlated with the induction of SOCS3 expression. Similar inhibition of CD28-mediated IL-2 production and augmentation of SOCS3 expression by IL-27 were observed in a T cell hybridoma cell line, 2B4. Forced expression of antisense SOCS3 or dominant negative SOCS3 in the T cell line blocked the IL-27-inudced inhibition of CD28-mediated IL-2 production. Furthermore, pretreatment with IL-27 inhibited IL-2-mediated cell proliferation and STAT5 activation, although IL-27 hardly affected the induction level of CD25 expression. These results suggest that IL-27 inhibits CD28-mediated IL-2 production and also IL-2 responses, and that SOCS3, whose expression is induced by IL-27, plays a critical role in the inhibitory effect in a negative feedback mechanism.     

Antigen receptor engagement selectively induces macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta chemokine production in human B cells

We show herein that B cell Ag receptor (BCR) triggering, but not stimulation by CD40 mAb and/or IL-4, rapidly induced the coordinated expression of two closely related T cell chemoattractants, macrophage inflammatory protein-1 beta (MIP-1 beta) and MIP-1 alpha, by human B cells. Naive, memory, and germinal center B cells all produced MIP-1 alpha/beta in response to BCR triggering. In contrast to MIP-1 alpha/beta, IL-8, which is spontaneously produced by germinal center B cells but not by naive and memory B cells, was not regulated by BCR triggering. Culturing follicular dendritic cell-like HK cells with activated B cells did not regulate MIP-1 alpha/beta production, but it did induce production of IL-8 by HK cells. Microchemotaxis assays showed that CD4+CD45RO+ T cells of the effector/helper phenotype actively migrated along a chemotactic gradient formed by BCR-stimulated B cells. This effect was partially blocked by anti-MIP-1 beta and anti-CC chemokine receptor 5 Ab, but not by anti-MIP-1 alpha Ab suggesting that MIP-1 beta plays a major role in this chemoattraction. Since maturation of the B cell response to a peptide Ag is mostly dependent on the availability of T cell help, the ability of Ag-stimulated B cells to recruit T cells via MIP-1 alpha/beta, may represent one possible mechanism enabling cognate interactions between rare in vivo Ag-specific T and B cells.     

Memory B cells and CD27

Following antigen activation in germinal centers, B cells develop into memory B cells or plasma cells. Triggering via B-cell immunoglobulin receptors by antigens, cytokines and direct cell-to-cell contact by B and T cells plays an important role in the B cell differentiation into memory or plasma cells. Adult human peripheral blood B cells are separated into three subtypes by the expression of IgD and CD27, which belong to the tumor necrosis factor receptor (TNFR) family: IgD+ CD27- naive B cells, IgD+ CD27+ and IgD- CD27+ B cells. CD27+ B cells are larger cells with abundant cytoplasm carrying somatic hypermutation, and have an ability to produce immunoglobulin, indicating that CD27 is a memory marker of B cells. The ligation of CD27 yields crucial signals that positively control the entry of B cells into the pathway to plasma cells. We review observations on subpopulations and differentiation of mature B-cells by T/B cell interaction via CD27/CD70 as compared with CD40/CD154 interaction, and discuss about memory B cells.     

Cutting edge: HIV-1 Tat protein differentially modulates the B cell response of naive, memory, and germinal center B cells.

Critical steps of B cell differentiation occur within lymphoid organs that are also major sites of HIV-1 replication. Because Tat can be released by infected cells, we investigated whether extracellular HIV-1 Tat modulates cell proliferation of B cells at critical stages of their differentiation. Here we show that extracellular Tat inhibited the proliferation of B cell receptor-triggered naive and memory B cells by >80% but had no effect on their CD40 mAb and IL-4-mediated proliferation. In striking contrast, Tat doubled the germinal center B cell proliferation induced by CD40 mAb and IL-4. These effects were dose dependent and required the addition of Tat at the initiation of the culture, suggesting that Tat acts on early stages of cell cycle progression. By its effects on B cell subsets, Tat might directly affect the normal B cell differentiation process in HIV-positive patients and favor the occurrence of AIDS-associated B cell lymphomas.     

Distinct effects of STAT5 activation on CD4+ and CD8+ T cell homeostasis: development of CD4+CD25+ regulatory T cells versus CD8+ memory T cells

Using transgenic mice that express a constitutively active version of STAT5b, we demonstrate that STAT5 plays a key role in governing B cell development and T cell homeostasis. STAT5 activation leads to a 10-fold increase in pro-B, but not pro-T, cells. Conversely, STAT5 signaling promotes the expansion of mature alphabeta T cells (6-fold increase) and gammadelta and NK T cells (3- to 4-fold increase), but not of mature B cells. In addition, STAT5 activation has dramatically divergent effects on CD8(+) vs CD4(+) T cells, leading to the selective expansion of CD8(+) memory-like T cells and CD4(+)CD25(+) regulatory T cells. These results establish that activation of STAT5 is the primary mechanism underlying both IL-7/IL-15-dependent homeostatic proliferation of naive and memory CD8(+) T cells and IL-2-dependent development of CD4(+)CD25(+) regulatory T cells.     

IL-27 induces the production of IgG1 by human B cells

It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human na?ve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to na?ve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by na?ve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.     

Human activation-induced cytidine deaminase is induced by IL-4 and negatively regulated by CD45: implication of CD45 as a Janus kinase phosphatase in antibody diversification

Activation-induced cytidine deaminase (AID) plays critical roles in Ig class switch recombination and V(H) gene somatic hypermutation. We investigated the role of IL-4 in AID mRNA induction, the signaling transduction involved in IL-4-mediated AID induction, and the effect of CD45 on IL-4-dependent AID expression in human B cells. IL-4 was able to induce AID expression in human primary B cells and B cell lines, and IL-4-induced AID expression was further enhanced by CD40 signaling. IL-4-dependent AID induction was inhibited by a dominant-negative STAT6, indicating that IL-4 induced AID expression via the Janus kinase (JAK)/STAT6 signaling pathway. Moreover, triggering of CD45 with anti-CD45 Abs can inhibit IL-4-induced AID expression, and this CD45-mediated AID inhibition correlated with the ability of anti-CD45 to suppress IL-4-activated JAK1, JAK3, and STAT6 phosphorylations. Thus, in humans, IL-4 alone is sufficient to drive AID expression, and CD40 signaling is required for optimal AID production; IL-4-induced AID expression is mediated via the JAK/STAT signaling pathway, and can be negatively regulated by the JAK phosphatase activity of CD45. This study indicates that the JAK phosphatase activity of CD45 can be induced by anti-CD45 Ab treatment, and this principle may find clinical application in modulation of JAK activation in immune-mediated diseases.