Renren Wen

Phospholipase Cγ2 Is Essential in the Functions of B Cell and Several Fc Receptors

Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent antibody production is absent. The similarity to Btk- or Blnk-deficient mice demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function. The results define a signal transduction pathway broadly utilized by immunoglobulin superfamily receptors.

Defective development and function of Bcl10-deficient follicular, marginal zone and B1 B cells.

Bcl10 is an intracellular protein essential for nuclear factor (NF)-κB activation after lymphocyte antigen receptor stimulation. Using knockout mice, we show that absence of Bcl10 impeded conversion from transitional type 2 to mature follicular B cells and caused substantial decreases in marginal zone and B1 B cells. Bcl10-deficient B cells showed no excessive apoptosis. However, both Bcl10-deficient follicular and marginal zone B cells failed to proliferate normally, although Bcl10-deficient marginal zone B cells uniquely failed to activate NF-κB efficiently after stimulation with lipopolysaccharide. Bcl10-deficient marginal zone B cells did not capture antigens, and Bcl10-deficient (Bcl10−/−) mice failed to initiate humoral responses, leading to an inability to clear blood-borne bacteria. Thus, Bcl10 is essential for the development of all mature B cell subsets.

STAT5 Protein Negatively Regulates T Follicular Helper (Tfh) Cell Generation and Function

Background: Tfh cells regulate B cell-mediated humoral immunity. Results: STAT5 regulated Blimp-1 expression, and STAT5 deficiency in CD4+ T cells resulted in an increase of Tfh generation and an impairment of B cell tolerance. Conclusion: STAT5 negatively regulates Tfh development by up-regulating Blimp-1 and thus controls the humoral immunity and B cell tolerance. Significance: These findings may help to find new ways to treat antibody-mediated autoimmune diseases. Recent work has identified a new subset of CD4+ T cells named as Tfh cells that are localized in germinal centers and critical in germinal center formation. Tfh cell differentiation is regulated by IL-6 and IL-21, possibly via STAT3 factor, and B cell lymphoma 6 (Bcl6) is specifically expressed in Tfh cells and required for their lineage specification. In the current study, we characterized the role of STAT5 in Tfh cell development. We found that a constitutively active form of STAT5 effectively inhibited Tfh differentiation by suppressing the expression of Tfh-associated factors (CXC motif) receptor 5 (CXCR5), musculoaponeurotic fibrosarcoma (c-Maf), Bcl6, basic leucine zipper transcription factor ATF-like (Batf), and IL-21, and STAT5 deficiency greatly enhanced Tfh gene expression. Importantly, STAT5 regulated the expression of Tfh cell suppressor factor B lymphocyte-induced maturation protein 1 (Blimp-1); STAT5 deficiency impaired Blimp-1 expression and resulted in elevated expression of Tfh-specific genes. Similarly, inhibition of IL-2 potentiated Tfh generation, associated with dampened Blimp-1 expression; Blimp-1 overexpression inhibited Tfh gene expression in Stat5-deficient T cells, suggesting that the IL-2/STAT5 axis functions to regulate Blimp-1 expression. In vivo, deletion of STAT5 in CD4+ T cells resulted in enhanced development of Tfh cells and germinal center B cells and led to an impairment of B cell tolerance in a well defined mouse tolerance model. Taken together, this study demonstrates that STAT5 controls Tfh differentiation.

Bcl10 plays a critical role in NF-κB activation induced by G protein-coupled receptors

G protein-coupled receptors (GPCRs) play pivotal roles in cell proliferation, differentiation, and survival. Although many studies indicate that the stimulation of GPCRs leads to NF-κB activation, the molecular mechanism by which GPCRs induced NF-κB activation remains largely unknown. Bcl10 is an essential adaptor molecule connecting antigen receptor signaling cascades to NF-κB activation in lymphocytes. However, the function of Bcl10 in nonlymphoid cells remains to be determined. In this study, we demonstrated that the deficiency of Bcl10 resulted in the defect in NF-κB activation induced by either expressing the constitutively active mutant of G protein or stimulation of cells with lysophosphatidic acid or endothelin-1, which activate their GPCR. In contrast, TNF-α-, LPS-, and integrin-induced NF-κB activation was not affected in Bcl10-deficient cells. Together, our results provide genetic evidence showing that Bcl10 is a key signaling component mediating NF-κB activation induced by GPCRs in nonlymphoid cells.

Phospholipase Cγ1 is essential for T cell development, activation, and tolerance

Phospholipase Cγ1 (PLCγ1) is an important signaling effector of T cell receptor (TCR). To investigate the role of PLCγ1 in T cell biology, we generated and examined mice with T cell–specific deletion of PLCγ1. We demonstrate that PLCγ1 deficiency affects positive and negative selection, significantly reduces single-positive thymocytes and peripheral T cells, and impairs TCR-induced proliferation and cytokine production, and the activation of ERK, JNK, AP-1, NFAT, and NF-κB. Importantly, PLCγ1 deficiency impairs the development and function of FoxP3+ regulatory T cells, causing inflammatory/autoimmune symptoms. Therefore, PLCγ1 is essential for T cell development, activation, and tolerance.

Jak3 Selectively Regulates Bax and Bcl-2 Expression To Promote T-Cell Development

ABSTRACT Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.

A critical role of TAK1 in B-cell receptor–mediated nuclear factor κB activation

The kinase TAK1 is essential for T-cell receptor (TCR)-mediated nuclear factor kappaB (NF-kappaB) activation and T-cell development. However, the role of TAK1 in B-cell receptor (BCR)-mediated NF-kappaB activation and B-cell development is not clear. Here we show that B-cell-specific deletion of TAK1 impaired the transition from transitional type 2 to mature follicular (FO) B cells and caused a marked decrease of marginal zone (MZ) B cells. TAK1-deficient B cells exhibited an increase of BCR-induced apoptosis and impaired proliferation in response to BCR ligation. Importantly, TAK1-deficient B cells failed to activate NF-kappaB after BCR stimulation. Thus, TAK1 is critical for B-cell maturation and BCR-induced NF-kappaB activation.

Phospholipase Cγ2 Is Essential for Specific Functions of FcεR and FcγR

Phospholipase Cγ2 (PLCγ2) plays a critical role in the functions of the B cell receptor in B cells and of the FcRγ chain-containing collagen receptor in platelets. Here we report that PLCγ2 is also expressed in mast cells and monocytes/macrophages and is activated by cross-linking of FcεR and FcγR. Although PLCγ2-deficient mice have normal development and numbers of mast cells and monocytes/macrophages, we demonstrate that PLCγ2 is essential for specific functions of FcεR and FcγR. While PLCγ2-deficient mast cells have normal mitogen-activated protein kinase activation and cytokine production at mRNA levels, the mutant cells have impaired FcεR-mediated Ca2+ flux and inositol 1,4,5-trisphosphate production, degranulation, and cytokine secretion. As a physiological consequence of the effect of PLCγ2 deficiency, the mutant mice are resistant to IgE-mediated cutaneous inflammatory skin reaction. Macrophages from PLCγ2-deficient mice have no detectable FcγR-mediated Ca2+ flux; however, the mutant cells have normal FcγR-mediated phagocytosis. Moreover, PLCγ2 plays a nonredundant role in FcγR-mediated inflammatory skin reaction.

Stat5 Is Essential for Early B Cell Development but Not for B Cell Maturation and Function

The two closely related Stat5 (Stat5A and Stat5B) proteins are activated by a broad spectrum of cytokines. However, with the complication of the involvement of Stat5A/5B in stem cell function, the role of Stat5A/5B in the development and function of lymphocytes, especially B cells, is not fully understood. In this study, we demonstrated that Stat5A/5B−/− fetal liver cells had severe diminution of B cell progenitors but clearly had myeloid progenitors. Consistently, the mutant fetal liver cells could give rise to hemopoietic progenitors and myeloid cells but not B cells beyond pro-B cell progenitors in lethally irradiated wild-type or Jak3−/− mice. Deletion of Stat5A/5B in vitro directly impaired IL-7-mediated B cell expansion. Of note, reintroduction of Stat5A back into Stat5A/5B−/− fetal liver cells restored their abilities to develop B cells. Importantly, CD19-Cre-mediated deletion of Stat5A/5B in the B cell compartment specifically impaired early B cell development but not late B cell maturation. Moreover, the B cell-specific deletion of Stat5A/5B did not impair splenic B cell survival, proliferation, and Ig production. Taken together, these data demonstrate that Stat5A/5B directly control IL-7-mediated early B cell development but are not required for B cell maturation and Ig production.

A critical role of TAK1 in B cell receptor-mediated NF-κB activation

The kinase TAK1 is essential for T-cell receptor (TCR)-mediated nuclear factor kappaB (NF-kappaB) activation and T-cell development. However, the role of TAK1 in B-cell receptor (BCR)-mediated NF-kappaB activation and B-cell development is not clear. Here we show that B-cell-specific deletion of TAK1 impaired the transition from transitional type 2 to mature follicular (FO) B cells and caused a marked decrease of marginal zone (MZ) B cells. TAK1-deficient B cells exhibited an increase of BCR-induced apoptosis and impaired proliferation in response to BCR ligation. Importantly, TAK1-deficient B cells failed to activate NF-kappaB after BCR stimulation. Thus, TAK1 is critical for B-cell maturation and BCR-induced NF-kappaB activation.