Guoping Fu

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.

Phosphorylation of Bcl10 Negatively Regulates T-Cell Receptor-Mediated NF-κB Activation

ABSTRACT Bcl10 (B-cell lymphoma 10) is an adaptor protein comprised of an N-terminal caspase recruitment domain and a C-terminal serine/threonine-rich domain. Bcl10 plays a critical role in antigen receptor-mediated NF-κB activation and lymphocyte development and functions. Our current study has discovered that T-cell activation induced monophosphorylation and biphosphorylation of Bcl10 and has identified S138 within Bcl10 as one of the T-cell receptor-induced phosphorylation sites. Alteration of S138 to an alanine residue impaired T-cell activation-induced ubiquitination and subsequent degradation of Bcl10, ultimately resulting in prolongation of TCR-mediated NF-κB activation and enhancement of interleukin-2 production. Taken together, our findings demonstrate that phosphorylation of Bcl10 at S138 down-regulates Bcl10 protein levels and thus negatively regulates T-cell receptor-mediated NF-κB activation.

An important role of phospholipase Cγ1 in pre‐B‐cell development and allelic exclusion

Phospholipase Cγ1 (PLCγ1) has been reported to be expressed predominantly in T cells and to play an important role in T‐cell receptor signaling. Here we show that PLCγ1 is expressed throughout B‐cell development, with high expression in B‐cell progenitors, and is involved in pre‐B‐cell receptor (pre‐BCR) signaling. Reduced expression of PLCγ1, in the absence of PLCγ2 (PLCγ1+/−PLCγ2−/−), impedes early B‐cell development at the pro‐B‐ to pre‐B‐cell transition and impairs immunoglobulin heavy chain allelic exclusion, hallmarks of defective pre‐BCR signaling. In contrast, early B‐cell development is largely normal, whereas late B‐cell maturation is impaired in the absence of PLCγ2 alone (PLCγ2−/−) and overexpression of PLCγ1 in PLCγ2−/− mice fails to restore BCR‐mediated B‐cell proliferation and maturation. These studies reveal an essential role of PLCγ1, distinct from that of PLCγ2, in B‐cell development.

Essential Role of Phospholipase Cγ2 in Early B-Cell Development and Myc-Mediated Lymphomagenesis

ABSTRACT Phospholipase Cγ2 (PLCγ2) is a critical signaling effector of the B-cell receptor (BCR). Here we show that PLCγ2 deficiency impedes early B-cell development, resulting in an increase of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B cells. PLCγ2 deficiency impairs pre-BCR-mediated functions, leading to enhanced interleukin-7 (IL-7) signaling and elevated levels of RAGs in the selected large pre-B cells. Consequently, PLCγ2 deficiency renders large pre-B cells susceptible to transformation, resulting in dramatic acceleration of Myc-induced lymphomagenesis. PLCγ2−/− Eμ-Myc transgenic mice mainly develop lymphomas of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B-cell origin, which are uncommon in wild-type Eμ-Myc transgenics. Furthermore, lymphomas from PLCγ2−/− Eμ-Myc transgenic mice exhibited a loss of p27Kip1 and often displayed alterations in Arf or p53. Thus, PLCγ2 plays an important role in pre-BCR-mediated early B-cell development, and its deficiency leads to markedly increased pools of the most at-risk large pre-B cells, which display hyperresponsiveness to IL-7 and express high levels of RAGs, making them prone to secondary mutations and Myc-induced malignancy.

Phospholipase Cγ2 Mediates RANKL-stimulated Lymph Node Organogenesis and Osteoclastogenesis

Phospholipase Cγ2 (PLCγ2) is an important signaling effector of multiple receptors in the immune system. Here we show that PLCγ2-deficient mice displayed impaired lymph node organogenesis but normal splenic structure and Peyers patches. Receptor activator of NF-κB ligand (RANKL) is a tumor necrosis factor family cytokine and is essential for lymph node organogenesis. Importantly, PLCγ2 deficiency severely impaired RANKL signaling, resulting in marked reduction of RANKL-induced activation of MAPKs, p38 and JNK, but not ERK. The lack of PLCγ2 markedly diminished RANKL-induced activation of NF-κB, AP-1, and NFATc1. Moreover, PLCγ2 deficiency impaired RANKL-mediated biological function, leading to failure of the PLCγ2-deficient bone marrow macrophage precursors to differentiate into osteoclasts after RANKL stimulation. Re-introduction of PLCγ2 but not PLCγ1 restores RANKL-mediated osteoclast differentiation of PLCγ2-deficient bone marrow-derived monocyte/macrophage. Taken together, PLCγ2 is essential for RANK signaling, and its deficiency leads to defective lymph node organogenesis and osteoclast differentiation.

Phospholipase Cγ2 Plays a Role in TCR Signal Transduction and T Cell Selection

One of the important signaling events following TCR engagement is activation of phospholipase Cγ (PLCγ). PLCγ has two isoforms, PLCγ1 and PLCγ2. It is known that PLCγ1 is important for TCR signaling and TCR-mediated T cell selection and functions, whereas PLCγ2 is critical for BCR signal transduction and BCR-mediated B cell maturation and functions. In this study, we report that PLCγ2 was expressed in primary T cells, and became associated with linker for activated T cells and Src homology 2-domain containing leukocyte protein of 76 kDa and activated upon TCR stimulation. PLCγ1/PLCγ2 double-deficient T cells displayed further block from CD4 and CD8 double-positive to single-positive transition compared with PLCγ1 single-deficient T cells. TCR-mediated proliferation was further impaired in PLCγ1/PLCγ2 double-deficient T cells compared with PLCγ1 single-deficient T cells. TCR-mediated signal transduction, including Ca2+ mobilization and Erk activation, was further impaired in PLCγ1/PLCγ2 double-deficient relative to PLCγ1 single-deficient T cells. In addition, in HY TCR transgenic mouse model, thymic positive and negative selections were reduced in PLCγ1 heterozygous- and PLCγ2 homozygous-deficient (PLCγ1+/−PLCγ2−/−) relative to wild-type, PLCγ2 single-deficient (PLCγ2−/−), or PLCγ1 heterozygous-deficient (PLCγ1+/−) mice. Taken together, these data demonstrate that PLCγ2 participates in TCR signal transduction and plays a role in T cell selection.

Critical Role of B Cell Lymphoma 10 in BAFF-Regulated NF-κB Activation and Survival of Anergic B Cells

Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-κB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimulation induced Bcl10 association with IκB kinase β, a key component of the canonical NF-κB pathway. Consistently, Bcl10-deficient B cells were impaired in BAFF-induced IκBα phosphorylation and formation of nuclear p50/c-Rel complexes. Bcl10-deficient B cells also displayed reduced expression of NF-κB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-xL, a BAFF-induced NF-κB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-κB activation directly and noncanonical NF-κB activation indirectly. The BAFF-R/Bcl10/NF-κB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells.

The adhesion molecule PECAM-1 enhances the TGF-β-mediated inhibition of T cell function.

Blocking an adhesion molecule on the surface of T cells may enhance antitumor activity. Sticking it to T cells Because of its immunosuppressive effects and abundance in tumor microenvironments, the cytokine transforming growth factor–β (TGF-β) inhibits the antitumor activities of T cells. Targeting TGF-β systemically would interfere with other functions of TGF-β; thus, therapies that block the effects of TGF-β specifically on T cells are needed. Newman et al. found that T cells without the adhesion molecule PECAM-1 were less sensitive to the TGF-β–mediated inhibition of their function than were PECAM-1–positive cells. In a tumor model, PECAM-1–deficient mice exhibited reduced tumor size compared to wild-type mice. Thus, targeting PECAM-1 may specifically enhance the antitumor activity of T cells. Transforming growth factor–β (TGF-β) is an immunosuppressive cytokine that inhibits the proinflammatory functions of T cells, and it is a major factor in abrogating T cell activity against tumors. Canonical TGF-β signaling results in the activation of Smad proteins, which are transcription factors that regulate target gene expression. We found that the cell surface molecule platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitated noncanonical (Smad-independent) TGF-β signaling in T cells. Subcutaneously injected tumor cells that are dependent on TGF-β–mediated suppression of immunity for growth grew more slowly in PECAM-1−/− mice than in their wild-type counterparts. T cells isolated from PECAM-1−/− mice demonstrated relative insensitivity to the TGF-β–dependent inhibition of interferon-γ (IFN-γ) production, granzyme B synthesis, and cellular proliferation. Similarly, human T cells lacking PECAM-1 demonstrated decreased sensitivity to TGF-β in a manner that was partially restored by reexpression of PECAM-1. Co-incubation of T cells with TGF-β and a T cell–activating antibody resulted in PECAM-1 phosphorylation on an immunoreceptor tyrosine–based inhibitory motif (ITIM) and the recruitment of the inhibitory Src homology 2 (SH2) domain–containing tyrosine phosphatase-2 (SHP-2). Such conditions also induced the colocalization of PECAM-1 with the TGF-β receptor complex as identified by coimmunoprecipitation, confocal microscopy, and proximity ligation assays. These studies indicate a role for PECAM-1 in enhancing the inhibitory functions of TGF-β in T cells and suggest that therapeutic targeting of the PECAM-1–TGF-β inhibitory axis represents a means to overcome TGF-β–dependent immunosuppression within the tumor microenvironment.

Kras Is Critical for B Cell Lymphopoiesis

The three major Ras members, Kras, Hras, and Nras, are highly homologous and individual Ras genes can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological functions of this Ras family member. In this study, we generated and examined mice with hematopoietic-specific deletion of Kras and bone marrow (BM) chimeric mice with B cell–specific targeted deletion of Kras. Hematopoietic-specific deletion of Kras impaired early B cell development at the pre–B cell stage and late B cell maturation, resulting in the reduction of BM pre–, immature, and mature B cells and peripheral follicular, marginal zone, and B1 mature B cells. In contrast, Kras deficiency did not affect T cell development. Studies of BM chimeric mice with B cell–specific deletion of Kras demonstrated that Kras deficiency intrinsically impaired B cell development. Kras deficiency reduced BCR-induced B cell proliferation and survival. Furthermore, Kras deficiency specifically impaired pre–BCR- and BCR-induced activation of the Raf-1/MEK/ERK pathway in pre–B and mature B cells, respectively. Thus, Kras is the unique Ras family member that plays a critical role in early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway.

Frontline Science: PECAM‐1 (CD31) expression in naïve and memory, but not acutely activated, CD8+ T cells

Inhibitory cell surface proteins on T cells are often dynamically regulated, which contributes to their physiologic function. PECAM‐1 (CD31) is an inhibitory receptor that facilitates TGF‐β‐mediated suppression of T cell activity. It is well established in CD4+ T cells that PECAM‐1 is expressed in naïve recent thymic emigrants, but is down‐regulated after acute T cell activation and absent from memory cells. The extent to which PECAM‐1 expression is similarly regulated in CD8+ T cells is much less well characterized. We evaluated T cells recovered from mice after infection with a model intracellular pathogen and determined that, in CD8+ T cells, PECAM‐1 expression was strongly down‐regulated during acute infection but re‐expressed to intermediate levels in memory cells. Down‐regulation of PECAM‐1 expression in CD8+ T cells was transcriptionally regulated and affected by the strength and nature of TCR signaling. PECAM‐1 was also detected on the surface of human activated/memory CD8+, but not CD4+ T cells. These data demonstrate that PECAM‐1 expression is dynamically regulated, albeit differently, in both CD4+ and CD8+ T cells. Furthermore, unlike memory CD4+ T cells, memory CD8+ T cells retain PECAM‐1 expression and have the potential to be modulated by this inhibitory receptor.