Established T cell-driven germinal center B cell proliferation is independent of CD28 signaling but is tightly regulated through CTLA-4

CD4 T cell activation is positively (CD28) and negatively (CTLA-4) regulated by the costimulatory ligands CD80 and CD86. A central question is how the balance between these two opposing forces is controlled as T cells differentiate. We have previously shown that CD28 signaling is absolutely required to prime naive CD4 T cells to differentiate into effectors that provide help for germinal centers and class-switched Ab responses. In this study, we show that the requirement for CD28 signaling is transient and effector CD4 T cells do not require CD28 signals to sustain their function. The CD28 independence of effector T cells within germinal centers suggested that a key function for CD80/CD86 under these circumstances might be to provide negative regulatory signals via the CD28 homologue CTLA-4. By examining germinal center responses in mice where the ability to signal through T cell CTLA-4 was compromised, we provide data that supports a critical role for CTLA-4 in down-regulating T cell help for germinal center B cells.     

Synergistic B cell activation by CD40 and the B cell antigen receptor: role of B lymphocyte antigen receptor-mediated kinase activation and tumor necrosis factor receptor-associated factor regulation

Optimal activation of B-lymphocytes depends both upon expression of various cell surface receptors and adequate integration of signaling pathways. This requires signals generated upon recognition of antigen by the B lymphocyte antigen receptor (BCR) as well as additional signals provided by cognate interaction with T helper cells, including the CD40-CD154 interaction. Engagement of both the BCR and CD40 results in synergistic activation of B cells. Previous studies identified tumor necrosis factor receptor-associated factor (TRAF)-2 and TRAF3 in the CD40-signaling pathway together with BCR-activated protein kinase D (PKD) as important cooperative factors in this synergy. To better understand the role of these factors in bridging the BCR and CD40 signaling pathways, BCR signal regulation of TRAF function was examined. Results show that phosphorylation of TRAF2 is increased upon BCR but not CD40 engagement and that of the potentially phosphorylated residues of TRAF2, tyrosine 484 is crucial for BCR-CD40 synergy. Additionally, wild type or constitutively active Bruton's tyrosine kinase (Btk) enhanced, whereas the xid mutant form of Btk prevented, BCR-CD40 synergy. These effects were dependent upon TRAF2 and PKD activity. These findings suggest a model in which Btk contributes to the enhancement of the CD40 response by TRAF2 in a PKD-dependent manner.     

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

The B cell antigen receptor （BCR） is the sensor on the B cell surface that surveys foreign molecules （antigen） in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR- mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling.     

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.     

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.     

SAP regulation of follicular helper CD4 T cell development and humoral immunity is independent of SLAM and Fyn kinase

Mutations in SH2D1A resulting in lack of SLAM-associated protein (SAP) expression cause the human genetic immunodeficiency X-linked lymphoproliferative disease. A severe block in germinal center development and lack of long-term humoral immunity is one of the most prominent phenotypes of SAP(-) mice. We show, in this study, that the germinal center block is due to an essential requirement for SAP expression in Ag-specific CD4 T cells to develop appropriate follicular helper T cell functions. It is unknown what signaling molecules are involved in regulation of SAP-dependent CD4 T cell help functions. SAP binds to the cytoplasmic tail of SLAM, and we show that SLAM is expressed on resting and activated CD4 T cells, as well as germinal center B cells. In addition, SAP can recruit Fyn kinase to SLAM. We have now examined the role(s) of the SLAM-SAP-Fyn signaling axis in in vivo CD4 T cell function and germinal center development. We observed normal germinal center development, long-lived plasma cell development, and Ab responses in SLAM(-/-) mice after a viral infection (lymphocytic choriomeningitis virus). In a separate series of experiments, we show that SAP is absolutely required in CD4 T cells to drive germinal center development, and that requirement does not depend on SAP-Fyn interactions, because CD4 T cells expressing SAP R78A are capable of supporting normal germinal center development. Therefore, a distinct SAP signaling pathway regulates follicular helper CD4 T cell differentiation, separate from the SLAM-SAP-Fyn signaling pathway regulating Th1/Th2 differentiation.     

The BTB-kelch protein KLHL6 is involved in B-lymphocyte antigen receptor signaling and germinal center formation

BTB-kelch proteins can elicit diverse biological functions but very little is known about the physiological role of these proteins in vivo. Kelch-like protein 6 (KLHL6) is a BTB-kelch protein with a lymphoid tissue-restricted expression pattern. In the B-lymphocyte lineage, KLHL6 is expressed throughout ontogeny, and KLHL6 expression is strongly upregulated in germinal center (GC) B cells. To analyze the role of KLHL6 in vivo, we have generated mouse mutants of KLHL6. Development of pro- and pre-B cells was normal but numbers of subsequent stages, transitional 1 and 2, and mature B cells were reduced in KLHL6-deficient mice. The antigen-dependent GC reaction was blunted (smaller GCs, reduced B-cell expansion, and reduced memory antibody response) in the absence of KLHL6. Comparison of mutants with global loss of KLHL6 to mutants lacking KLHL6 specifically in B cells demonstrated a B-cell-intrinsic requirement for KLHL6 expression. Finally, B-cell antigen receptor (BCR) cross-linking was less sensitive in KLHL6-deficient B cells compared to wild-type B cells as measured by proliferation, Ca2+ response, and activation of phospholipase Cgamma2. Our results strongly point to a role for KLHL6 in BCR signal transduction and formation of the full germinal center response.     

Cell-cell interactions in synovitis: Interactions between T cells and B cells in rheumatoid arthritis

In rheumatoid arthritis, T cells and B cells participate in the immune responses evolving in the synovial lesions. Interaction between T cells and B cells is probably antigen specific because complex microstructures typical of secondary lymphoid organs are generated. Differences between patients in forming follicles with germinal centers, T-cell–B-cell aggregates without germinal center reactions, or loosely organized T-cell–B-cell infiltrates might reflect the presence of different antigens or a heterogeneity in host response patterns to immune injury. Tertiary lymphoid microstructures in the rheumatoid lesions can enhance the sensitivity of antigen recognition, optimize the collaboration of immunoregulatory and effector cells, and support the interaction between the tissue site and the aberrant immune response. The molecular basis of lymphoid organogenesis studied in gene-targeted mice will provide clues to why the synovium is a preferred site for tertiary lymphoid tissue. B cells have a critical role in lymphoid organogenesis. Their contribution to synovial inflammation extends beyond antibody secretion and includes the activation and regulation of effector T cells.     

Activation—induced cell death in B lymphocytes

Upon encountering the antigen(Ag),the immune system can either develop a specific immune response of enter a specific state of unresponsiveness,tolerance.The response of B cells to their specific Ag can be activation and proliferation,leading to the immune response,or anergy and activation-induced cell death(AICD),leading to tolerance.AICD in B lymphocytes is a highly regulated event initiated by crosslinking of the B cell receptor (BCR).BCR engagement initiates several signaling events such as activation of PLCγ,Ras,and PI3K,which generally speaking,lead to survival.However,in the absence of survival signals(CD40 or IL-4R engagement),BCR crosslinking can also promote apoptotic signal transduction pathways such as activation of effector caspases,expression of pro-apoptotic genes,and inhibition of pro-survival genes.The complex interplay between survival and death signals determines the B cell fate and, consequently,the immune response.     

Differential effects of IL-27 on human B cell subsets

IL-27 is a novel heterodimeric cytokine of the IL-12 family that plays an important role in the regulation of T cell responses. Its role on human B cells has not been previously studied. In this study, we show that both chains of the IL-27 receptor complex, IL-27R and gp130, are constitutively expressed at the surface of naive and memory human tonsillar B cells, and are induced on germinal center B cells following CD40 stimulation. In naive B cells, IL-27 induced strong STAT1 and STAT3 phosphorylation, whereas it induced moderate STAT1 and low STAT3 activation in memory B cells. IL-27 induced T-bet expression in naive and memory B cells stimulated by CD40 or surface Ig engagement, but induced significant IL-12Rbeta2 surface expression in anti-Ig-stimulated naive B cells only. In anti-Ig-stimulated naive or memory B cells, IL-27 also induced CD54, CD86, and CD95 surface expression. In addition, IL-27 increased proliferation of anti-Ig-activated naive B cells and of anti-CD40-activated naive and germinal center B cells, but not of CD40-activated memory B cells. These data indicate that the B cell response to IL-27 is modulated during B cell differentiation and varies depending on the mode of B cell activation.