SLAM family receptors control pro-survival effectors in germinal center B cells to promote humoral immunity

Expression of the signaling lymphocytic activation molecule (SLAM)–associated protein (SAP) is critical for the germinal center (GC) reaction and T cell–dependent antibody production. However, when SAP is expressed normally, the role of the associated SLAM family receptors (SFRs) in these processes is nebulous. Herein, we established that in the presence of SAP, SFRs suppressed the expansion of the GC reaction but facilitated the generation of antigen-specific B cells and antibodies. SFRs favored the generation of antigen-reactive B cells and antibodies by boosting expression of pro-survival effectors, such as the B cell antigen receptor (BCR) and Bcl-2, in activated GC B cells. The effects of SFRs on the GC reaction and T cell–dependent antibody production necessitated expression of multiple SFRs, both in T cells and in B cells. Hence, while in the presence of SAP, SFRs inhibit the GC reaction, they are critical for the induction of T cell–mediated humoral immunity by enhancing expression of pro-survival effectors in GC B cells.     

Disruption of the Bcl6 Gene Results in an Impaired Germinal Center Formation

The Bcl6 gene has been identified from the chromosomal translocation breakpoint in B cell lymphomas, and its products are expressed highly in germinal center (GC) B cells. To investigate the function of Bcl6 in lymphocytes, we have generated RAG1-deficient mice reconstituted with bone marrow cells from Bcl6-deficient mice (Bcl6^−/−RM). Lymphogenesis in primary lymphoid tissues of Bcl6^−/−RM is normal, and Bcl6^−/−RM produced control levels of primary IgG1 antibodies specific to T cell–dependent antigens. However, GCs were not found in these mice. This defect was mainly due to the abnormalities of B cells. Therefore, Bcl6 is essential for the differentiation of GC B cells.     

TGF-β prevents T follicular helper cell accumulation and B cell autoreactivity

T follicular helper (Tfh) cells contribute to the establishment of humoral immunity by controlling the delivery of helper signals to activated B cells; however, Tfh development must be restrained, as aberrant accumulation of these cells is associated with positive selection of self-reactive germinal center B cells and autoimmunity in both humans and mice. Here, we show that TGF-β signaling in T cells prevented Tfh cell accumulation, self-reactive B cell activation, and autoantibody production. Using mice with either T cell–specific loss or constitutive activation of TGF-β signaling, we demonstrated that TGF-β signaling is required for the thymic maturation of CD44⁺CD122⁺Ly49⁺CD8⁺ regulatory T cells (Tregs), which induce Tfh apoptosis and thus regulate this cell population. Moreover, peripheral Tfh cells escaping TGF-β control were resistant to apoptosis, exhibited high levels of the antiapoptotic protein BCL2, and remained refractory to regulation by CD8⁺ Tregs. The unrestrained accumulation of Tfh cells in the absence of TGF-β was dependent on T cell receptor engagement and required B cells. Together, these data indicate that TGF-β signaling restrains Tfh cell accumulation and B cell–associated autoimmunity and thereby controls self-tolerance.     

Repression of B7.2 on Self-reactive B Cells Is Essential to Prevent Proliferation and Allow Fas-mediated Deletion by CD4+ T Cells

Peripheral tolerance mechanisms normally prevent delivery of T cell help to anergic self-reactive B cells that accumulate in the T zones of spleen and lymph nodes. Chronic exposure to self-antigens desensitizes B cell antigen receptor (BCR) signaling on anergic B cells so that they are not stimulated into clonal expansion by CD4⁺ T cells but instead are eliminated by Fas (CD95)-induced apoptosis. Because a range of BCR-induced signals and responses are repressed in anergic B cells, it is not known which of these are critical to regulate for Fas-mediated peripheral tolerance. Display of the costimulatory molecule, B7.2 (CD86), represents a potentially important early response to acute BCR engagement that is poorly induced by antigen on anergic B cells. We show here that restoring B7.2 expression on tolerant B cells using a constitutively expressed B7.2 transgene is sufficient to prevent Fas-mediated deletion and to trigger extensive T cell–dependent clonal expansion and autoantibody secretion in the presence of specific T cells. Dysregulated expression of B7.2 on tolerant B cells caused a more extreme reversal of peripheral tolerance than that caused by defects in Fas or Fas ligand, and resulted in T cell–dependent clonal expansion and antibody secretion comparable in magnitude to that made by foreign antigen-specific B cells. These findings demonstrate that repression of B7.2 is critical to eliminate autoreactive B cells by Fas in B cell–T cell interactions. The possible role of B7.2 dysregulation in systemic autoimmune diseases is discussed.     

B Lymphocyte Chemotaxis Regulated in Association with Microanatomic Localization, Differentiation State, and B Cell Receptor Engagement

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein–coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell– derived factor (SDF)-1α is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1α also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1α by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1α is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.     

Paracrine Regulation of Germinal Center B Cell Adhesion through the c-Met–Hepatocyte Growth Factor/Scatter Factor Pathway

T cell–dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell migration and interaction with FDC critically depend on integrin-mediated adhesion. To date, the physiological regulators of this adhesion were unkown. In the present report, we have identified the c-met–encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signaling pathway regulating B cell adhesion. We observed that c-Met is predominantly expressed on CD38⁺CD77⁺ tonsillar B cells localized in the dark zone of the GC (centroblasts). On tonsil B cells, ligation of CD40 by CD40-ligand, induces a transient strong upregulation of expression of the c-Met tyrosine kinase. Stimulation of c-Met with HGF/SF leads to receptor phosphorylation and, in addition, to enhanced integrin-mediated adhesion of B cells to both VCAM-1 and fibronectin. Importantly, the c-Met ligand HGF/SF is produced at high levels by tonsillar stromal cells thus providing signals for the regulation of adhesion and migration within the lymphoid microenvironment.     

Human Dendritic Cells Skew Isotype Switching of CD40-activated Naive B Cells towards IgA1 and IgA2

Within T cell–rich areas of secondary lymphoid organs, interdigitating dendritic cells recruit antigen-specific T cells that then induce B cells to secrete Igs. This study investigates the possible role(s) of dendritic cells in the regulation of human B cell responses. In the absence of exogenous cytokines, in vitro generated dendritic cells (referred to as Dendritic Langerhans cells, D-Lc) induced surface IgA expression on ~10% of CD40-activated naive sIgD⁺ B cells. In the presence of IL-10 and TGF-β, a combination of cytokines previously identified for its capacity to induce IgA switch, D-Lc strongly potentiated the induction of sIgA on CD40-activated naive B cells from 5% to 40–50%. D-Lc alone did not induce the secretion of IgA by CD40-activated naive B cells, which required further addition of IL-10. Furthermore, D-Lc skewed towards the IgA isotype at the expense of IgG, the Ig production of CD40-activated naive B cells cultured in the presence of IL-10 and TGF-β. Importantly, under these culture conditions, both IgA₁ and IgA₂ were detected. In the presence of IL-10, secretion of IgA2 by CD40-activated naive B cells could be detected only in response to D-Lc and was further enhanced by TGF-β. Collectively, these results suggest that in addition to activating T cells in the extrafollicular areas of secondary lymphoid organs, human D-Lc also directly modulate T cell–dependent B cell growth and differentiation, by inducing the IgA isotype switch.     

Negative selection of human germinal center B cells by prolonged BCR cross-linking

The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist signals leading either to activation/survival or anergy/death. The outcome of B lymphocyte antigen receptor (BCR) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCR occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti- immunoglobulin kappa and lambda light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross-linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR (kappa + gamma) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCR cross-linking induces death of CD40- activated GC B cells but enhances proliferation of naive or memory cells. Anti-kappa antibody only kills kappa + GC B cells without affecting surrounding gamma + GC B cells, thus demonstrating that BCR- mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the addition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selection of GC B cells. This report provides evidence that in human, susceptibility to BCR killing is regulated along peripheral B cell differentiation pathway.     

Uncovering a novel role of PLCβ4 in selectively mediating TCR signaling in CD8+ but not CD4+ T cells

Because of their common signaling molecules, the main T cell receptor (TCR) signaling cascades in CD4⁺ and CD8⁺ T cells are considered qualitatively identical. Herein, we show that TCR signaling in CD8⁺ T cells is qualitatively different from that in CD4⁺ T cells, since CD8α ignites another cardinal signaling cascade involving phospholipase C β4 (PLCβ4). TCR-mediated responses were severely impaired in PLCβ4-deficient CD8⁺ T cells, whereas those in CD4⁺ T cells were intact. PLCβ4-deficient CD8⁺ T cells showed perturbed activation of peripheral TCR signaling pathways downstream of IP₃ generation. Binding of PLCβ4 to the cytoplasmic tail of CD8α was important for CD8⁺ T cell activation. Furthermore, GNAQ interacted with PLCβ4, mediated double phosphorylation on threonine 886 and serine 890 positions of PLCβ4, and activated CD8⁺ T cells in a PLCβ4-dependent fashion. PLCβ4-deficient mice exhibited defective antiparasitic host defense and antitumor immune responses. Altogether, PLCβ4 differentiates TCR signaling in CD4⁺ and CD8⁺ T cells and selectively promotes CD8⁺ T cell–dependent adaptive immunity.     

B cell intrinsic TLR signals amplify but are not required for humoral immunity

Although innate signals driven by Toll-like receptors (TLRs) play a crucial role in T-dependent immune responses and serological memory, the precise cellular and time-dependent requirements for such signals remain poorly defined. To directly address the role for B cell–intrinsic TLR signals in these events, we compared the TLR response profile of germinal center (GC) versus naive mature B cell subsets. TLR responsiveness was markedly up-regulated during the GC reaction, and this change correlated with altered expression of the key adaptors MyD88, Mal, and IRAK-M. To assess the role for B cell–intrinsic signals in vivo, we transferred MyD88 wild-type or knockout B cells into B cell–deficient μMT mice and immunized recipient animals with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma globulin. All recipients exhibited similar increases in NP-specific antibody titers during primary, secondary, and long-term memory responses. The addition of lipopolysaccharide to the immunogen enhanced B cell-intrinsic, MyD88-dependent NP-specific immunoglobulin (Ig)M production, whereas NP-specific IgG increased independently of TLR signaling in B cells. Our data demonstrate that B cell–intrinsic TLR responses are up-regulated during the GC reaction, and that this change significantly promotes antigen-specific IgM production in association with TLR ligands. However, B cell–intrinsic TLR signals are not required for antibody production or maintenance.     

Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation

We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21–mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (T_FH cells) provide a local source of BLyS. Whereas T cell–derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on T_FH-derived BLyS for their persistence.The affinity maturation of antibodies involves competition among B cells expressing novel specificities generated by somatic hypermutation (SHM). This process occurs in germinal centers (GCs), transient structures formed during T cell–dependent immune responses that enable the preferential survival of B cells producing higher affinity antibodies. Ultimately, this competitive selection process preserves GC B cells with improved antigen affinity and eliminates those that lose specificity or gain autoreactivity. The mechanisms responsible for differential survival remain uncertain but involve tripartite interactions between the GC B cells, FO DCs (FDCs), and T FO helper (T_FH) cells. How the B cell receptor (BCR) drives this affinity-dependent selection process is debated. Although loss of BCR-associated signals disrupt GC kinetics (Wang and Carter, 2005; Huntington et al., 2006), recent findings suggest that antigen capture may be its primary function because BCR signaling is damped in most GC B cells by negative regulatory mechanisms (Khalil et al., 2012). This is consistent with models whereby GC B cells compete for antigen displayed on FDCs to mediate effective MHCII-restricted antigen presentation, thereby fostering sustained T_FH interactions, which in turn promote GC B cell survival (Allen and Cyster, 2008; McHeyzer-Williams et al., 2009; Victora and Nussenzweig, 2012). This idea is further supported by observations indicating that cognate T_FH interactions are a limiting factor in affinity maturation (Schwickert et al., 2011). Thus, higher affinity GC B cells can capture and present antigen more effectively, enabling their preferential access to T_FH cells and facilitating positive selection (Victora et al., 2010; Schwickert et al., 2011).Despite mounting evidence for this model, the mechanism whereby T_FH interactions mediate selective survival of higher affinity GC B cells remains unclear. T–B interactions via receptors such as co-stimulatory molecules, death receptor ligands, and soluble survival factors are probably involved. However, the precise identities and relative roles of these molecules remain obscure because most potential candidates also play roles in GC initiation or maintenance on their own. Therefore, separating these functions from direct roles in the preferential selection of high affinity clones has proven difficult. For example, the initiation and maintenance of GCs rely on sustained CD40/CD40L signals, and death receptors such as Fas/FasL interactions act to limit GC responses (Foy et al., 1993; Han et al., 1995; Hao et al., 2008). Similarly, soluble mediators such as IL-21 are essential for maintenance of GC B cell character as well as fate choices (Linterman et al., 2010; Zotos et al., 2010).The B lineage survival cytokine, B lymphocyte stimulator (BLyS, also termed B cell activating factor [BAFF]), plays a key role in setting thresholds for BCR-mediated selection among naive B cells (Cancro, 2004), making it an attractive candidate for mediating analogous processes in the GC. Consistent with this notion, GC responses prematurely terminate in mice with either global BLyS deficiency or defects in BLyS receptor 3 (BR3, also known as BAFFR) signaling (Rahman et al., 2003). Straightforward interpretation of these findings is difficult, because both BLyS-deficient and BR3 mutant mice are severely B lymphopenic (Moore et al., 1999; Schneider et al., 1999; Yan et al., 2001a). Thus, deficits in naive B cell numbers might explain an inability to sustain GC reactions because GCs are resupplied from the naive pools (Schwickert et al., 2007). Moreover, defects in FDC network maturation and T_FH function also occur in B lymphopenic environments (Rahman et al., 2003; Johnston et al., 2009). Thus, whether BLyS plays a direct role in GC B cell selection and affinity maturation has remained unclear.To better understand how BLyS influences GC function, we investigated the distribution and expression of BLyS and its receptors during GC responses in normal mice. We find that BLyS is spatially segregated between the follicles and GCs, as well as within the GCs, where it is found mainly in the light zone (LZ). Thus, in contrast to FO B cells, GC B cells lack appreciable surface-bound BLyS. This results from profound down-regulation of the BLyS receptor, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which occurs as FO B cells adopt GC B character after IL-21 signals in the context of BCR cross-linking and CD40 co-stimulation. However, in the LZ BLyS is highly expressed by and associated with FO T cells, both helper (T_FH) and regulatory (T_FR). Mixed BM chimeras that lack T cell–derived BLyS have normal GC cellularity and low-affinity IgM and IgG1 antibodies but exhibit significant reductions in high affinity antibody. Moreover, although SHM occurs under these conditions, the patterns of mutation are broadly distributed and show a lower strength of positive selection. Together, these findings indicate that T_FH-derived BLyS is required to preserve high affinity clones among antigen binding GC B cells.