Search

CD22 is an inhibitory B cell coreceptor that regulates B cell development and activation by downregulating BCR signaling through activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1). CD22 recognizes α2,6 sialic acid as a specific ligand and interacts with α2,6 sialic acid-containing membrane molecules, such as CD45, IgM, and CD22, expressed on the same cell. Functional regulation of CD22 by these endogenous ligands enhances BCR ligation-induced signaling and is essential for normal B cell responses to Ags. In this study, we demonstrate that CD45 plays a crucial role in CD22-mediated inhibition of BCR ligation-induced signaling. However, disruption of ligand binding of CD22 enhances CD22 phosphorylation, a process required for CD22-mediated signal inhibition, upon BCR ligation in CD45−/− as well as wild-type mouse B cells but not in mouse B cells expressing a loss-of-function mutant of SHP-1. This result indicates that SHP-1 but not CD45 is required for ligand-mediated regulation of CD22. We further demonstrate that CD22 is a substrate of SHP-1, suggesting that SHP-1 recruited to CD22 dephosphorylates nearby CD22 as well as other substrates. CD22 dephosphorylation by SHP-1 appears to be augmented by homotypic CD22 clustering mediated by recognition of CD22 as a ligand of CD22 because CD22 clustering increases the number of nearby CD22. Our results suggest that CD22 but not CD45 is an endogenous ligand of CD22 that enhances BCR ligation-induced signaling through SHP-1–mediated dephosphorylation of CD22 in CD22 clusters.

Autoimmunity and inflammation are controlled in part by regulatory B cells, including a recently identified IL-10-competent CD1dhighCD5+ B cell subset termed B10 cells that represents 1–3% of adult mouse spleen B cells. In this study, pathways that influence B10 cell generation and IL-10 production were identified and compared with previously described regulatory B cells. IL-10-competent B cells were predominantly CD1dhighCD5+ in adult spleen and were the prevalent source of IL-10, but not other cytokines. B10 cell development and/or maturation in vivo required Ag receptor diversity and intact signaling pathways, but not T cells, gut-associated flora, or environmental pathogens. Spleen B10 cell frequencies were significantly expanded in aged mice and mice predisposed to autoimmunity, but were significantly decreased in mouse strains that are susceptible to exogenous autoantigen-induced autoimmunity. LPS, PMA, plus ionomycin stimulation in vitro for 5 h induced B10 cells to express cytoplasmic IL-10. However, prolonged LPS or CD40 stimulation (48 h) induced additional adult spleen CD1dhighCD5+ B cells to express IL-10 following PMA plus ionomycin stimulation. Prolonged LPS or CD40 stimulation of newborn spleen and adult blood or lymph node CD1dlow and/or CD5− B cells also induced cytoplasmic IL-10 competence in rare B cells, with CD40 ligation uniformly inducing CD5 expression. IL-10 secretion was induced by LPS signaling through MyD88-dependent pathways, but not following CD40 ligation. LPS stimulation also induced rapid B10 cell clonal expansion when compared with other spleen B cells. Thereby, both adaptive and innate signals regulate B10 cell development, maturation, CD5 expression, and competence for IL-10 production.

Because pathogens induce infectious symptoms in a time-dependent manner, a rapid immune response is beneficial for defending hosts from pathogens, especially those inducing acute infectious diseases. However, it is largely unknown how the time course of immune responses is regulated. In this study, we demonstrate that B cells deficient in the inhibitory coreceptor CD22 undergo accelerated cell division after Ag stimulation, resulting in rapid generation of plasma cells and Ab production. This finding indicates that CD22 regulates the time course of B cell responses and suggests that CD22 is a good target to shorten the time required for Ab production, thereby augmenting host defense against acute infectious diseases as “universal vaccination.”

Several studies indicate that CD4+ T cells, macrophages, and dendritic cells initially mediate intestinal inflammation in murine models of human inflammatory bowel disease. However, the initial role of B cells in the development of intestinal inflammation remains unclear. In this study we present evidence that B cells can trigger intestinal inflammation using transgenic (Tg) mice expressing CD40 ligand (CD40L) ectopically on B cells (CD40L/B Tg). We demonstrated that CD40L/B Tg mice spontaneously developed severe transmural intestinal inflammation in both colon and ileum at 8–15 wk of age. In contrast, CD40L/B Tg×CD40−/− double-mutant mice did not develop colitis, indicating the direct involvement of CD40-CD40L interaction in the development of intestinal inflammation. The inflammatory infiltrates consisted predominantly of massive aggregated, IgM-positive B cells. These mice were also characterized by the presence of anti-colon autoantibodies and elevated IFN-γ production. Furthermore, although mice transferred with CD4+ T cells alone or with both CD4+ T and B220+ B cells, but not B220+ cells alone, from diseased CD40L/B Tg mice, develop colitis, mice transferred with B220+ B cells from diseased CD40L/B Tg mice and CD4+ T cells from wild-type mice also develop colitis, indicating that the Tg B cells should be a trigger for this colitis model, whereas T cells are involved as effectors. As it has been demonstrated that CD40L is ectopically expressed on B cells in some autoimmune diseases, the present study suggests the possible contribution of B cells in triggering intestinal inflammation in human inflammatory bowel disease.

Ag-induced B cell proliferation in vivo requires a costimulatory signal through CD40, whereas B cell Ag receptor (BCR) ligation by anti-Ig H chain Abs, such as anti-Ig μ H chain Ab and anti-Ig δ H chain Ab, alone induces proliferation of B cells in vitro, even in the absence of CD40 ligation. In this study, we demonstrate that CD40 signaling is required for survival and proliferation of B cells stimulated by protein Ags in vitro as well as in vivo. This indicates that the in vitro system represents B cell activation in vivo, and that protein Ags generate BCR signaling distinct from that by anti-Ig H chain Abs. Indeed, BCR ligation by Ags, but not by anti-Ig H chain Abs, efficiently phosphorylates the inhibitory coreceptors CD22 and CD72. When these coreceptors are activated, anti-Ig H chain Ab-stimulated B cells can survive and proliferate only in the presence of CD40 signaling. Conversely, treatment of Ag-stimulated B cells with anti-CD72 mAb blocks CD72 phosphorylation and induces proliferation, even in the absence of CD40 signaling. These results strongly suggest that activation of B cells by anti-Ig H chain Abs involves their ability to silence the inhibitory coreceptors, and that the inhibitory coreceptors install requirement of CD40 signaling for survival and proliferation of Ag-stimulated B cells.

Activation signals of lymphocytes are negatively regulated by the membrane molecules carrying the immunoreceptor tyrosine-based inhibition motif (ITIM). Upon tyrosine phosphorylation, ITIMs recruit SH2-containing phosphatases such as SHP-1, resulting in down-modulation of cell activation. We showed that the cytoplasmic domain of the CD72 molecule carries an ITIM and is associated in vitro with SHP-1 upon tyrosine phosphorylation. Moreover, cross-linking of B cell Ag receptor (BCR) enhances both tyrosine phosphorylation of CD72 and association of CD72 with SHP-1 in B cell line WEHI-231. These results indicate that CD72 recruits SHP-1 upon tyrosine phosphorylation induced by BCR signaling, suggesting that CD72 is a negative regulator of BCR signaling.

CD40LBTg mice, expressing a CD40 ligand (CD40L) transgene on B cells, represent a model for human diseases where B cells aberrantly express CD40L or receive excess CD40/CD40L signaling under inflammatory conditions. Here, we show that B cells expressing transgenic CD40L are capable of priming CD8 T cells and generate strong antigen-specific cytotoxicity. Adoptively transferred SIINFEKL peptide-loaded B cells from CD40LBTg but not wild type mice were able to activate self-reactive OT-I CD8 T cells upon immunization with OVA plus alum and trigger diabetes in RIP-OVA mice by enhancing the help of endogenous self-reactive CD4 T cells. CD40L expressing B cells also trigger spontaneous activation of splenic CD8 T cells, which rapidly up-regulate PD-1, Blimp-1 and LAG-3 and lose cytotoxicity along with IL-10 expression via interaction with PDL-1hi CD11c+ dendritic cells in T cell zones. Thus, CD11c+ dendritic cells from CD40LBTg mice exhibit regulatory phenotype, which block granzyme B expression and preferentially induce IL-10 expression in activated CD8 T cells. These results demonstrate that constitutive CD40 signaling on B cells under inflammation increases the risk of breaking peripheral CD8 T cell tolerance. However, the presence of CD40-activated B cells results in PDL-1hi CD11c+ dendritic cells, which exhibit a regulatory role to dampen harmful self-reactive cytotoxicity by promoting IL-10 expression in CD8 T cells.

To test the pathogenic role of B cells under excess inflammatory CD40/CD40L signaling, experimental autoimmune encephalomyelitis (EAE) was induced in B cell specific CD40L transgenic (CD40LBTg) and C57BL/6 control mice by immunization with MOG35-55 peptide. Clinical symptoms were monitored using a 0-5 scoring system during days 10-30 after induction. Alternatively, MOG-specific CD4 TCR Tg (2D2) mice were crossed onto a CD40LBTg background and the incidence of spontaneous encephalomyelitis monitored at 4-35 weeks of age. Both CD40LBTg and control mice developed EAE with similar kinetics, with disease onset at around day 15-17 and the peak of disease score at around day 21-25. However, CD40LBTg mice showed significantly more severe disease than wild type mice with average peak clinical scores of 3.0 and 1.5, respectively. Higher frequencies of IL-17+ CD4 T cells were also observed in CD40LBTg mice compared to wild type mice at the EAE disease peak. Also, 60% (6/10) of 2D2 CD40LB double Tg mice showed spontaneous encephalomyelitis-like symptoms (severe hind and/or fore limb paralysis) between 4-6 weeks of age, with IL-17+ and IFNγ+ CD4 T cells in spleen and inguinal lymph nodes. Furthermore, even those without apparent EAE symptoms died before 30 weeks of age. None of the 2D2 or CD40LBTg mice showed such symptoms between 4-35 weeks of age. These results support the notion that excess B cell CD40/CD40L signaling promotes MOG-specific pathogenic CD4 T cell response.