B cells can suppress autoimmunity by secreting interleukin-10 (IL-10). Although subpopulations of splenic B lineage cells are reported to express IL-10 in vitro, the identity of IL-10-producing B cells with regulatory function in vivo remains unknown. By using IL-10 reporter mice, we found that plasmablasts in the draining lymph nodes (dLNs), but not splenic B lineage cells, predominantly expressed IL-10 during experimental autoimmune encephalomyelitis (EAE). These plasmablasts were generated only during EAE inflammation. Mice lacking plasmablasts by genetic ablation of the transcription factors Blimp1 or IRF4 in B lineage cells developed an exacerbated EAE. Furthermore, IRF4 positively regulated IL-10 production that can inhibit dendritic cell functions to generate pathogenic T cells. Our data demonstrate that plasmablasts in the dLNs serve as IL-10 producers to limit autoimmune inflammation and emphasize the importance of plasmablasts as IL-10-producing regulatory B cells.

A chief Ca(2+) entry pathway in immune cells is store-operated Ca(2+) (SOC) influx, which is triggered by depletion of Ca(2+) from the endoplasmic reticulum (ER). However, its physiological role in B cells remains elusive. Here, we show that ER calcium sensors STIM1- and STIM2-induced SOC influx is critical for B cell regulatory function. B cell-specific deletion of STIM1 and STIM2 in mice caused a profound defect in B cell receptor (BCR)-induced SOC influx and proliferation. However, B cell development and antibody responses were unaffected. Remarkably, B cells lacking both STIM proteins failed to produce the anti-inflammatory cytokine IL-10 because of defective activation of nuclear factor of activated T cells (NFAT) after BCR stimulation. This resulted in exacerbation of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Our data establish STIM-dependent SOC influx as a key signal for B cell regulatory function required to limit autoimmunity.

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SUMMARYCellular immune mechanisms resulting in interferon-gamma (IFN-g) production are essential for protection against cutaneous leishmaniasis. Antigens of the intracellular amastigote form of the parasite, found in mammalian hosts, are likely to be good candidates for the induction of T cell response and protection from development of leishmaniasis. We purified a stage-specific antigen from amastigote soluble antigen (A-SLA) of Leishmania major by immunoaffinity chromatography. The purified protein was characterized as a cysteine proteinase with enzymatic activity which is inhibited by E-64, and it was named the amastigote cysteine proteinase (ACP). BALB/c mice were immunized by two intraperitoneal injections, at a month interval, of 5 m g of ACP or A-SLA in Freund's complete adjuvant (FCA). Animals were challenged 4 weeks later with 10 6 L. major promastigotes and examined 4 months after the last injection. The immunized animals developed significantly smaller or no lesions compared with controls. Spleen cells from immunized mice showed a significant proliferative response and produced a high level of IFN-g in response to ACP, suggesting the induction of Th1 cells after immunization. These results make 24-kD ACP a possible component for an eventual cocktail vaccine against L. major infection.

The Cre/loxP system is a powerful tool for generating conditional gene knockout (KO) mice and elucidate gene function in vivo. CD19-Cre and Mb1-iCre transgenic mice are commonly used for generating B cell-specific KO mice and investigate the development, as well as the physiological and pathophysiological roles of B cells. However, the CD19-Cre line low efficiency and the Mb1-iCre line occasional ectopic recombination represent challenges for their use. Thus, we developed a CD19-codon-improved Cre (CD19-iCre) knock-in mouse with the T2A-iCre sequence inserted into the Cd19 locus, just before the stop codon. The CD19-iCre mice were compared with existing models, crossed with the Rosa26-EYFP reporter mice, and their recombination activity in B cells carrying different Cre alleles was assessed. CD19-iCre mice showed more effective Cre recombination in the early B cell developmental stages compared with the CD19-Cre mice. The efficiencies of the CD19-iCre and Mb1-iCre lines were similar; however, the B lineage-specific recombination was more stringent in the CD19-iCre line. Furthermore, the utility value of the CD19-iCre model was superior than that of the CD19-Cre mice regarding deletion efficiency in IL10-floxed mice. Thus, the CD19-iCre line is a valuable tool for highly efficient gene targeting specific to the B cell compartment.