Aims/hypothesis NOD.Igµ null mice lacking mature B cells are highly resistant to diabetes and display poor CD4 T cell responses to autoantigens. Nevertheless, the degree to which different B cell subsets contribute to diabetes in NOD mice remains unresolved. Due to their role in the recognition of microbial and autoantigens, peritoneal B cell characteristics were examined in NOD mice to see if they differ developmentally, phenotypically or functionally in aspects relevant to diabetogenesis. Methods The population dynamics, activation state, migratory behaviour and antigen presentation function were investigated in NOD peritoneal B cells. Results NOD peritoneal B cells were found to express abnormally high levels of co-stimulatory molecules (CD40, CD86 and CD69). In contrast, the expression of L-selectin and integrin α4β1 was markedly reduced in NOD mice compared with BALB/c and C57BL/6 mice. The number of B cells in the peritoneum was lower in NOD than in control mice throughout development; migration of B cells from the peritoneum to the pancreatic lymph nodes in NOD mice was enhanced tenfold. NOD B cells showed no chemotactic response to sphingosine-1-phosphate, which normally acts to retain B cells in the peritoneum. Peritoneal B cells of NOD mice also presented insulin autoantigen to CD4 T cells, inducing T cell proliferation. Conclusions/interpretation NOD peritoneal B cells are hyperactivated, migrate to the pancreatic lymph nodes and are capable of driving insulin-specific CD4 T cell activation. These characteristics could make them important for inducing or amplifying T cell responses against islet-antigens.

Two monoclonal antibodies, termed nnIE11 and nnIG11, were generated against the murine thymic stromal lymphopoietin receptor, mTSLPR, using traditional hybridoma technology. The antibody-producing hybridoma clones were obtained by fusing P3X63-Ag8.653 myeloma cells with splenocytes from Balb/c mice immunized with anti-FLAG M2 affinity-purified FLAG-tagged mTSLPR from pSVL-mTSLPR-FLAG-transfected COS cells and Ni-NTA-purified his-tagged mTSLPR from recombinant FastBacHisB-mdelta1 baculovirus-infected Sf9 cells. Several monoclonal anti-mTSLPR-specific hybridoma clones were obtained and two of these clones are further characterized here. The generated antibodies could in an immunoblotting identify baculovirus-expressed mTSLPR proteins with a molecular weight corresponding to 50 kDa. Both immunoblotting and ELISA with recombinant mouse TSLPR/Fc chimera as antigen, having only the N-terminal domain of mTSLPR present, indicated that the generated monoclonal antibodies identify the C-terminus of mTSLPR. Although sandwich ELISAs performed with a goat anti-mTSLPR antiserum as capture antibody and nnIE11 as indicator antibody were able to detect mTSLPR in the range of 5 ng/ml, no souble mTSLPR could be observed in serum samples from CBA/H, Balb/c and C57Bl/6 mice.