3c), and occurred in all major splenic B cell populations (Fig. and pancreatic lymph 6-Acetamidohexanoic acid nodes prior to disease development. Conclusions/interpretation These findings are consistent with genetic determination of the escape of high-affinity IBCs from anergy and their early contribution to the development of type 1 diabetes. test was used to compare differences between groups for Figs 1, ?,22 and ?and3.3. MannCWhitney tests were used to compare differences between groups for Figs 4 and ?and5.5. ANOVA was used to analyse data means with repeated measures when appropriate. Data are expressed as means (SEM). A value of 0.5 was considered significant. Randomisation and blinding was not carried out. No animals or data were excluded from these experiments. Open in a separate window Fig. 1 Detection and enrichment of IBCs in IKBA disease-resistant and disease-prone mice. (a) Diagram of the absorbent used for the magnetic-particle-based staining and enrichment of IBCs. Splenic cells from VH125.NOD mice were subjected to the enrichment protocol in the presence of 50 unlabelled insulin, or in the absence of biotinylated insulin. (b) Representative cytograms comparing the total and enriched populations of splenic IBCs from female 8- to 12-week-old VH125.C57BL/6-H2g7, VH125.NOD, VH281.NOD and NOD mice. (c) MFI of IBCs compared across the four strains (test Open in a separate window Fig. 2 BCR specificity for insulin contributes to development of diabetes in NOD mice. (a) Disease development in female VH125.NOD (tests; *tests Open in a separate window Fig. 4 IBCs in VH125.NOD are functionally responsive. (a) Gating strategy to identify IBCs in VH125.C57BL/6-H2g7 and VH125.NOD mice for assay of calcium mobilisation. Cytograms depicting gates when insulin is omitted are shown for comparison and to demonstrate the specificity of the binding. IBCs in VH125.C57BL/6-H2g7 mice are probably Insulinhi due to decreased mIgM expression and do not show a mobilisation of intracellular calcium ([Ca2+]i) following stimulation (black line) compared with non-IBCs (light grey line), indicative of anergy. IBCs in VH125.NOD mice show increased mIgM expression and equal calcium mobilisation (dark grey line) compared with non-IBCs (light grey line), suggesting they 6-Acetamidohexanoic acid are not anergic. (b) Representative histograms of pSyk before (dotted lines) and after (solid lines) stimulation in non-IBCs (Insulin?) and IBCs (Insulin+) from VH125.C57BL/6-H2g7 (B6) and VH125.NOD (NOD) mice. Gating on IBCs and non-IBCs is similar to that shown in Fig. 1b. Stim, stimulated; unstim, unstimulated. (c) Change in geometric MFI (gMFI) of phosphorylated Syk following stimulation in IBCs and non-IBCs in the two strains. (d) Representative basal PTEN levels in non-IBCs and IBCs from the two strains. Gating on IBCs and non-IBCs is similar to that shown in Fig. 1b. (e) Basal PTEN levels in IBCs vs non-IBCs in VH125.C57BL/6-H2g7 and VH125.NOD mice. Results are representative of at least three independent experiments with at least three mice per group. Results from the other two experiments demonstrated a greater than 70% decrease in calcium flux in IBCs from all VH125.C57BL/6-H2g7 mice assayed compared 6-Acetamidohexanoic acid with IBCs in VH125.NOD mice. In the experiments not shown, the gMFI of pSyk in IBCs from VH125.C57BL/6-H2g7 mice was on average (mean) 145 and 128 times less than that for insulin? cells (test. Data in (c) and (e) are mean SEM Open in a separate window Fig. 5 IBCs in VH125.NOD mice accumulate in the 6-Acetamidohexanoic acid pLNs and pancreas, express activation markers and produce autoantibodies. (a) Representative cytograms of Insulinlo, Insulinhi and Insulin+ B cells in the spleen, pLNs and pancreas from unenriched tissue samples. Staining from VH281.NOD mice is shown as a negative control for insulin binding. (b) IBCs as a percentage of B220+ B cells are compared for each organ in the two strains. IBCs accumulate in the pLNs and pancreas of VH125.NOD (NOD) but not VH125.C57BL/6-H2g7 (B6) mice. (c, d) Relative CD86 and CD69 geometric MFI (gMFI) of IBCs compared with non-IBCs in each organ is compared for the two strains. Insulinhi B cells in VH125.NOD mice upregulate both activation markers. (e) Representative gating strategy for IBC plasmablasts (PBs) in the pLNs of both strains. (f) The percentage of IBC PBs is increased in the pLNs of VH125.NOD compared with VH125.C57BL/6-H2g7 mice. The percentage of IBC PBs was determined by multiplying the frequency of B220lo IBC+ cells in the lymphocyte gate in the pLNs by the percentage that were also CD138+. (g) The absolute number of IBC PBs is also increased in the pLNs of VH125.NOD compared with VH125.C57BL/6-H2g7 mice..