Fig. 5.

CD140a is highly expressed in Tlr9^−/− islets. (a, b) CD140a expression on islet beta cells from Tlr9^+/+ NOD and Tlr9^−/− NOD mice. Pancreatic islets were isolated from ~4-week-old female Tlr9^+/+ NOD and Tlr9^−/−NOD mice and beta cells were harvested after treatment with Cell Dissociation Solution (Sigma). The cells were stained with monoclonal antibodies to CD45, CD140a and FluoZin-AM and analysed by flow cytometry. CD45⁻ cells (non-haematopoietic cells) were gated and the expression of CD140a on FluoZin⁺ beta cells is shown (a). Quantification of CD140a⁺ beta cells in Tlr9^+/+ NOD and TLR9^−/− NOD mice (b) (p = 0.0002; n = 4 mice [pooled into two samples] per group per experiment), with two experiments being performed). (c, d) CD140a expression on islet beta cells from Tlr9^+/+ NOD mice treated with control (Ctrl) ODN or antagonist ODN. Isolated islets from Tlr9^+/+ NOD mice were cultured with TLR9 antagonist ODN or control ODN (both at 10 μg/ml) overnight. Islet beta cells were harvested after dissociation and stained with monoclonal antibodies to CD45, CD140a and FluoZin-AM. CD140a expression is shown on FluoZin⁺ beta cells after gating on CD45⁻ cells (c). Quantification of CD140a⁺ beta cells after treatment with TLR9 antagonist ODN or control ODN (d) (p < 0.001; n = 7 mice per group per experiment; data are from two experiments). (e) Insulin release assay of islets treated with control ODN (white circles; n = 7 mice) or antagonist ODN (black circles; n = 7 mice) (p < 0.001). Two-tailed unpaired Student’s t test was used for (a–d) and two-way ANOVA was used for (e). Data in (b) and (d) are expressed as the actual numbers, with the mean indicated as the line in the figures. Data in (e) are expressed as means (SD). ***p < 0.001