Fig. 4.

Impaired APC function and attenuated activation of CD11c⁺ DCs in TRIF^−/−NOD mice. (A) Percentage of CD80-expressing CD11c⁺ DCs. Immune cells from spleen, PLN, MLN and PP of 2-month-old female WT NOD and TRIF^−/−NOD mice were stained with fluorochrome-conjugated anti-CD11c, anti-CD80, anti-CD11b, anti-B220 and anti-TCRβ antibodies. Representative FACS plots are shown on the left and the summary of CD80-expressing CD11c⁺ DCs is shown on the right. Data are shown as mean±SEM. The experiment was repeated more than three times. (B–D): Splenocytes from 2-month-old female WT NOD and TRIF^−/−NOD mice were stimulated with PMA and ionomycin in the presence of Golgi plug followed by ICC staining. (B) IL-6; (C) TNF-α and (D) TGF-β. Data are shown as mean±SEM and pooled from two independent experiments. (E) Proliferation of BDC2.5 CD4⁺ T cells. Purified NOD BDC2.5 CD4⁺ T cells were cultured with irradiated T cell-depleted splenocytes from 2-month-old female WT NOD and TRIF^−/−NOD mice in the presence of mimotope peptide. Proliferation was assessed using ³H-thymidine incorporation. n=3–5/mice/group/experiments from more than three experiments. (F) Proliferation of BDC2.5 CD4⁺ T cells. Purified NOD BDC2.5 CD4⁺ T cells were cultured with irradiated splenic CD11c⁺ DCs from 2-month-old female WT NOD and TRIF^−/−NOD mice in the presence of mimotope peptide. Data were pooled from two independent experiments. (G) Diabetes incidence following adoptive transfer. Purified splenic T cells (7×10⁶ cells/mouse) from diabetic WT NOD mice were i.v. injected into Rag−/−NOD or NOD.scid mice with T cell-depleted splenic APCs (7×10⁶ cells/mouse) from either female WT NOD or TRIF^−/−NOD mice. Data were pooled from two independent experiments. Log-rank test for survival was used for analysis of diabetes incidence. Two-way ANOVA was used for the comparison in (E) and (F). Two-tailed Student’s t test was used for statistical analysis in (B–D).