Fig. 2.

Nlrp12-mediated protection against uveitis occurs independent of an inherent T cellular mechanism. The T cell infiltrate in uveitic eyes of WT and Nlrp12^−/− mice was evaluated by flow cytometry 21 day post-immunization (A). CD4⁺ cells were identified from gated live, singles that were CD45⁺ and identified as CD4⁺ CD8⁻. Data are of pooled eyes (5 mice/group) and representative of 3 independent experiments. B, C Rag1^−/− mice injected with CD4⁺ T cells purified from naïve, WT or Nlrp12^−/− donors. The reconstituted Rag1^−/− mice were then immunized for EAU and evaluated for uveitis. Splenocytes of CD4⁺ T cell-reconstituted Rag1^−/− recipients harvested 28 day post-immunization were analyzed by flow cytometry for proportions of indicated T cell subsets (DN, double negative) (B). Clinical uveitis 28 day post-immunization was assessed by fundoscopy. Representative fundus photographs (C, left) and clinical uveitis scores are shown (C, right). D Autologous criss-cross cultures were performed to evaluate Nlrp12 function within APCs in induction of Th1/Th17 immunity. IRBP-reactive CD4⁺ T cells (purified from immunized donors) were cultured with naïve, WT or Nlrp12^−/− APCs, and stimulated with IRBP_1–20 peptide. As a negative control, APCs were stimulated with IRBP_1–20 peptide in the absence of CD4⁺ T cells. Cytokine production was measured 18 h later by ELISA. Data are combined from 3 experiments and are mean ± SEM; *p < 0.05. E Th-effector responses were evaluated in IRBP_1–20 peptide-stimulated splenocyte cultures from WT vs. Nlrp12^−/− mice at 21 d post immunization. Representative dot plot (left) and summary statistics (right, of 3 independent experiments, each with n = 5 mice pooled/group) showing frequencies of IL-17A and IFNγ-producing cells of live CD4⁺ T cells. F Splenocytes of immunized WT vs. Nlrp12^−/− mice were stimulated with IRBP_1–20 peptide and cytokine levels in culture supernatants were measured 18 h later by ELISA. Data are mean ± SEM combined from 3 experiments; in each experiment splenocytes from immunized mice were pooled (5 mice/group) and cytokine measured in triplicate wells