Figure 2: Modest effects of Tiam1 genetic deficiency on IL-17A expression and EAE.

(a) Naive CD4⁺ T cells were isolated from Tiam1 deficient mice (Tiam1−/−) or control (Tiam1+/+) littermates and were stimulated in vitro under Th17 cell polarizing conditions and cytokine profile was analyzed by intracellular cytokine staining. (b) Flow cytometry cytokine expression of Tiam1−/− and control Tiam1+/+ CD4⁺ T cells differentiated under Th1, Th2 and iTregs. (c) Analysis of cytokine expression by Taqman PCR in Tiam1−/− Th17 cells 4 days after cell differentiation. (d) Tiam1 genetic deficiency ameliorates clinical EAE clinical score. EAE was induced in Tiam1−/− and control Tiam1+/+ littermates with MOG_35–55/CFA and mice were monitored for clinical EAE symptoms for up to 25 days post-immunization (DPI). Mean maximal EAE score (±s.e.m.) of Tiam1−/− was compared with that of control Tiam1+/+ mice by two-tailed Mann–Whitney U test. Statistical differences (*P<0.05) between the two groups on day 13–16 are shown (n=12–15 mice per group). (e) ELISPOT data of the frequency of IL-17A and IFNγ-producing cells in MOG_35–55-activated spleen cells of immunized Tiam1−/− and Tiam1+/+ mice on day 8–10 after immunization (n=3–5 mice per group). (f) Cytokine expression of MOG_35–55-activated spleen cells analyzed by Luminex assay. Results are representative of three independent experiments with three mice per group. (g) On day 10 post-immunization, splenocytes of Tiam1−/− and Tiam1+/+ mice were harvested, CD4⁺ T cells and CD11c⁺ dendritic cells were criss-crossed (DC:CD4 1:4 ratio) and tested for MOG_35–55-specific proliferation. Data are shown as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001 by Student t-test. NS, non-significant. (h) Mean EAE scores (±s.e.m.) after adoptive transfer of MOG_35–55-restimulated WT or Tiam1−/− CD4⁺ T cells. Tiam1−/− and Tiam1+/+ mice were immunized, LN cells were isolated and re-activated with MOG_35–55 peptide (20 μg) in the presence of WT DCs and IL-23 for 2 days, and 0.75 million CD4⁺ T cells were injected into Rag1 deficient mice. The mean maximal score of recipients of Tiam1−/− compared with control Tiam1+/+ cells was compared by two-tailed Mann–Whitney U test. Statistical differences (*P<0.05) between the two groups on day 11–15 are shown (n=5 mice per group, representative of two independent experiments of total 10 mice per group).