Supplementary Figure 2: Characterization of T cells from Taz^f/fLck-Cre mice.

(a) Flow cytometric analysis (top panel) and quantification (lower panel) of thymocytes from 8-week-old Taz^f/f mice or Taz^f/f Lck-Cre mice, stained with anti-CD4 and anti-CD8 antibodies. (b-d) Flow cytometric analysis (b) and quantification of B220⁺ B cells, CD3⁺CD4⁺ or CD3⁺CD8⁺ T cell (c), the naïve T cell (CD62L^highCD44^low) and the effector T cell (CD62L^lowCD44^high) (d) populations in the spleen or lymph node of Taz^f/f mice and Taz^f/f Lck-Cre mice, with the indicated antibodies. (e-f) Flow cytometry analysis of the activation markers CD69, CD25, CD62L, and CD44 on cell surfaces (e) and enzyme-linked immunosorbent assay of IL-2 or IFNγ production (f) in culture medium of CD4⁺ T cells isolated from 8-week-old Taz^f/f mice or Taz^f/f Lck-Cre mice, stimulated with anti-CD3 or anti-CD3 plus anti-CD28 antibodies for 24 hours. Data were assessed with Student’s t-test and are represented as mean ± s.d. ns, not significant. (g) CFSE proliferation assay of CD4⁺ T cells isolated from 8-week-old Taz^f/f mice or Taz^f/f Lck-Cre mice, stimulated with anti-CD3 plus anti-CD28 antibodies for 48 hours. (h) CFSE proliferation assay of Th17 or Treg cells differentiated from Taz^f/f mice or Taz^f/f Lck-Cre naïve T cells, stimulated with anti-CD3 plus anti-CD28 antibodies for indicated time. (i and j) Quantification of intracellular or ELISA assay of IL-17F expression (i) or Real-time quantitative PCR analysis of the expression levels of indicated genes in CD4⁺ naïve T cells from Taz^f/f mice or Taz^f/f Lck-Cre mice differentiated under T_H17. (k and l) H&E (k) and anti-CD3 (l) staining for lymphocyte infiltration in the spinal cord of Taz^f/f mice and Taz^f/f Lck-Cre mice subjected to EAE by MOG immunization. Scale bars, 100μM. (m and n) Proliferation rate (m) and IL-2 production (n) of responder CD4+ naïve T cells cocultured with CD4⁺CD25⁺ T_reg cells from Taz^f/f mice or Taz^f/f Lck-Cre mice and wild-type irradiated spleen cells and stimulated with anti-CD3 antibodies. The data are representative of three independent experiments with similar results. The data represent the mean ± s.d. (n=3). ns, not significant (P > 0.05); *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control, Student’s t-test.