FIGURE 4.

Antigen-specific cytokine production, myelin-specific gene expression, and T cell receptor repertoire unaltered in Aire-deficient HLA-DR tg mice. Mice immunized for EAE were euthanized during disease remission (day 34 – 36), splenocytes of Aire^−/− and Aire^+/+ HLA-DR2b tg (A) or HLA-DR4 tg mice (B) were harvested and recalled with peptides MOG35–55 (HLA-DR2b tg) or MOG97–108 (HLA-DR4 tg) and tested by cytokine ELISPOT assay to quantify IFN-γ, IL-17 and GM-CSF producing cells as described in Materials and Methods. Shown is mean ± SEM of the number of cytokine forming cells. Data are the representative of 3–4 independent experiments (n= 3 – 4 mice/group; * indicates significant difference between Aire^−/− group and Aire^+/+ group). C) naïve Aire^+/+ and Aire^−/− HLA-DR2b or (D) naïve Aire^+/+ and Aire^−/− HLA-DR4 mice were euthanized, their thymus was harvested, and RNA was extracted and used for performing qPCR against myelin-specific genes (MOG, PLP, MBP) as described in Materials and Methods. Data are representative for two independent experiments (n= 3 – 4 mice/groups), (mean ± SEM of mRNA expression). (E–H) Shown is the flow cytometry analysis of TCR Vβ and Vα distribution of splenic CD4⁺ (E, F) and CD8⁺ T cells (G, H) of Aire^−/− vs Aire^+/+ HLA-DR2b tg mice. (I–L) Flow cytometry analysis of TCR Vβ and Vα distribution of splenic CD4⁺ (I, J) and CD8⁺ T cells (K, L) of Aire^−/− vs Aire^+/+ HLA-DR4 tg mice. Flow cytometry analysis was performed using panels of mAbs for TCR families as described in Materials and Methods. Shown are pooled data of two independent experiments (n = 2 – 4 mice/group, Mean ± SEM).