PKCθ^−/− CD4⁺ T cells are more potent IFN-γ producers in an autoimmune disease mouse model.

<p>A) Experimental autoimmune encephalomyelitis (EAE) disease progression in wild-type and PKCθ^−/− mice. The graph summarizes data from three independent experiments with total n>20 for each genotype. B) and C) Infiltrating mononuclear cells (MNC) from brain and spinal cord (SC) and lymph node (LN) cells were isolated at onset of the disease (day 11 after EAE-inducing immunisation). The cells were activated with phorbol 12,13-dibutyrate (PDBu)/ionomycin for 4 h and stained intracellulary for cytokine (IFN-γ, IL-17A) and T-bet expression. B) Representative flow cytometric plots of brain-infiltrating CD4⁺ cells. C) Quantification of cytokine-producing CD4⁺ T cell. Graphs depict results from two independent experiments with n = 3 per genotype for brain samples and from one experiment with n = 3 per genotype for SC and LNs. D) Cells from the lymph nodes and spleens were isolated at onset of the disease (day 11 after EAE-inducing immunisation). Cells were cultured <i>in vitro</i> in the presence of MOG_35–55 peptide for 3 days, re-stimulated with PDBu/ionomycin and analyzed by intracellular flow cytometric staining for IFN-γ, IL-17A and T-bet expression. Data from one experiment with n = 12 per genotype are shown. All graphs present mean values with error bars indicating +/− SEM. Statistical significance was assessed by a two-way repeated measures ANOVA (disease progression), a two-way ANOVA with Bonferroni post hoc test (cytokines and T-bet expression in C) or by a two tailed unpaired Student's t-test (gene expression in D).