Fig. 4

Early activation but not survival of PKCθ-E2^mut CD4⁺ T cells is impaired. a Viability of CD4⁺ T cells after overnight culture (total spleen/LN cells), either non-stimulated or stimulated with anti-CD3 and anti-CD28 antibodies, was determined by Annexin V/7AAD staining and flow cytometry. b CD25 and CD69 expression by CD4⁺ T cells of the three different genotypes was analyzed by flow cytometry upon overnight stimulation with anti-CD3 and anti-CD28 antibodies (total spleen/LN cells, gated on CD4⁺). The results are depicted as mean fluorescence intensity, MFI, relative to wt. c Interleukin-2 expression of CD4⁺ T cells of wt, PKCθ-deficient (ko) and PKCθ-E2^mut mice was analyzed by intracellular staining and flow cytometry (IC FACS, see Additional file 4: Figure S4 whole LN/spleen stimulated with anti-CD3/CD28 for 7 h, in the presence of GolgiPlug). IL-2 expression of MACS-sorted CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 antibodies for the indicated time was analyzed by RT-PCR on mRNA level (normalized with gapdh and depicted relative to wt) and on protein level by luminex analysis of the supernatant. Each symbol represents values obtained on an individual mouse (n ≥ 6), the solid line shows the mean value. Statistical significance was determined by one-way Anova and Bonferroni’s multiple comparison test