A, Defectiveproliferation of Erk3^−/− T lymphocytes after anti-CD3 stimulation. Splenocytes from Erk3^+/+or Erk3^−/− hematopoietic chimeras were labeled with CFSE and stimulated with different doses of anti-CD3 Ab for 72 h. CFSE profiles gated on CD4⁺ or CD8⁺ T cells lacking or not ERK3 are shown for the different anti-CD3 Ab concentrations. One representative experiment is shown. B, Quantification of T cell proliferation. T cell proliferation, measured as in A, was quantified by determining the percentage of cells that have divided (one division and more; CFSE^lo). Each dot represents the results from one mouse. Unpaired Student’s t test (two-sided) was used to determine statistical significance. * p<0.05. C, Addition of anti-CD28 Abs does not rescue the proliferation of ERK3-deficient CD4⁺ T cells. Splenocytes were stimulated with a sub-optimal dose of anti-CD3 Ab (0.3 µg/ml) in the presence (bottom) or absence (top) of soluble anti-CD28 Ab (5 µg/ml). CFSE profiles gated on CD4⁺ T cells lacking or not ERK3 are shown. D, Reduced production of IL-2 and IFN-γ by ERK3-deficient T cells after anti-CD3 stimulation. After 72 h of anti-CD3 stimulation, activated T cells were stimulated with PMA and ionomycin for 4 h. Brefeldin A was added for the last 2 h of culture. IL-2 and IFN-γ production was detected using intracellular cytokine staining. CFSE/IL-2 and CFSE/IFN-γ profiles gated on CD4⁺ or CD8⁺ T lymphocytes deficient or not for ERK3 are shown for the different anti-CD3 Ab concentrations. Numbers in parenthesis represent the % of proliferating and cytokine producing cells. The results in this figure are representative of at least three independent experiments with mice from independent hematopoietic chimeras

https://doi.org/10.1371/journal.pone.0086681.g005