Figure 2.

Cytokine production by wild-type vs. CDK2-deficient CD4+ T cells. CD8-depleted spleen and lymph node cells from wild-type (dark gray) or CDK2-deficient (light gray) mice were stimulated with soluble anti-CD3 plus either anti-CD28 or CTLA-4Ig, and supernatants were tested for IL-2 (A) and IFNγ (B) by ELISA. Wild-type cells were stimulated with anti-CD3 in the presence of three CDK2-selective inhibitory drugs (roscovitine, NU6140 & alsterpaullone) or the CDK1-selective drug indolylmethylene-2-indolinone (I2I), and IL-2 secretion was measured by ELISA (C). Purified wild-type CD4+ T cells were transduced with retroviruses encoding either a control shRNA that targets human cyclin D3, or shRNA targeting two distinct regions of the CDK2 transcript, and CDK2 protein levels were measured by immunoblotting (D, top left panel), and IL-2 and IFNγ in the supernatants were quantified by ELISA (D, lower panels). Wild-type or CDK2-deficient cells were stimulated as in A and B, and IL-2 (E) and IFNγ (G) in primary supernatants were measured by ELISA. Cultures were rested, CD4+ T cells were purified and restimulated on plate-bound anti-CD3 and anti-CD28, and IL-2 (F) and IFNγ (H) in secondary supernatants were measured ELISA. Error bars in A–F depict the standard error of the mean. The data depicted are representative of 3–4 independent experiments.