FIG. 4.

T-cell functions in Cybr-deficient mice. (A) CD3⁺ T cells were purified from spleen, and TCR-induced calcium mobilization in response to CD3 cross-linking (at 60 s) or ionomycin (at 300 s) was measured by flow cytometry in cells that had been loaded with the calcium-sensitive dye Indo. (B) CD4⁺ T cells were analyzed for their abilities to proliferate and produce cytokines in response to stimulation through CD3 in the presence or absence of CD28 costimulation. Proliferation was measured by incorporation of [³H]thymidine. Cytokine production was measured in 24-h cell culture supernatants by cytometric bead array (BD Pharmingen). (C) Wild-type or Cybr-deficient naive CD4⁺ T cells were polarized under Th1 or Th2 conditions and assayed for the ability to secrete IFN-γ or IL-4, respectively. IFN-γ mRNA (mIFN-γ) or IL-4 mRNA (mIL-4) levels were determined by real-time PCR with or without a 4-h restimulation with plate-bound anti-CD3 (upper panels). Lower panels show intracellular cytokine staining on polarized cells after restimulation with anti-CD3. Error bars indicate standard deviations. WT, wild type; KO, knockout; unstim., unstimulated; stim.; stimulated.