Figure 5

Attenuation of TCR-mediated responses and mTOR signaling transduction in GNMT-deficient CD4⁺ T cells. (A) Attenuation of TCR-mediated cell proliferation in Gnmt^−/− CD4⁺ cells. The purified CD4⁺ T cells isolated from Gnmt^−/− mice and wild-type mice were stimulated with anti-CD3/CD28 (1 µg/mL). For proliferation, the (³H)thymidine was pulsed at 24 h and 48 h after stimulation, and the incorporated radioactivity was measured after 12 h as described in Materials and Methods. Data are representative of two independent experiments, presented as the mean ± SD. (B) TCR-induced IL-2 cytokine production was not affected by GNMT deficiency. The purified CD4⁺ T cells were stimulated with anti-CD3/CD28 (1 µg/mL) for 48 h. The supernatants were harvested and measured for IL-2 concentrations by ELISA. Data are representative of two independent experiments, presented as the mean ± SD. (C,D) GNMT deficiency did not affect TCR-induced IL-2 and IL-2Rα (CD25) mRNA expression. The purified CD4⁺ T cells were stimulated with anti-CD3/CD28 (1 µg/mL) for 24 h and the expression of IL-2 and CD25 were analyzed by q-PCR. Data are representative of two independent experiments, presented as the mean ± SD. (E and F) Attenuation of TCR-induced IFN-γ and IL-17A production in Gnmt^−/− CD4⁺ T cells. The purified CD4⁺ T cells were stimulated with anti-CD3/CD28 (1 µg/mL) for 48 h. The supernatants were collected and measured for IFN-γ and IL-17A concentrations by ELISA. Data are representative of two independent experiments, presented as the mean ± SD. (G) The purified CD4⁺ T cells from Gnmt^−/− mice or wild-type mice were stimulated with anti-CD3 and anti-CD28 (1 µg/mL) for 48 h. The cell extracts were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (H) Average densitometric quantification of immunoblots of GRAIL. Data are representative of at least three experiments. Statistics were calculated by the Student t test. *p < 0.05; **p < 0.01. N.D., nondetermined.