Figure S1.

Enhancement of proliferation and activation of CD4⁺ T cells by LN-TRCs regardless of CD25 expression. (A) Naive CD4⁺ T cells (2 × 10⁵) were cultured for 72 h with or without TRCs (ratio of T cells to TRCs is 10:1, 5:1, and 2.5:1) and treated with (black bars) or without (white bars) anti-CD3 and CD28 antibodies (3 and 1 μg/ml) to stimulate T cells with irradiated splenocytes as APCs (2 × 10⁵). Cell viability was assessed by flow cytometry with viability dye staining. **, P < 0.01; ***, P < 0.001 compared with naive CD4⁺ T cells in the absence of stimulation or antibody stimulation. Error bars indicate the mean ± SEM (n = 4 per each group). (B) Comparison of the proliferation of CFSE-labeled naive CD4⁺ T cells (2 × 10⁵) cultured for 72 h with or without TRCs (2 × 10⁴) in the presence or absence of anti-CD3 and CD28 antibodies (3 and 1 μg/ml) with irradiated APCs (2 × 10⁵). The proliferation of CFSE-labeled naive CD4⁺ T cells was analyzed by flow cytometry (1 × 10⁵). (C) The production of cytokines in supernatants naive CD4⁺ T cells (2 × 10⁵) cultured for 72 h with (black bars) or without (white bars) TRCs (2 × 10⁴) in the presence of anti-CD3 and CD28 antibodies (3 and 1 μg/ml) with irradiated APCs (2 × 10⁵) was quantified in the culture supernatants by ELISA. *, P < 0.05; **, P < 0.01 compared with naive CD4⁺ T cells in the absence of stimulation or antibody-stimulated CD4⁺ T cells. Error bars indicate the mean ± SEM (n = 4 per each group). Data are representative of three independent experiments.