Fig. 3.

Iron overload is detrimental to naive CD4 T cells in response to TCR stimulation. Sorted naive CD4 T cells from the spleens of WT and KO mice were labeled with CellTrace™ Violet (CTV) and then activated with anti-CD3 and anti-CD28 antibodies for 3 d as described in Materials and Methods. (A) Graphs show levels of heme (Left) and HO-1 (Right) in activated CD4 T cells (n = 4 to 5). (B and C) The graphs show the expression of the indicated parameters (n = 3 to 5). (D) The representative histograms and the summary graph show the percentages of Annexin V⁺ in activated CD4 T cells from WT and KO mice (n = 4). (E) Cells were stained with a lipid-peroxidation reagent. The graph shows the ratio of fluorescence intensity of FITC over PE. (F) The representative histograms and the summary graph show cell proliferation (n = 5). (G) Cells were stimulated with PMA and ionomycin for 4 h and then analyzed for intracellular IL-2 expression. Representative dot plots show the percentage of IL-2⁺ CD4 T cells (n = 3). The summary graph shows the amount of IL-2 secreted into the media at indicated time points as measured by ELISA (n = 9). (H) Enriched naive CD4 T cells from OT-II and OT-II KO mice were stimulated with dendritic cells in the presence of 100 mg/mL of Ova protein for 3 d. Cells were also activated with anti-CD3 and anti-CD28 antibodies. The representative histograms depict cell proliferation (n = 4). (I) Cells in (H) were restimulated with PMA and ionomycin for 4 h and then assessed for the expression of IL-2 cytokine. Representative dot plots and the summary graph show the percentages of IL-2⁺ OT-II CD4 T cells (n = 3). (J) The graph shows CD25 expression in activated CD4 T cells (n = 4). (K) Activated CD4 cells were subjected to intracellular staining of pSTAT5 and pS6. The data are representative of three independent experiments. Error bars represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.