Cutting Edge: Activation-Induced Iron Flux Controls CD4 T Cell Proliferation by Promoting Proper IL-2R Signaling and Mitochondrial Function

Abstract

Iron has long been established as a critical mediator of T cell development and proliferation. However, the mechanisms by which iron controls CD4 T cell activation and expansion remain poorly understood. In this study, we show that stimulation of CD4 T cells from C57BL/6 mice not only decreases total and labile iron levels but also leads to changes in the expression of iron homeostatic machinery. Additionally, restraining iron availability in vitro severely inhibited CD4 T cell proliferation and cell cycle progression. Although modulating cellular iron levels increased IL-2 production by activated T lymphocytes, CD25 expression and pSTAT5 levels were decreased, indicating that iron is necessary for IL-2R-mediated signaling. We also found that iron deprivation during T cell stimulation negatively impacts mitochondrial function, which can be reversed by iron supplementation. In all, we show that iron contributes to activation-induced T cell expansion by positively regulating IL-2R signaling and mitochondrial function.

Figure 1.

Iron homeostasis is dynamically regulated during T cell activation. (A) Both unstimulated (D0) and stimulated (D3) CD4 T cells were subjected to ICP-MS analysis. The graph shows the fold change in total iron level [reported in parts per billion (ppb) per 1 x 10⁶ cells] relative to D0. (n=6) (B) Enriched CD4 T cells were stimulated for 3 days and total iron levels were measured in both fresh media (D0) and media collected from cell cultures (D3). (n=3) (C) Splenocytes were stained with 0.02 µM Calcein-AM dye and analyzed via flow cytometry. Representative histograms show the LIP levels in CD4 and CD8 T cells as a function of mean fluorescence intensity (MFI) of Calcein. (n=3) (D) Splenocytes were stimulated with αCD3/αCD28 antibodies after B cell depletion with αCD19 magnetic beads. Representative graphs show the LIP levels in CD4 and CD8 T cells over time post-activation relative to D0. (E) Bar graph illustrates the LIP level in human CD4 T cells at day 5 (D5) relative to that at D0. (n=4) (F) Enriched CD4 T cells were stimulated for up to 3 days and stained for TfR1, ferritin, and Fpn as described in the Materials and Methods. Graphs shows the change in expression of each of the aforementioned proteins relative to D0. (n=3) (G) Bar graphs represent the relative gene expression from both unstimulated and stimulated CD4 T cells over time, which was calculated as described in the Materials and Methods. (n=3). Error bars represent Mean ± SEM. All statistics were performed by comparing each time point to D0. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

Figure 2.

T cell proliferation correlates with the downregulation of intracellular iron levels. (A) The bar graph represents total iron levels in CD4 T cells that were freshly isolated or stimulated for 3 days with either 0.25 µg/mL or 5 µg/mL αCD3. (n=3) (B) CD4 T cells were stained with CellTrace Violet (CTV) and stimulated for 3 days with the indicated concentrations of αCD3. Representative histograms show both the change in Calcein signal and cell proliferation as measured by CTV dilutions under each of the indicated stimulation conditions. (n=3) (C) CD4 T cells were stimulated for 3 days with either αCD3 only or both αCD3/αCD28. The bar graph shows relative LIP levels. (n=3) (D) CD4 T cells were stimulated for 3 days in the presence (20 µM) or absence (0 µM) of DFO. The bar graph shows the total iron levels of cells from each of the aforementioned culture conditions. (n=3) (E) CD4 T cells were stained with CTV and stimulated for 3 days in the presence of the indicated concentrations of DFO. Representative histograms show both the change in Calcein signal and cell proliferation as measured by CTV dilutions at each of the indicated DFO concentrations. (n=3) (F) Representative histograms show human CD4 T cell proliferation using CTV dilutions with (+) or without (−) 10 µM of DFO after 4 days of activation. (n=4) (G) The bar graph shows the LIP level in human CD4 T cells at day 4 post-activation with and without DFO. All graphs are cumulative of at least 3 independent experiments. Error bars represent Mean ± SEM. **p<0.005, ***p<0.0005, ****p<0.0001, ns: not significant.

Figure 3.

Iron chelation does not affect IL-2 production by activated T cells. (A-C) Enriched CD4 T cells were stimulated for 3 days in the presence (5, 10, 20 µM) or absence (0 µM) of DFO. (A) The graph represents the fold change in mean fluorescence intensity (MFI) of CD25 and CD69 at each of the indicated DFO concentrations relative to untreated cells. Statistics in (A) were performed by comparing each DFO concentration to untreated cells (0 µM). (n=3) (B) Graphs show IL-2 levels (µg/mL) in media collected from CD4 T cells as measured by ELISA. (n=3) (C) Representative dot plots show the % IL-2⁺ CD4 T cells after 3 days of stimulation followed by re-stimulation with PMA and Ionomycin for 4 h. The bar graph shows the cumulative percentages of IL-2⁺ cells from 3 independent experiments. (D) Enriched CD4 T cells were stained with CellTrace Violet (CTV) and stimulated in the absence (0 µM) or presence (5, 10, 20 µM) of DFO either with or without 50 units of recombinant IL-2. Representative histograms show cell proliferation at day 3 post-activation as measured by CTV dilutions. (n=3) E) Enriched CD4 T cells were stimulated for 48 h in the presence (10 µM, 20 µM) or absence (0 µM) of DFO and stained for pSTAT5. The pooled graph shows pSTAT5 levels at each treatment condition. Statistics in (E) were performed by comparing DFO-treated conditions to the untreated (0 µM) condition. (n=3). Error bars represent Mean ± SEM. *p<0.05, **p<0.005, ****p<0.0001.

Figure 4.

Intracellular iron stores promote T cell proliferation by controlling mitochondrial function. (A) Enriched CD4 T cells were stimulated in the presence or absence of 10 µM DFO for 48 h. Representative histograms show the percentage of cells in the G0/G1, S, and G2 phases of the cell cycle using PI. The bar graph shows the cumulative percentages of cells in each phase either with or without DFO treatment. (n=4) (B) Graphs show the pooled mean fluorescence intensities (MFI) of MitoTracker green, TMRM, or MitoSOX in CD4 T cells stimulated for 3 days in the presence or absence of 10 µM DFO. (n=3) (C-E) CD4 T cells were stimulated either with or without 10 µM DFO. After 24 h of stimulation, 10 µM FAC was added to the DFO-treated cultures. (C) Representative histograms depict cell proliferation of CellTrace Violet (CTV)-stained CD4 T cells at day 3 post-activation. (n=3) (D) The bar graph depicts the cumulative percentages of CD4 T cells in the different phases of the cell cycle after 48 h of culture. (n=4) (E) Bar graphs show the fold change in MFI of MitoTracker, TMRM, and MitoSOX in CD4 T cells after 3 days of stimulation. Data are cumulative of 3 independent experiments. Error bars represent Mean ± SEM. *p<0.05, **p<0.005, ***p<0.0005, ns: not significant.