Figure 7. Calcium-mediated shaping of the CD4 T_N-cell pTreg-cell differentiation potential in vivo.

Purified CD4 T cells from CD45.1/.2⁺ C57BL/6 Foxp3-GFP OT-II mice were cultured in IL-7 (10 ng/ml) with or without TG (4 nM). After 5 days live CD4 T_N (CD44^lo CD25^lo CD8β^- CD11b^- CD11c^- NK1.1^- TCRγδ^- Foxp3-GFP^-) cells were flow-cytometry sorted and injected intravenously (0.5−1 × 10⁶ cells) into sex-matched CD45.1⁺ C57BL/6 Foxp3-GFP recipient mice fed with Ovalbumin (OVA; 1.5% w/v) in the drinking water. One week after transfer, peripheral and mesenteric LNs (pLNs and mLNs, respectively), Peyer’s Patches (PPs) and spleen were recovered separately and donor-derived CD4 T cells were analyzed. (A) Diagram illustrating the experimental model. (B) Absolute numbers of donor-derived OT-II CD4 T (CD45.1⁺ CD45.2⁺ CD4⁺ TCRβ⁺ Vβ5⁺) cells recovered from pLNs, mLNs, PPs and spleen of recipient mice are shown as means ± s.e.m. for three independent experiments with two or three mice per group. (C) Representative Foxp3/Vβ5 contour-plots and proportions of Foxp3-GFP⁺ cells for gated donor-derived OT-II CD4 T (CD45.1⁺ CD45.2⁺ CD4⁺ TCRβ⁺ Vβ5⁺) cells recovered from mLNs are shown. (D–E) Percentages (D) and absolute numbers (E) of Foxp3-GFP⁺ among donor-derived OT-II CD4 T (CD45.1⁺ CD45.2⁺ CD4⁺ TCRβ⁺ Vβ5⁺) cells recovered from pLNs, mLNs, PPs and spleen of recipient mice are shown as means ± s.e.m. for three independent experiments with two or three mice per group. (B, D, E) Significance of differences were assessed using a two-tailed unpaired Student’s t-test. Values of p<0.05 were considered as statistically significant (*p<0.05; ns, not significant). figure supplement legends.

Figure 7—figure supplement 1. OT-II CD4 T_N cells are Ly-6C⁺and can be Ca²⁺-converted and tested for their ability to convert into pTreg cells in vivo.

(A) Ly-6C fluorescence histograms of gated CD4 T_N (CD4⁺ CD8α^- TCRβ⁺ CD44^lo CD25^lo Foxp3-GFP^-) cells recovered from LNs of C57BL/6 Foxp3-GFP and C57BL/6 Foxp3-GFP OT-II mice are shown. (B, C) Purified CD4 T cells from CD45.1/.2⁺ C57BL/6 Foxp3-GFP OT-II mice were cultured in IL-7 (10 ng/ml) with or without TG (4 nM). After 5 days, live CD4 T_N (CD44^lo CD25^lo CD8β^- CD11b^- CD11c^- NK1.1^- TCRγδ^- Foxp3-GFP^-) cells were flow-cytometry sorted, analyzed for their cell surface expression of Ly-6C (B) and injected intravenously (0.5−1 × 10⁶ cells) into sex-matched CD45.2⁺ C57BL/6 Foxp3-GFP recipient mice fed with Ovalbumin (+OVA; 1.5% w/v) or not (-OVA) in the drinking water (C). One week after transfer, mesenteric LNs (mLNs) were recovered and donor-derived CD4 T cells were analyzed. (C) Gating strategy and representative CD44/Foxp3 dot-plots for gated donor-derived OT-II CD4 T (CD45.1⁺ CD45.2⁺ CD4⁺ TCRβ⁺ Vβ5⁺) cells recovered from mLNs are shown. Percentages of Foxp3-GFP⁺ cells are indicated.

Figure 7—figure supplement 2. Calcium-mediated shaping of the CD4 T_N-cell phenotype increases the pTreg-cell differentiation potential of OT-II CD4 T_N cells in vivo.

Purified CD4 T cells from CD45. 2⁺ and CD45.1/.2⁺ C57BL/6 Foxp3-GFP OT-II mice were cultured in IL-7 (10 ng/ml) without or with TG (4 nM), respectively. After 5 days live CD4 T_N (CD44^lo CD25^lo CD8β^- CD11b^- CD11c^- NK1.1^- TCRγδ^- Foxp3-GFP^-) cells were flow-cytometry sorted, mixed at a 1:1 ratio and injected intravenously (0.5−1 × 10⁶ cells) into sex-matched CD45.1⁺ C57BL/6 Foxp3-GFP-recipient mice gavaged with Ovalbumin (OVA; 50 mg) 4 and 24 hr later. 10 days after transfer, peripheral and mesenteric LNs (pLNs and mLNs, respectively), Peyer’s Patches (PPs) and spleen were recovered separately and donor-derived CD4 T cells were analyzed. (A) Diagram illustrating the experimental model and Ly-6C surface expression of transferred cells. (B) Representative CD45.1/Foxp3 dot-plots for gated donor-derived OT-II CD4 T (CD45.2⁺ CD4⁺ TCRβ⁺) cells recovered from Spleen, pLNs, mLNs and PP of recipient mice are shown. Absolute numbers of donor-derived Foxp3-GFP⁺ OT-II CD4 T cells recovered from pLNs, mLNs, PPs and spleen of recipient mice are shown as means ± s.e.m. for a representative experiment with four mice per group. (C) Total (pool of Spleen, pLNs, mLNs and PPs cells) absolute numbers of Foxp3-GFP⁺ OT-II CD4 T cells recovered from recipient mice are shown as means ± s.e.m. (D) Percentages of CD45.1⁺ cells among donor-derived Foxp3-GFP⁺ OT-II CD4 T (CD45.2⁺ CD4⁺ TCRβ⁺) cells recovered from pLNs, mLNs, PPs and spleen of recipient mice are shown as means ± s.e.m. (B, C) Significance of differences were assessed using a two-tailed paired Student’s t-test. Values of p<0.05 were considered as statistically significant (**p<0.01; ***p<0.001; ns, not significant).