Figure 5. The calcium-calcineurin pathway shapes the phenotype of the CD4 T_N-cell compartment in vivo.

(A) Flow-cytometry sorted Ly-6C⁺ CD4 T_N cells from C57BL/6 Foxp3-GFP mice were cultured in IL-7 (10 ng/mL) alone or in the presence of either TG (4 nM), TG and Cyclosporin A (CsA; 50 nM) or TG and FK506 (FK; 200 nM). Flow-cytometry sorted Ly-6C^- CD4 T_N cells rested in IL-7 were used as control. After 5 days, cells were analyzed for their expression of Ly-6C, CD5, CD73, CD122, CD200 and Izumo1r. Representative contour-plots of cell surface markers are shown for gated CD4 T_N cells (CD4⁺ TCRβ⁺ CD44^lo CD25^lo Foxp3-GFP^-) as a function of culture condition. (B–F) C57BL/6 Foxp3-GFP mice were daily injected intraperitoneally with Prograf (FK506; 2.5 mg/kg) or diluent (PBS). Two weeks after treatment LNs (pooled pLNs and mLNs) and spleen were recovered and CD4 T cells were analyzed. (B) Diagram illustrating the experimental procedure. (C) Ly-6C and Izumo1r fluorescence histograms for gated CD4 T_N cells (CD4⁺ TCRβ⁺ CD44^lo CD25^lo Foxp3-GFP^-) recovered from LNs of PBS (white) and FK506 (grey) treated mice. (D) Percentage of Ly-6C⁺ cells among CD4 T_N (CD4⁺ TCRβ⁺ CD44^lo CD25^lo Foxp3-GFP^-) cells are shown for LNs and spleens of PBS (white) and FK506 (grey) treated mice. (E) Ly-6C Mean fluorescence intensities (MFIs), for gated Ly-6C⁺ CD4 T_N (Ly-6C⁺ CD4⁺ TCRβ⁺ CD44^lo CD25^lo Foxp3-GFP^-) cells recovered from LNs of PBS (white) and FK506 (grey) treated mice, are shown as means ± s.e.m. for two independent experiments with three mice per group. (F) Izumo1r and CD200 mean fluorescence intensities (MFIs), for gated Ly-6C⁺ CD4 T_N (Ly-6C⁺ CD4⁺ TCRβ⁺ CD44^lo CD25^lo Foxp3-GFP^-) cells recovered from LNs of PBS (white) and FK506 (grey) treated mice, are shown as means ± s.e.m. for a representative experiment with three mice per group. (G–I) 1 × 10⁶ flow-cytometry sorted Ly-6C^- CD4 T_N cells from CD45.1⁺ C57BL/6 Foxp3-GFP mice were adoptively transferred into sex-matched CD45.2⁺ C57BL/6 Foxp3-GFP recipient mice daily injected intraperitoneally with Prograf (FK506; 2.5 mg/kg) or diluent (PBS). Two weeks after transfer and treatment, LNs (pooled pLNs and mLNs) and spleen were recovered and donor-derived CD45.1⁺ CD4 T cells were analyzed. (G) Diagram illustrating the experimental model. (H) Absolute numbers of donor-derived CD4 T_N (CD45.1⁺ CD45.2^- CD4⁺ TCRβ⁺ CD44^lo CD25^lo Foxp3-GFP^-) cells recovered from LNs and spleen of recipient mice are shown as means ± s.e.m. for two independent experiments with three mice per group. (I) Percentage of Ly-6C⁺ among donor-derived CD4 T_N (CD45.1⁺ CD45.2^- CD4⁺ TCRβ⁺ CD44^lo CD25^lo Foxp3-GFP^-) cells recovered from LNs and spleen of recipient mice are shown as means ± s.e.m. for two independent experiments with three mice per group. (D, H, I) Each dot represents an individual mouse. (D-F; H, I) 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<0.01; ***p<0.001; ns, not significant).

Figure 5—figure supplement 1. The calcium-calcineurin cascade drives NFAT nuclear translocation in CD4 T_N cells in vivo.

(A) C57BL/6 Foxp3-GFP mice were injected intraperitoneally with Prograf (FK506; 2.5 mg/kg; two times) or diluent (PBS). 18 hr later LN cells were isolated from C57BL/6 mice and fixed in 4% paraformaldehyde immediately. CD4 T_N cells (CD4⁺ CD44^lo CD25^lo Foxp3^-) were sorted by flow cytometry and stained for Ly-6C, NFAT1, and DNA (DRAQ5). Cells were then analyzed by imaging flow cytometry. (A) Scheme depicting the experimental procedure. (B) NFAT1 nuclear localization was calculated as similarity score between NFAT1 and DRAQ5 intensities. Data are representative of one of two independent experiments.