Figures and data in Calcium-mediated shaping of naive CD4 T-cell phenotype and function

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7 figures, 1 table and 1 additional file

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Figure 1 with 1 supplement

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Cell-intrinsic enhanced ability of Ly-6C^- CD4 T_N cells to commit into iTreg cells.

(**A–E**) Flow-cytometry sorted Ly-6C^- and Ly-6C⁺ CD4 T_N cells from C57BL/6 Foxp3-GFP mice were stained with CTv (Ly-6C^-) or CTv and CTfr (Ly-6C⁺) and stimulated separately or together for 4 days with coated αCD3 and αCD28 Abs (4 µg/mL), in the presence of graded doses of TGFβ1. (A) Diagram illustrating the experimental protocol. (B) Representative CTv/CTfr dot-plots for gated CD4⁺ cells recovered after 4 days of culture. Ly-6C^- and Ly-6C⁺ CD4 T_N cells were either cultured separately (top left and right panels, respectively) or together (bottom panel) (C) Representative Foxp3/CD4 contour-plots and proportions of Foxp3⁺ cells for gated CD4⁺ cells are shown at a dose of 0.25 ng/mL TGFβ1. (D) Proportions of Foxp3⁺ cells among CD4⁺ cells are shown as a function of TGFβ1 concentration. Mean ± s.e.m of four independent experiments are shown. (E) Concentrations of TGFβ1 needed to obtain 50% of the maximal percentages of iTreg-cell polarization (EC50) were calculated for each CD4 T_N cell subset in separated or mixed cultures. Each dot represents an independent experiment. 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; ns, not significant).

https://doi.org/10.7554/eLife.27215.003

Figure 1—figure supplement 1

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Cytokine-independent enhanced ability of Ly-6C^- CD4 T_N cells to commit into iTreg cells.

(**A–B**) Flow-cytometry sorted Ly-6C^- and Ly-6C⁺ CD4 T_N cells from C57BL/6 Foxp3-GFP mice were stimulated separately with coated αCD3 and αCD28 Abs (4 µg/mL), in the presence of TGFβ1 (4 ng/mL) or not. 24 hr later, culture supernatants were collected and analyzed for the presence of interferon-gamma (IFN-γ) and interleukins (IL) −4, −17A/F and −10. (A) Diagram illustrating the experimental protocol. (B) IFN-γ, IL-4, IL-17A/F and IL-10 concentrations in the culture supernatants of both Ly-6C^- and Ly-6C⁺ CD4 T_N cells after 24 hr culture are shown. (**C–E**) Flow-cytometry sorted Ly-6C^- and Ly-6C⁺ CD4 T_N cells from C57BL/6 mice were stimulated separately with coated αCD3 and αCD28 Abs (4 µg/mL), in the presence of αIFN-γ and αIL-4 blocking antibodies and graded doses of TGFβ1. (C) Diagram illustrating the experimental protocol. (D) Proportions of Foxp3⁺ cells among CD4⁺ cells are shown as a function of TGFβ1 concentration. Mean ± s.e.m of two independent experiments are shown. (E) Concentrations of TGFβ1 needed to obtain 50% of the maximal percentages of iTreg-cell polarization (EC50) were calculated for each CD4 T_N-cell subset in separated or mixed cultures. Each dot represents an independent experiment. 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; ns, not significant).

https://doi.org/10.7554/eLife.27215.004

Figure 2 with 1 supplement

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Transcriptional profiling identifies a set of differentially regulated genes between Ly-6C^- and Ly-6C⁺ CD4 T_N-cell subsets.

(**A–E**) Microarray analysis was performed on Ly-6C^- and Ly-6C⁺ CD4 T_N cells sorted from LNs of C57BL/6 Foxp3-GFP mice. (A) ‘Volcano plot’ representation (Log2 (ratio) versus Log10 (t test p-value)). Genes expressed >1.3 fold higher or lower in Ly-6C^- CD4 T_N cells compared to Ly-6C⁺ CD4 T_N cells with a p-value of <0.05 are highlighted in red. The number of genes up- or down-regulated (1.3-fold cut-off) for each comparison is indicated. (B) Scheme depicting the selection of genes that were included in the 6CSign (list of the genes differentially expressed between Ly-6C^- and Ly-6C⁺ CD4 T_N cells, at a 1.3-fold cut-off). (C) Heat map of selected differentially expressed genes between Ly-6C^- and Ly-6C⁺ CD4 T_N cells. The scaled expression of each replicate, denoted as the row z-score, is plotted in yellow-blue color scale with yellow indicating high expression and blue indicating low expression.

https://doi.org/10.7554/eLife.27215.005

Figure 2—figure supplement 1

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Validation of the 6CSign at the protein level.

(**A–B**) CD5, CD73, CD122, CD200, Ikzf3, IL18Rα, Izumo1r and Sca-1 expression at the cell surface of Ly-6C^- and Ly-6C⁺ CD4 T_N cells was analysed by FACS. (A) Fluorescence histograms of gated Ly-6C^- and Ly-6C⁺ CD4 T_N (CD4⁺ CD8α^- TCRβ⁺ CD44^lo CD25^lo Foxp3-GFP^-) cells recovered from pLNs are shown for a representative C57BL/6 Foxp3-GFP mouse. (B) Mean fluorescence intensities (MFIs) are shown as means ± s.e.m. for a representative experiment. Each dot represents an individual mouse.

https://doi.org/10.7554/eLife.27215.006

Figure 3

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Transcriptomic signature of Ly-6C^- CD4 T_N cells reveals both their active TCR signaling and their bias toward iTreg-cell polarization.

(**A–E**) 6CSign was compared to several public Geo Datasets. (A) Diagram illustrating the analysis protocol. (B) Ratio vs ratio representation comparing gene expression ratio between Ly-6C^- CD4 T_N cells and Ly-6C⁺ CD4 T_N cells (6CSign) with either CD5^hivslo cell signature (Richards et al., 2015) (ratio of CD5^hi CD4 T_N cells to CD5^lo CD4 T_N cells, left panel), anti-CD3 activated CD4 T_N-cell signature (Wakamatsu et al., 2013) (ratio of CD4 T_N cells stimulated for 24–48 hr with anti-CD3 coated Ab to unstimulated CD4 T_N cells, middle panel) and PMA-activated CD4 T_N-cell signature (Bevington et al., 2016) (ratio of CD4 T_N cells stimulated for 2 hr with PMA to unstimulated CD4 T_N cells, right panel). (C) Ratio vs ratio representation comparing gene expression ratio between Ly-6C^- CD4 T_N cells and Ly-6C⁺ CD4 T_N cells (6CSign) with in-vitro-induced T_H1, T_H2, T_H17, iTreg and ex vivo purified Treg cell signatures that have been identified by Wei et al. (2009) (ratio of CD4 T_H-cell subsets to CD4 T_N cells). (D) Ratio vs ratio representation comparing gene expression ratio between Ly-6C^- CD4 T_N cells and Ly-6C⁺ CD4 T_N cells (6CSign) with TCR-signaling-dependent CD4-Treg-specific signature (Vahl et al., 2014) (ratio between TCR⁺ Treg cells and TCR-ablated (TCR^-) Treg cells). (**B–D**) Correlation analyses were performed using Pearson’s correlation test. (E) ‘Volcano plot’ representation (Log₂ (ratio) versus Log₁₀ (t-test p-value)) between TCR⁺ Treg cells and TCR-ablated (TCR^-) Treg cells (Vahl et al., 2014), for 6CSign genes upregulated (upper panel) or downregulated (lower panel) in Ly-6C^- CD4 T_N cells. (**B–E**) Datasets were filtered to common probes between the two arrays.

https://doi.org/10.7554/eLife.27215.007

Figure 4 with 2 supplements

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Ly-6C^- CD4 T_N-cell phenotype relies upon calcium signaling pathway in vitro.

(**A–B**) Flow-cytometry sorted Ly-6C⁺ CD4 T_N cells from C57BL/6 Foxp3-GFP mice were labelled with CTv and cultured in IL-7 (10 ng/ml) in the presence of either coated αCD3/28 (4µg/ml), PMA and TG (1.25 ng/ml and 4 nM, respectively), PMA alone (1.25 ng/ml) or TG (4 nM). Cells were recovered and analyzed after 5 days of culture. (A) Representative Ly-6C/CTv contour-plots are shown. (B) Ly-6C Mean fluorescence intensities (MFIs), for gated CD4⁺ TCR⁺ cells, are shown as means ± s.e.m. for a representative experiment with three mice per group (upper panel). Mean cell cycle numbers were calculated (lower panel). (C) Ex-vivo-purified CD4 T cells from C57BL/6 Foxp3-GFP mice were stained with Indo-1 and cell surface molecules CD44, Ly-6C and lineage markers (CD11c, CD11b, CD8β, CD25, TCRγδ and NK1.1). Intracellular calcium concentration was assessed before and after stimulation with 4 or 200 nM TG to the extracellular medium and monitored by flow cytometry for 20 min; results are presented as normalized ratio of Indo-1 emission at 405 nm to that at 510 nm (405/510) for gated Ly-6C⁺ CD4 T_N cells (Lineage^- Foxp3-GFP^- CD44^lo Ly-6C⁺ cells). Normalized Indo-1 ratio at the *Plateau* are shown as means ± s.e.m. for a representative experiment (out of two independent experiments) with three mice per group (Each dot represents an individual mouse). (**D–G**) Flow-cytometry sorted Ly-6C⁺ CD4 T_N cells from C57BL/6 Foxp3-GFP mice were cultured in the presence of IL-7 (10 ng/mL) with TG (4 nM) or not. (D) Diagram illustrating the experimental protocol. (E) Basal intracellular calcium concentration was assessed, as in C, after 5 days of culture. Raw Indo-1 ratio are shown as means ± s.e.m. for a representative experiment (out of two independent experiments) with three mice per group (each dot represents an individual mouse). (F) After 1, 3 and 5 days of culture, cells were analyzed by imaging flow cytometry. NFAT1 nuclear localization was calculated as similarity score between NFAT1 and DRAQ5 intensities. Data are representative of one of two independent experiments. (G) Cells were analyzed after 0, 1, 2, 3, 4 and 7 days of culture. Representative Ly-6C contour plots are shown for gated CD4 T_N cells (CD4⁺ CD44^lo CD25^lo Foxp3-GFP^-) are shown. (**H, I**) LN cells were isolated from C57BL/6 mice and fixed in 4% paraformaldehyde immediately (Ex vivo) or after 30 min of culture in the presence of 200 nM of TG (TG) or not (Resting). Ly-6C^- and Ly-6C⁺ CD4 T_N cells (CD4⁺ CD44^lo CD25^lo Foxp3^-) were sorted by flow cytometry and stained for NFAT1, and DNA (DRAQ5). (H) Cells were analyzed by confocal microscopy; CD4 (Red), Ly-6C (Magenta), NFAT1 (pseudocolor) and DNA (DRAQ5, cyan) fluorescence are shown for ‘ex vivo’ purified Ly-6C^- (upper panel) and Ly-6C⁺ (lower panel) CD4 T_N cells. Original magnification ×63. (I) Cells were analyzed by imaging flow cytometry and NFAT1 nuclear localization assessed as in F. Data are representative of one of three independent experiments. (**B, C, E, F, 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).

https://doi.org/10.7554/eLife.27215.008

Figure 4—figure supplement 1

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‘Ca²⁺-converted’ Ly-6C⁺CD4 T_N cells keep a naive phenotype.

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 TG (4 nM) or αCD3 and αCD28 coated antibodies (4 µg/mL). After 5 days, cells were analyzed for their forward scatter profile (FSC) and their expression of CD44 and CD62L.

https://doi.org/10.7554/eLife.27215.009

Figure 4—figure supplement 2

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NFAT2 localization is more nuclear in Ly-6C^- CD4 T_N cells than in their Ly-6C⁺-cell counterparts.

LN cells were isolated from C57BL/6 mice and fixed in 4% paraformaldehyde immediately (Ex vivo) or after 30 min of culture alone (Resting) or in the presence of 200 nM of TG (TG). Ly-6C^- and Ly-6C⁺ CD4 T_N cells (CD4⁺ CD44^lo CD25^lo Foxp3^-) were sorted by flow cytometry and stained for NFAT2, and DNA (DRAQ5). Cells were then analyzed by imaging flow cytometry. (A) Scheme depicting the experimental procedure. (B) NFAT2 nuclear localization was calculated as similarity score between NFAT2 and DRAQ5 intensities. Data are representative of one of three independent experiments.

https://doi.org/10.7554/eLife.27215.010

Figure 5 with 1 supplement

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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).

https://doi.org/10.7554/eLife.27215.011

Figure 5—figure supplement 1

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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.

https://doi.org/10.7554/eLife.27215.012

Figure 6

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Ly-6C^- CD4 T_N-cell sensitization to iTreg-cell polarization signals relies upon calcium signaling pathway in vitro.

Flow-cytometry sorted Ly-6C^- and Ly-6C⁺ CD4 T_N cells from C57BL/6 Foxp3-GFP mice were cultured in IL-7 (10 ng/mL) with or without TG (4 nM) and CsA (50 nM). After 5 days, live cells were flow-cytometry sorted and stimulated with coated αCD3 and αCD28 Abs (4 µg/ml) in the presence of graded doses of TGFβ1. Cells were analyzed after 4 days of stimulation. (A) Diagram illustrating the experimental procedure. (B) Representative Foxp3/CD4 contour-plots and proportions of Foxp3⁺ cells for gated CD4⁺ cells are shown at a dose of 0.25 ng/mL TGFβ1, as a function of pre-culture condition. (C) Concentrations of TGFβ1 needed to obtain 50% of the maximal percentage of iTreg-cell polarization (EC50) were calculated for each CD4 T_N-cell subset and each pre-culture condition. Each dot represents an independent experiment. 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<0.05; **p<0.01; ***p<0.001; ns, not significant).

https://doi.org/10.7554/eLife.27215.013

Figure 7 with 2 supplements

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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.

https://doi.org/10.7554/eLife.27215.014

Figure 7—figure supplement 1

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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.

https://doi.org/10.7554/eLife.27215.015

Figure 7—figure supplement 2

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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).

https://doi.org/10.7554/eLife.27215.016

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers
Strain, strain background (Mus musculus)	B6.Cg-Foxp3tm1Mal/J	Wang et al. (2008) (PMID: 18209052)	IMSR Cat# JAX:018628, RRID:IMSR_JAX:018628
Strain, strain background (Mus musculus)	B6.Cg-Tg(TcraTcrb)425Cbn/J	Barnden et al., 1998(PMID: 9553774)	IMSR Cat# JAX:004194, RRID:IMSR_JAX:004194
Antibody	Alexa Fluor 700-conjugated anti CD45.2 (104)	BD Biosciences	Cat# 560693
Antibody	Allophycocyanin (APC)-conjugated anti-CD25 (PC61)	BD Biosciences	Cat# 561048
Antibody	Allophycocyanin (APC)-conjugated anti-CD44 (IM7)	BD Biosciences	Cat# 561862
Antibody	Brilliant Violet (BV) 421-conjugated anti Ly-6C (AL-21)	BD Biosciences	Cat# 562727
Antibody	BV 510-conjugated anti-CD4 (RM4-5)	BD Biosciences	Cat# 563106
Antibody	BV 786-conjugated anti-CD25 (PC61)	BD Biosciences	Cat# 564023
Antibody	Phycoerythrin (PE)-conjugated anti-CD25 (PC61)	BD Biosciences	Cat# 561065
Antibody	Phycoerythrin (PE)-conjugated anti-CD69 (H1.2F3)	BD Biosciences	Cat# 553237
Antibody	Phycoerythrin (PE)-conjugated anti-Izumo1r (TH6)	BD Biosciences	Cat# 560320
Antibody	Phycoerythrin (PE)-conjugated anti-TCRgd (GL3)	BD Biosciences	Cat# 553178
Antibody	Phycoerythrin (PE)-conjugated anti-Vb5.1/5.2 (MR9-4)	BD Biosciences	Cat# 553190
Antibody	PE-Cy7-conjugated anti-CD44 (IM7)	BD Biosciences	Cat# 560569
Antibody	PE-Cy7-conjugated anti-CD45.1 (A20)	BD Biosciences	Cat# 560578
Antibody	Biotinylated anti-CD5 (53–7,3)	BD Biosciences	Cat# 553019
Antibody	Biotinylated anti-CD62L (MEL14)	BD Biosciences	Cat# 553149
Antibody	Biotinylated anti-Ly-6C (AL-21)	BD Biosciences	Cat# 557359
Antibody	Biotinylated anti-Sca1 (E13-161.7)	BD Biosciences	Cat# 553334
Antibody	Alexa Fluor 647-conjugated anti-IL18ra (BG/IL18ra)	BioLegend	Cat# 132903
Antibody	APC-conjugated streptavidin	BioLegend	Cat# 405207
Antibody	BV 421-conjugated anti-Ly-6C (HK1.4)	BioLegend	Cat# 128032
Antibody	PE-conjugated anti-Ly-6C (HK1.4)	BioLegend	Cat# 128008
Antibody	Alexa 448-conjugated anti-NFAT2 (7A6)	BioLegend	Cat# 649603
Antibody	Alexa 448-conjugated anti-NFAT1 (D43B1)	Cell Signaling	Cat# 14324
Antibody	PE-conjugated anti-CD200 (OX-90)	eBioscience	Cat# 12-5200-82
Antibody	PE-conjugated anti-Ikzf3 (8B2)	eBioscience	Cat# 12-5789-80
Antibody	PE-conjugated anti-Nur77 (12.14)	eBioscience	Cat# 12-5965-82
Antibody	PerCP-Cy5.5-conjugated anti-TCRb (H57-597)	eBioscience	Cat# 45-5961-82
Antibody	Biotinylated anti-CD73 (eBioTY/11.8)	eBioscience	Cat# 14-0731-82
Antibody	APC-conjugated anti-Foxp3 (FJK-165)	eBioscience	Cat# 12-5773-82
Antibody	PE-conjugated anti-Foxp3 (FJK-165)	eBioscience	Cat# 17-5773-82
Antibody	Pacific Blue-conjugated streptavidin	Invitrogen	Cat# S11222
Antibody	APC-Vio770-conjugated anti-CD8a (53–6.7)	Miltenyi Biotec	Cat# 130-102-305
Antibody	PE-conjugated anti-CD122 (TM-b1)	Miltenyi Biotec	Cat# 130-102-569
Chemical compound, drug	Phorbol 12-myristate 13-acetate (PMA)	Calbiochem	CAS 16561-29-8
Chemical compound, drug	FK506 (tacrolimus)	Sigma Aldrich	CAS 109581-93-3
Chemical compound, drug	CellTrace Violet	Invitrogen	Cat# C34557
Chemical compound, drug	CellTrace Far Red	Invitrogen	Cat# C34564
Chemical compound, drug	Recombinant Mouse IL-7	R and D Systems	Cat# 407 ML-025
Chemical compound, drug	Thapsigargin	Calbiochem	CAS 67526-95-8
Chemical compound, drug	TGFβ1	Invitrogen	Cat# PHG9204
Chemical compound, drug	DRAQ5	Cell Signaling	Cat# 4084
Chemical compound, drug	Indo-1, AM	Invitrogen	Cat# I1223
Software, algorithm	Illustrator CS5	Adobe Systems Inc.	http://www.graphpad.com
Software, algorithm	GeneChip Scanner 3000 7G	Affymetrix	N/A
Software, algorithm	Expression Console	Affymetrix	https://imagej.nih.gov/ij/
Software, algorithm	DIVA8.0.1	BD Biosciences	N/A
Software, algorithm	R	Bioconductor	N/A
Software, algorithm	Prism 7	GraphPad	N/A
Software, algorithm	ImageJ	NIH	https://www.bioconductor.org/
Software, algorithm	Partek Genomics Suite	Partek	N/A
Deposited data	GSE14308	Wei et al. (2009), PMID: 19144320	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE14308
Deposited data	GSE42276	Wakamatsu et al. (2013), PMID: 23277554	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE42276
Deposited data	GSE67464	Bevington et al., 2016, PMID: 26796577	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67464
Deposited data	GSE70154	Richards et al. (2015), PMID: 26195815	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE70154
Deposited data	GSE62532	Vahl et al. (2014), PMID: 25464853	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE62532
Deposited data	GSE97477	This paper	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE97477

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Vincent Guichard
Nelly Bonilla
Aurélie Durand
Alexandra Audemard-Verger
Thomas Guilbert
Bruno Martin
Bruno Lucas
Cédric Auffray

(2017)

Calcium-mediated shaping of naive CD4 T-cell phenotype and function

eLife 6:e27215.

https://doi.org/10.7554/eLife.27215

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Cell-intrinsic enhanced ability of Ly-6C- CD4 TN cells to commit into iTreg cells.

Cytokine-independent enhanced ability of Ly-6C- CD4 TN cells to commit into iTreg cells.

Transcriptional profiling identifies a set of differentially regulated genes between Ly-6C- and Ly-6C+ CD4 TN-cell subsets.

Validation of the 6CSign at the protein level.

Transcriptomic signature of Ly-6C- CD4 TN cells reveals both their active TCR signaling and their bias toward iTreg-cell polarization.

Ly-6C- CD4 TN-cell phenotype relies upon calcium signaling pathway in vitro.

‘Ca2+-converted’ Ly-6C+CD4 TN cells keep a naive phenotype.

NFAT2 localization is more nuclear in Ly-6C- CD4 TN cells than in their Ly-6C+-cell counterparts.

The calcium-calcineurin pathway shapes the phenotype of the CD4 TN-cell compartment in vivo.

The calcium-calcineurin cascade drives NFAT nuclear translocation in CD4 TN cells in vivo.

Ly-6C- CD4 TN-cell sensitization to iTreg-cell polarization signals relies upon calcium signaling pathway in vitro.

Calcium-mediated shaping of the CD4 TN-cell pTreg-cell differentiation potential in vivo.

OT-II CD4 TN cells are Ly-6C+and can be Ca2+-converted and tested for their ability to convert into pTreg cells in vivo.

Calcium-mediated shaping of the CD4 TN-cell phenotype increases the pTreg-cell differentiation potential of OT-II CD4 TN cells in vivo.

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Cell-intrinsic enhanced ability of Ly-6C^- CD4 T_N cells to commit into iTreg cells.

Cytokine-independent enhanced ability of Ly-6C^- CD4 T_N cells to commit into iTreg cells.

Transcriptional profiling identifies a set of differentially regulated genes between Ly-6C^- and Ly-6C⁺ CD4 T_N-cell subsets.

Transcriptomic signature of Ly-6C^- CD4 T_N cells reveals both their active TCR signaling and their bias toward iTreg-cell polarization.

Ly-6C^- CD4 T_N-cell phenotype relies upon calcium signaling pathway in vitro.

‘Ca²⁺-converted’ Ly-6C⁺CD4 T_N cells keep a naive phenotype.

NFAT2 localization is more nuclear in Ly-6C^- CD4 T_N cells than in their Ly-6C⁺-cell counterparts.

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

The calcium-calcineurin cascade drives NFAT nuclear translocation in CD4 T_N cells in vivo.

Ly-6C^- CD4 T_N-cell sensitization to iTreg-cell polarization signals relies upon calcium signaling pathway in vitro.

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

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.

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.