Calcium-mediated shaping of naive CD4 T-cell phenotype and function
- Institut Cochin, Paris Descartes Université, CNRS UMR8104, INSERM U1016, France
- Paris Diderot Université, France
Figures
Figure 1 with 1 supplement
Cell-intrinsic enhanced ability of Ly-6C- CD4 TN cells to commit into iTreg cells.
(A–E) Flow-cytometry sorted Ly-6C- and Ly-6C+ CD4 TN 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 …
Figure 1—figure supplement 1
Cytokine-independent enhanced ability of Ly-6C- CD4 TN cells to commit into iTreg cells.
(A–B) Flow-cytometry sorted Ly-6C- and Ly-6C+ CD4 TN 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. …
Figure 2 with 1 supplement
Transcriptional profiling identifies a set of differentially regulated genes between Ly-6C- and Ly-6C+ CD4 TN-cell subsets.
(A–E) Microarray analysis was performed on Ly-6C- and Ly-6C+ CD4 TN cells sorted from LNs of C57BL/6 Foxp3-GFP mice. (A) ‘Volcano plot’ representation (Log2 (ratio) versus Log10 (t test p-value)). …
Figure 2—figure supplement 1
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 TN cells was analysed by FACS. (A) Fluorescence histograms of gated Ly-6C- and …
Figure 3
Transcriptomic signature of Ly-6C- CD4 TN 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 TN …
Figure 4 with 2 supplements
Ly-6C- CD4 TN-cell phenotype relies upon calcium signaling pathway in vitro.
(A–B) Flow-cytometry sorted Ly-6C+ CD4 TN 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 …
Figure 4—figure supplement 1
‘Ca2+-converted’ Ly-6C+CD4 TN cells keep a naive phenotype.
Flow-cytometry sorted Ly-6C+ CD4 TN 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 …
Figure 4—figure supplement 2
NFAT2 localization is more nuclear in Ly-6C- CD4 TN 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+ …
Figure 5 with 1 supplement
The calcium-calcineurin pathway shapes the phenotype of the CD4 TN-cell compartment in vivo.
(A) Flow-cytometry sorted Ly-6C+ CD4 TN 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 …
Figure 5—figure supplement 1
The calcium-calcineurin cascade drives NFAT nuclear translocation in CD4 TN 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% …
Figure 6
Ly-6C- CD4 TN-cell sensitization to iTreg-cell polarization signals relies upon calcium signaling pathway in vitro.
Flow-cytometry sorted Ly-6C- and Ly-6C+ CD4 TN 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 …
Figure 7 with 2 supplements
Calcium-mediated shaping of the CD4 TN-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 TN (CD44lo CD25lo CD8β- CD11b- CD11c- NK1.1- TCRγδ-…
Figure 7—figure supplement 1
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.
(A) Ly-6C fluorescence histograms of gated CD4 TN (CD4+ CD8α- TCRβ+ CD44lo CD25lo Foxp3-GFP-) cells recovered from LNs of C57BL/6 Foxp3-GFP and C57BL/6 Foxp3-GFP OT-II mice are shown. (B, C) …
Figure 7—figure supplement 2
Calcium-mediated shaping of the CD4 TN-cell phenotype increases the pTreg-cell differentiation potential of OT-II CD4 TN 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 TN (CD44lo CD25lo CD8β- …
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 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.27215.017
