Sustained store-operated calcium entry utilizing activated chromatin state leads to instability in iTregs
- Institute for Immunology, Beijing Key Lab for Immunological Research on Chronic Diseases, Tsinghua University, China
- Tsinghua-Peking Center for Life Sciences, Tsinghua University, China
- IDG/McGovern Institute for Brain Research and School of Pharmaceutical Sciences, Tsinghua University, China
- MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Pharmaceutical Sciences, Tsinghua University, China
- Department of Basic Medical Sciences, School of Medicine, Tsinghua University, China
- Department of Microbiology, Immunology and Infectious Diseases, Snyder Institute, University of Calgary, Canada
- Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
Figures
Figure 1 with 1 supplement
iTreg share similar suppressive mechanism to tTregs.
(A) Comparison of suppressive activity between tTreg and iTreg. CFSE (carboxy fluoroscein succinimidyl Eester) -labeled OT-II T cells were stimulated with OVA-pulsed DC, then Foxp3-GFP+ iTregs and …
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Figure 1—source data 1
The suppression activity, AFM force of iTreg and nTreg, and calcium oscillation during iTreg induction.
- https://cdn.elifesciences.org/articles/88874/elife-88874-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Expression of Ryr2 in iTreg and the effect of Ryr2 on iTreg induction.
(A) QPCR for Ryr2 mRNA expression in iTregs, tTregs, and Tconvs. n = 3, N = 4. (B) The effect of Ryr2 expression on iTreg induction. Naive Tconv cells were sorted from Ryr2 fl/fl; CD4 cre mice and …
Figure 2 with 1 supplement
Diminished store-operated calcium entry (SOCE) signal and NFAT translocation in tTregs, but not in iTregs.
(A) Comparison of Treg stability between tTregs and iTregs. iTregs and tTregs were sorted and restimulated with anti-CD3 and anti-CD28 antibodies. Cells were harvested after 2-day restimulation and …
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Figure 2—source data 1
Treg stability, early store-operated calcium entry (SOCE) signal, long-term SOCE signal in iTreg and nTreg.
- https://cdn.elifesciences.org/articles/88874/elife-88874-fig2-data1-v1.xlsx
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Figure 2—source data 2
Original imaging data of long-term store-operated calcium entry (SOCE) signal in Tconv, nTreg, and iTreg, related to Figure 2C.
- https://cdn.elifesciences.org/articles/88874/elife-88874-fig2-data2-v1.zip
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Figure 2—source data 3
Original western data of NFAT nuclear translocation in Tconv, nTreg, and iTreg, related to Figure 2D.
- https://cdn.elifesciences.org/articles/88874/elife-88874-fig2-data3-v1.zip
Figure 2—figure supplement 1
TCR responsiveness and NFAT translocation in tTregs and iTregs.
(A) Calcium oscillation were recorded in steady state and after anti-CD3 and anti-CD28 activation. Tconv cells were loaded with Fluo-4 AM, and left in steady state without anti-CD3 and anti-CD28 …
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Figure 2—figure supplement 1—source data 1
Original western data of NFAT nuclear translocation upon early activation in Tconv, nTreg, and iTreg, related to Figure 2—figure supplement 1D.
- https://cdn.elifesciences.org/articles/88874/elife-88874-fig2-figsupp1-data1-v1.zip
Figure 3 with 1 supplement
iTregs display highly open chromatin state at the activation and differentiation-related genes.
(A) Principal component analysis (PCA) visualization of transcriptional profiles of Tconvs, tTregs, and iTregs with or without TCR stimulation. Color indicates cell types. (B) PCA visualization of …
Figure 3—figure supplement 1
iTreg has highest proliferation state and partial Treg feature.
(A) Left, heatmap showing the top genes that positively contribute to PC1. Right, heatmap showing the top genes that negatively contribute to PC1. As shown, genes contributing positively to PC1 …
Figure 4 with 1 supplement
Store-operated calcium entry (SOCE) signaling and NFAT can disrupt iTreg stability.
(A) Impact of calcium signal and NFAT on iTreg stability. Sorted Foxp3-GFP+ iTregs were rested for 1 day, then restimulated by anti-CD3 and CD28 in the presence of cyclosporine A (CsA) and CM-4620. …
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Figure 4—source data 1
iTreg stability, QPCR of related gene expression upon activation after CsA and CM-4620 blockade.
- https://cdn.elifesciences.org/articles/88874/elife-88874-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Store-operated calcium entry (SOCE) signaling and NFAT cause instability and downregulate Treg-related gene.
(A) Stability of iTreg treated with CM-4620, cyclosporine A (CsA), and BTP-2 upon restimulation. iTregs were restimulated by anti-CD3 and CD28 in the presence of CsA, CM-4620, and BTP-2. Percentages …
Figure 5 with 1 supplement
NFAT disrupt iTreg stability by upregulating prime-opened TH genes.
(A) Model of CUT&Tag experiments to capture the binding sites of NFATc1. (B) The number of NFAT Cut&Tag peaks in mock control, resting iTregs, and restimulated iTregs. (C) Normalized counts of …
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Figure 5—source data 1
IL-21 secretion in tTreg and iTreg upon activation.
- https://cdn.elifesciences.org/articles/88874/elife-88874-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
iTregs have highest accessibility in TH-associated genes.
(A) Sorted Foxp3-RFP+ naive Tconvs were induced into iTregs in the presence of IL-2 and TGF-β for 4 days, on the second and third days, iTreg cells were infected with retrovirus packaging with …
Figure 6 with 1 supplement
Manipulation of store-operated calcium entry (SOCE) can enhance iTreg stability.
(A) Manipulation of SOCE signal by dominant negative ORAI (DN-ORAI). SOCE was recorded in DN-ORAI iTreg cells loaded with Fura-Red by flow cytometry, TG was added after 1 min to induce ER depletion, …
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Figure 6—source data 1
Store-operated calcium entry (SOCE) signal, iTreg stability in vitro and in vivo after over-expression DN-ORAI.
- https://cdn.elifesciences.org/articles/88874/elife-88874-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Different optimization of iTreg have various impact on activation and Treg regions.
(A) Line plots (top) and heatmaps (bottom) of activation regions in anti-CD3, anti-CD28, IL-2, and TGF-β-induced iTregs, treated with retinoic acid, rapamycin, vitamin C, removal of CD28, and …
Author response image 1
Restimulation with TGFb will persist iTreg inducing environment, resulting in less pronounced instability.
Sorted Foxp3-GFP+ iTregs were rested for 1d, and then rested or restimulated in the presence of TGF-β for 2 d. Percentages of Foxp3+ cells were analyzed by intracellular staining of Foxp3 after 2 d.
Author response image 2
Comparing effects of NFAT, NF-kB and c-Jun/c-Fos inhibitors on iTreg instability.
Sorted Foxp3-GFP+ iTregs were rested for 1d, then restimulated by anti-CD3 and CD28 in the presence of listed inhibitors. Percentages of Foxp3+ cells were analyzed by intracellular staining after 2d …
Author response image 3
The NFAT binding and histone modification on Foxp3 gene locus.
Genome track visualization of NFAT binding profiles and H3K4me3 profiles in Foxp3 CNS3 locus in two batches of dataset.
Author response image 4
Effect of inhibiting NFAT and calcium on human iTreg stability.
Human naïve CD4 cells from PBMC were subjected to a two-week induction process to generate human iTreg. Subsequently, human iTreg were restimulated for 2 days with dynabeads followed by 2 days of …
Author response image 5
Chromatin accessibility of Rest, Retimulated, CsA/ORAIinh treated restimulated iTreg.
PCA visualization of chromatin accessibility profiles of different cell types. Color indicates cell type.
Author response image 6
Stability of DN-ORAI iTreg in vivo with or without OVA immunization.
WT-ORAI/DN-ORAI-GFP+-transfected CD45.2+ Foxp3-RFP+ OT-II iTregs were transferred i.v. into CD45.1 mice. Recipients were left or immunized with OVA323-339 in Alum adjuvant. On day 5, mLN were …
Author response image 7
Compare multiple dose of Tconv:Treg ratio in suppression functionCFSE-labelled OT-II T cells were stimulated with OVA-pulsed DC, then different number of Foxp3-GFP+ iTregs and tTregs were added to the culture to suppress the OT-II proliferation.
After 4 days, CFSE dilution were analyzed. Left, Representative histograms of CFSE in divided Tconvs. Right, graph for the percentage of divided Tconvs.
Author response image 8
Chromatin accessibility of some “Activation Region”.
Genomic track showing chromatin accessibility of Irf4, Atf3, Lta, Tnfsf8, Tnfsf4, Tnsfsf14, Il12rb2, Il9, Gzmc in activated Tconv and iTreg.
Author response image 9
Relationship of cell death and Foxp3 stability in restimulated iTregs.
Sorted Foxp3-GFP+ iTregs were rested for 1d, then restimulated by anti-CD3 and CD28 in the presence of CsA or CM-4620. After 2d restimulation, live cell percentage were analyzed by staining of …
Author response image 10
IL-21 secretion in tTreg and iTreg upon activation.
iTregs and tTregs were sorted and restimulated with anti-CD3 and anti-CD28 antibodies, in the presence of CsA and CM-4620. Cell culture supernatant were harvested after 2 d restimulation and IL-21 …
Videos
Video 1
Imaging of store-operated calcium entry (SOCE) signal in Tconvs.
Video 2
Imaging of store-operated calcium entry (SOCE) signal in tTregs.
Video 3
Imaging of store-operated calcium entry (SOCE) signal in iTregs.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL6/J | Jackson Laboratory | Strain #:000664 from Jackson Laboratory | |
| Strain, strain background (Mus musculus) | Foxp3-GFP (B6.Cg-Foxp3tm2Tch/J) | Hai Qi of School of Medicine, Tsinghua University | Strain #:006772 from Jackson Laboratory | |
| Strain, strain background (Mus musculus) | Foxp3-RFP | Zhou Xuyu of Institute of Microbiology, Chinese Academy of Sciences | Foxp3-RFP | |
| Strain, strain background (Mus musculus) | CD45.1 | Jackson Laboratory | Strain #:002014 from Jackson Laboratory | |
| Strain, strain background (Mus musculus) | OT-II | Hai Qi of School of Medicine, Tsinghua University | Strain #:004194 from Jackson Laboratory | |
| Antibody | Ultra-LEAF Purified anti-mouse CD3ε Antibody, Armenian Hamster monoclonal | Biolegend | 100340 | 0.5 μg/ml |
| Antibody | Ultra-LEAF Purified anti-mouse CD28 Antibody, Syrian Hamster monoclonal | Biolegend | 102116 | 1 μg/ml |
| Antibody | NFATc1 Antibody (7A6), mouse monoclonal | Santa Cruz | sc-7294 | 1:2000 |
| Antibody | NFATc2 Antibody (4G6-G5), mouse monoclonal | Santa Cruz | sc-7296 | 1:2000 |
| Antibody | Lamin A/C (4C11) Mouse mAb | CST | 4777 | 1:2000 |
| Antibody | β-Actin (13E5) Rabbit mAb | CST | 4970 | 1:1000 |
| Antibody | GAPDH (D4C6R) Mouse mAb | CST | 97166 | 1:1000 |
| Antibody | Monoclonal ANTI-FLAG M2 antibody produced in mouse | Sigma | F1804 | 1:50 |
| Antibody | Goat Anti-Mouse IgG Secondary Antibody, monoclonal | Sino Biological | SSA021 | 1:50 |
| Antibody | Histone H3 (D2B12) XP Rabbit mAb (ChIP Formulated) | CST | 4620 | 1:50 |
| Antibody | Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade, Rabbit polyclonal | Abcam | ab8580 | 1:50 |
| Antibody | Goat Anti-Rabbit IgG Secondary Antibody, monoclonal | Sino Biological | SSA018 | 1:50 |
| Antibody | Biotin anti-mouse CD3ε Antibody, Armenian Hamster monoclonal | Biolegend | 100303 | 0.5 μg/ml |
| Antibody | FOXP3 Monoclonal Antibody (150D/E4), PE, mouse monoclonal | eBioscience | 12-4774-42 | 1:500 |
| Antibody | FOXP3 Monoclonal Antibody (150D/E4), Alexa Fluor 488, mouse monoclonal | eBioscience | 53-4774-41 | 1:500 |
| Antibody | CD4 Monoclonal Antibody (GK1.5), APC, rat monoclonal | eBioscience | 17-0041-82 | 1:500 |
| Antibody | CD25 Monoclonal Antibody (PC61.5), PE, rat monoclonal | eBioscience | 12-0251-82 | 1:500 |
| Antibody | CD44 Monoclonal Antibody (IM7), APC, rat monoclonal | eBioscience | 17-0441-83 | 1:500 |
| Antibody | PE anti-mouse CD62L, rat monoclonal | Biolegend | 104408 | 1:500 |
| Peptide, recombinant protein | Recombinant Human TGF-beta 1 Protein | R&D | 240-B-010 | |
| Peptide, recombinant protein | Recombinant Human IL-2 | R&D | 202-IL | |
| Peptide, recombinant protein | Purified Streptavidin | Biolegend | 405150 | |
| Commercial assay, kit | LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation | Invitrogen | L34965 | |
| Commercial assay, kit | Propidium Iodide | Beyotime | C1062M-3 | |
| Commercial assay, kit | Mouse CD4+ T Cell Isolation Kit | Stem cell | 19852 | |
| Commercial assay, kit | Mouse CD25 Treg Cell positive selection Kit | Stem cell | 18782 | |
| Commercial assay, kit | Foxp3/Transcription Factor Staining Buffer Set | eBioscience | 00-5523-00 | |
| Commercial assay, kit | NE-PER Nuclear and Cytoplasmic Extraction Reagents | Thermo | 78835 | |
| Commercial assay, kit | TruePrep DNA Library Prep Kit V2 for Illumina | Vazyme | TD501 | |
| Commercial assay, kit | Hyperactive Universal CUT&Tag Assay Kit for Illumina | Vazyme | TD903 | |
| Commercial assay, kit | TruePrep Index Kit V2 for Illumina | Vazyme | TD202 | |
| Commercial assay, kit | VAHTS DNA Clean Beads | Vazyme | N411 | |
| Commercial assay, kit | Mouse IL-21 Uncoated ELISA | Invitrogen | 88–8210 | |
| Chemical compound, drug | Cyclosporin A | MCE | HY-B0579 | |
| Chemical compound, drug | CM-4620 | MCE | HY-101942 | |
| Chemical compound, drug | Thapsigargin | Invitrogen | T7459 | |
| Chemical compound, drug | Ionomycin | beyotime | S1672 | |
| Chemical compound, drug | Caffeine | Aladdin | C106953 | |
| Chemical compound, drug | 4-CMC | Sigma | C55402 | |
| Chemical compound, drug | Fluo-4, AM, cell permeant | Thermo | F14201 | |
| Chemical compound, drug | Cal RedTM R525/650 AM | AAT Bioquest | 20591 | |
| Chemical compound, drug | Indo-1 AM | BD | 565879 | |
| Chemical compound, drug | Pluronic F-127 | Sigma | P2443 | |
| Chemical compound, drug | CellTak 1 MG WI, 1/CS | BD | 354240 | |
| Chemical compound, drug | Hanks’ Solution | Coolaber | SL6080 | |
| Chemical compound, drug | HBSS, 10× (without Calcium) | Macgene | CC016 | |
| Chemical compound, drug | Imject Alum | Thermo | 77161 | |
| Chemical compound, drug | OVA Peptide (323-339) | GenScript | RP10610-1 | |
| Chemical compound, drug | OVA | Sigma | A5503 | |
| Chemical compound, drug | Cell Trace CFSE | Thermo | C34554 | |
| Chemical compound, drug | Retinoic Acid | Sigma | R2625 | |
| Chemical compound, drug | InSolution Rapamycin | Sigma | 553211 | |
| Chemical compound, drug | L-Ascorbic acid (Vitamin C) | Sigma | A4403 | |
| Chemical compound, drug | AS2863619 | MCE | HY-126675A | |
| Chemical compound, drug | BAY 11-7082 | MCE | HY-13453 | |
| Chemical compound, drug | SP600125 | MCE | HY-12041 | |
| Chemical compound, drug | T-5224 | MCE | HY-12270 | |
| Software, algorithm | FlowJo | FlowJo LLC | ||
| Software, algorithm | Prism | GraphPad | ||
| Software, algorithm | IGV | Broad Institute | ||
| Software, algorithm | Imaris | Andor | ||
| Software, algorithm | R | R studio | ||
| Software, algorithm | JPK Data Processing | JPK |
