Induction of the IL-1RII decoy receptor by NFAT/FOXP3 blocks IL-1β-dependent response of Th17 cells
- Laboratory of Autoimmunity and Inflammation (LAI), Department of Biomedical Sciences, Seoul National University College of Medicine, Republic of Korea
- Department of Microbiology and Immunology, Seoul National University College of Medicine, Republic of Korea
- Cancer Research Institute and Institute of Infectious Diseases, Seoul National University College of Medicine, Republic of Korea
- Department of Internal Medicine, Chungnam National University School of Medicine, 282 Munhwa-ro, Jung-gu, Republic of Korea
- Department of Life Science, College of Natural Sciences and Research Institute for Natural Sciences, Hanyang University, Republic of Korea
- Department of Biochemistry and Molecular Biology, Department of Biomedical Sciences, and Cancer Research Institute, Seoul National University College of Medicine, Republic of Korea
- Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine; Seoul National University Hospital Biomedical Research Institute, Republic of Korea
Figures
Figure 1 with 1 supplement
IL-1β is crucial for promoting human Th17 responses.
(A) Representative flow cytometric plot of IL-17 and/or IFN-γ production by human memory CD4+ T cells obtained from HCs and stimulated for 7 days with anti-CD3/28-coated microbeads with or without rhIL-1β (5 ng/ml). (B) Frequency (%) of IL-17-producing cells and IL-17/IFN-γ double-producing cells (flow cytometry) and the amount of IL-17 in the culture supernatant of (A) (ELISA). (C) Frequency (%) of IFN-γ-producing cells and IL-10-producing cells (flow cytometry). (D) The amount of IL-17 in the culture supernatant under the indicated cytokine conditions (ELISA): rhIL-6 (25 ng/ml), rhIL-23 (25 ng/ml), and rhL-1β (5 ng/ml). (E) Purifed naive CD4+ T cells were stimulated for 7 days with anti-CD3/28-coated microbeads in serum-free X-VIVO 10 medium under the indicated cytokine conditions. IL-17 in the culture supernatant (ELISA): rhIL-6 (25 ng/ml), rhIL-23 (25 ng/ml), rhL-1β (5 ng/ml), and rhTGF-β (10 ng/ml). Bar graphs show the mean ± SEM of six (A–D), and five (E) independent experiments. * = p<0.05, ** = p<0.01, and *** = p<0.001 by two-tailed paired t-test.
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Figure 1—source data 1
Figure 1B IL-1β significantly enhance IL-17 & IFN-γ producing memory CD4+ T cells.
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Figure 1—source data 2
Figure 1C IL-1β regulated IL-10 producing memory CD4+ T cells.
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Figure 1—source data 3
Figure 1D Th17-polarizing cytokines intensify the Th17 response of memory CD4+ T cells.
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Figure 1—source data 4
Figure 1E IL-1β is important for promoting Th17 differentiation form naïve CD4+ T cells.
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Figure 1—figure supplement 1
IL-1β is a crucial cytokine for promoting Th17 responses in humans.
Purified human memory CD4+ T cells were stimulated for 7 days with anti-CD3/28-coated microbeads under the indicated cytokine conditions: rhIL-6 (25 ng/ml) and rhIL-23 (25 ng/ml). (A) Amount of IL-17A in culture supernatants was quantified by conventional ELISA. (B) mRNA expression of the indicated genes was analyzed by qRT-PCR on day 7 post-stimulation. The relative gene expression under different concentrations of IL-1β is displayed using a heatmap. (C) Expression of pathogenic Th17 cell-associated genes in IL-1β (5 ng/ml)-treated CD4+ T cells was presented relative to their expression in PBS-treated CD4 T cells.
Figure 2 with 2 supplements
Dynamic regulation of IL-1β receptors by memory CD4+ T cells upon TCR stimulation.
(A) Representative flow cytometric plot and the frequency (%) of functional IL-1RI- and decoy IL-1RII-expressing CD4+ T cells of HCs (n = 13). (B) Representative flow cytometric plot and the frequency (%) of IL-1RI+ cells in naive (CD45RA+CCR7+) or memory (CD45RA-) CD4+ T-cell subset of HCs (n = 13). (C) Representative flow cytometric plot of change in IL-1RI and IL-1RII expression by TCR-stimulated memory CD4+ T cells (n = 7). (D) Time kinetics of IL-1RI and IL-1RII expression on TCR-stimulated memory CD4+ T cells (n = 5). (E) Representative flow cytometric plot and the frequency (%) of IL-1RI and IL-1RII expression by memory CD4 + T cells in response to stimulation with different concentrations of anti-CD3 and 1.5 μg/ml of anti-CD28 Ab for 48 hr (n = 5). (F) The amounts of IL-17A (left) and IFN-γ (right) in culture supernatants of memory CD4+ T cells stimulated with anti-CD3/28-coated microbeads for 7 days with or without IL-1β (n = 5). Anti-IL-1RII neutralizing Ab or control isotype Ab was added into the culture at day 2 post-stimulation. Bar graphs and line graphs show the mean ± SEM. * = p<0.05, ** = p<0.01, and *** = p<0.001 by two-tailed paired t-test.
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Figure 2—source data 1
Figure 2A Ex vivo expression of IL-1RI & IL-1RII on CD4+ T cells.
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Figure 2—source data 2
Figure 2B Ex vivo expression of IL-1RI between naïve and memory CD4+ T cells.
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Figure 2—source data 3
Figure 2B Ex vivo expression of IL-1RI between naïve and memory CD4+ T cells.
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Figure 2—source data 4
Figure 2D Time kinetics of IL-1RI & IL-1RII.
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Figure 2—source data 5
Figure 2E Effect of TCR signaling strength on expression of IL-1RI & IL-1RII.
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Figure 2—figure supplement 1
The differential expression of IL-23R on IL-1RI+ and IL-1RI- memory CD4+ T cells in humans.
Bar graphs show the mean ± SEM. *** = p<0.001 by two-tailed paired t-test.
Figure 2—figure supplement 2
The expression of receptors for IL-1β is dynamically changed by various stimulations of CD4+ T cells.
(A) Purified human memory CD4+ T cells were stimulated with anti-CD3/28-coated microbeads. mRNA expression of IL-1RI and IL-1RII was analyzed by qRT-PCR at the indicated time-points (B) Purified human naive and memory CD4+ T cells were stimulated for 48 hr with anti-CD3/28-coated microbeads under the indicated cytokine conditions. The expression of IL-1RI and II was analyzed by flow cytometry (n = 6 for memory, n = 5 for naive CD4+ T cells). Bar graphs and line graphs show the mean ± SEM. * = p<0.05, ** = p<0.01, and *** = p<0.001 by two-tailed paired t-test.
Figure 3 with 4 supplements
Differential expression pattern of IL-1Rs defines immunological features of CD4+ T cells.
(A) Treg-depleted memory CD4+ T cells were stimulated with anti-CD3/28-coated microbeads for 48 hr. The expression of IL-1RI and IL-1RII were analyzed by flow cytometry (n = 12). (B) The frequency (%) of Treg-related marker-expressing cells in IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- memory CD4+ T cell populations at day 2 post-stimulation (n = 12). (C) Representative flow cytometric plot and the frequency (%) of Th1 (CCR6-CD161-CXCR3+), Th17 (CCR6+CD161+CXCR3-), and ex-Th17 (CCR6+CD161+CXCR3+) cells in IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- memory CD4+ T cell populations (n = 9). (D) Intracellular cytokine staining (ICS) of IL-17- and IFN-γ in IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- memory CD4+ T cell populations (n = 4). Bar graphs and pie charts show the mean ± SEM. * = p<0.05, ** = p<0.01, and *** = p<0.001 by two-tailed paired t-test. †, ‡, and ¶ = p<0.05: compared between indicated subsets by two-tailed paired t-test (C).
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Figure 3—source data 1
Figure 3A Frequency of IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- subset of Treg-depleted memory CD4+ T cells.
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Figure 3—source data 2
Figure 3B Expression of Treg related markers on L-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- subsets.
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Figure 3—source data 3
Figure 3C Frequency of ex-Th17, Th17, and Th1 of L-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- subsets.
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Figure 3—source data 4
Figure 3D Expression of IL-17 & IFN-γ in L-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- subsets.
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Figure 3—figure supplement 1
CD4+ T cells expressing GARP, a marker of activated Tregs, preferentially, but not exclusively, increase IL-1RII expression.
Purifed memory CD4+ T cells were stimulated with anti-CD3/28-coated microbeads. (A) Representative flow cytometric plot (A) and the frequencies (B) of IL-1RI and IL-1RII expression on GARP+ or GARP- memory CD4+ T cells at day 2 post-stimulation (n = 4). (C) The frequencies (%) of Treg- or Th17-related marker-expressing cells on IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- memory CD4+ T cells at day 2 post-stimulation (n = 12). Bar graphs show the mean ± SEM. * = p<0.05, ** = p<0.01, and *** = p<0.001 by two-tailed paired t-test.
Figure 3—figure supplement 2
Efficiency of sorting-based Treg depletion from CD4+ T cells.
(A) Expression of CD127 and CD25 before and after sorting-based depletion of CD25highCD127dim/- Treg from memory CD4+ T cells. (B) Expression of Foxp3 before and after sorting-based depletion as performed in (A). (C) Expression of CD45RA and Foxp3 before and after sorting-based depletion as performed in (A) (n = 4). Faction II and III indicate activated Treg cells and non-suppressive cytokine-producing Foxp3low T cells, respectively, according to the definition of the Sakaguchi group.
Figure 3—figure supplement 3
Critical roles of Smad3 and NFAT for Foxp3 expression in non-Treg memory CD4+ T cells.
Total or Treg-depleted memory CD4+ T cells were stimulated for 2 days with anti-CD3/28-coated microbeads in the presence of various signaling inhibitors. The expression of Foxp3 was analyzed by flow cytometry. Bar graphs show the mean ± SEM. *** = p<0.005 by two-tailed paired t-test.
Figure 3—figure supplement 4
The correlation of IL-23R with IL-1RI+IL-1RII+ and IL-1RI+IL-1RII- subset of memory CD4+ T cells.
Purified memory CD4+ T cells were stimulated for 48 hr to induce the expression of IL-1RI and IL-1RII. The expression of IL-23R were compared among IL-1RI-IL-1RII-, IL-1RI+IL-1RII-, and IL-1RI+IL-1RII+ cells. MFI indicates mean fluorescent intensity. Bar graphs show the mean ± SEM. * = p<0.05 and ** = p<0.01 by two-tailed paired t-test.
Figure 4
NFAT and Foxp3 are essential for IL-1RII expression in memory CD4+ T cells.
(A and B) Representative flow cytometric plot (A) and the frequency (B) of IL-1RI and IL-1RII expression on TCR-stimulated memory CD4+ T cells treated with the indicated concentration of CsA, a chemical inhibitor of NFAT at day 2 post-stimulation (n = 5). (C) The expression of IL-1RII mRNA in TCR-stimulated or unstimulated memory CD4+ T cells in the presence or absence of CPP-VIVIT (1 μM), NFAT-specific peptide inhibitor (n = 4). (D) The expression of CD25, IL-1RI, and IL-1RII on TCR-stimulated memory CD4 T cells in the presence or absence of CPP-VIVIT (n = 3). (E) Representative histogram plot of Foxp3 in TCR-stimulated memory CD4+ T cells in the presence or absence of 1,25-dihydroxyvitamin D3 (VD3; 10 μM) and rhIL-2 (250 IU/ml) with or without CsA. MFI indicates mean fluorescent intensity. (F) Flow cytometric analysis of IL-1RI and IL-1RII on memory CD4+ T cell under same conditions as in (E). (G and H) The frequencies (%) of IL-1RI+ and IL-1RII+ cells and the ratio of IL-1RII+ to IL-1RI+ cells under same conditions in (E) (n = 6). Bar graphs show the mean ± SEM. * = p<0.05, ** = p<0.01, and *** = p<0.001 by two-tailed paired t-test.
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Figure 4—source data 1
Figure 4A NFAT inhibitor CsA selectivley repress the expression of IL-1RII.
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Figure 4—source data 2
Figure 4B NFAT inhibiton peptide VIVIT repress the expression of IL-1RII.
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Figure 4—source data 3
Figure 4G expression of IL-1RII significantly upregulated by treatment with 1,25(OH)2VD3 and IL-2 in memory CD4+ T cells.
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Figure 4—source data 4
Figure 4H Ratio of IL-1RII+/IL-1RI+.
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Figure 5 with 3 supplements
NFAT/FOXP3 interaction is responsible for expression of IL-1RII by human memory CD4+ T cells.
(A) The frequency (%) of CD25, a NFAT-dependent molecule, on TCR-stimulated memory CD4+ T cells in the presence of Foxp3 393–403 peptide (100 μM), NFAT/FOXP3 interaction inhibition peptide, or control Foxp3 399 peptide (100 μM) at day 2 post-stimulation (n = 4). (B and C) Representative flow cytometric plot (B) and the frequencies (C) of IL-1RI and IL-1RII expression on TCR-stimulated memory CD4+ T cells in the presence of Foxp3 393–403 peptide or control Foxp3 399 peptide at day 2 post-stimulation (n = 4). (D) Foxp3-expressing Jurkat cells were transfected with the indicated pGL4.10 luciferase vectors (pGL4/IL-1RII-a1414, pGL4/IL-1RII-a814, and pGL4/IL-1RII-a215) and pGL4.74 control renilla vector as internal control, followed by stimulation with PMA and ionomycin for 48 hr (upper panel: n = 4). Comparison of luciferase activity between pGL4/IL-1RII-a1414 and pGL4/IL-1RII-a1414mu (−1209 to −1188 NFAT-binding motif mutant) transfected Foxp3-expressing Jurkat cells (lower panel: n = 4). Luciferase activity was determinded using dual lucifease assay system. (E and F) Purified memory CD4+ T cells were stimulated with plate-bound anti-CD3/28 mAbs for 24 hr. ChIP qPCR was perfomed using anti-NFATc2 or anti-Foxp3 Ab at the IL-1RII promoter region. Enrichment of NFAT within four putative NFAT binding motifs (E) and enrichment of Foxp3 within three putative Foxp3-binding motifs (F) was analyzed by qPCR (n = 5 or 6). There are two (−1209 ~ −1188 of IL-1RII promoter), one (−649 ~ −639), and one (+10 ~ +18) binding sites for NFAT B1, B2 and B3, respectively. NFAT BC (binding control) has a binding site in human IL-2 promoter region. There are three (−4652 ~ −4611), one (−1236 ~ −1230), and one (−425 ~ −418) binding sites for Foxp3 B1, B2 and B3, respectively. Foxp3 BC (binding control) has a binding site in the human IL-2Rα promoter region (Zhang et al., 2013). Bar graphs show the mean ± SEM. * = p<0.05, ** = p<0.01, and *** = p<0.001 by two-tailed paired t-test.
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Figure 5—source data 1
Figure 5A The inhibitory effect of FOXP3 393–403, a specific inhibitor of the NFAT/FOXP3 interaction.
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Figure 5—source data 2
Figure 5C NFAT/Foxp3 interaction inhibitor significantly repress the IL-1RII expression.
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Figure 5—source data 3
Figure 5D IL-1RII promoer activity measured via luciferase assay.
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Figure 5—source data 4
Figure 5E and F Result of NFAT & Foxp3 ChIP-qPCR via IL-1RII promter.
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Figure 5—figure supplement 1
Analysis of potential binding sites of NFAT and Foxp3 in the minimal promoter of the human IL-1RII gene and schematic diagram of three luciferase reporter constructs.
CNS (conserved noncoding sequences).
Figure 5—figure supplement 2
Overexpression of Foxp3 in Jurkat cells leads to an increase in the expression of IL-1RII.
(A) Flow cytometric analysis of Foxp3 expression in Puro-control Jurkat (mock transfected: hereafter Jurkat) or Foxp3-expressing Jurkat cells. The numbers indicate the frequecy of Foxp3+ cells (B) Representative flow cytometric plot of IL-1RII and Foxp3 expression in Jurkat or Foxp3-expressing Jurkat cells upon stimulation with PMA and ionomycin. Histogram plot shows MFIs of IL-1RII expression on Jurkat cells under the indicated condtions. (C) mRNA expression of Foxp3 and IL-1RII in PMA/Ionomycin-stimulated Jurkat cells or Foxp3-expressing Jurkat cells (n = 3).
Figure 5—figure supplement 3
Cooperation of NFAT/Foxp3 via binding of NFAT on NFAT B1 site.
Control Jurkat or Foxp3-expressing Jurkat cells were stimulated with or without PMA (50 ng/ml)/ionomycin (1 μg/ml) for 2 hr. ChIP qPCR was performed using anti-NFATc2 at the IL-1RII promoter region. NFAT enrichment at NFAT B1 (−1209 ~ −1188 of IL-1RII promoter) in IL-1RII gene or NFAT BC in IL-2 gene was analyzed by qPCR. Bar graphs show the mean ± SEM. * = p<0.05 by two-tailed paired t-test.
Figure 6 with 2 supplements
Differential expression of IL-1RI and IL-1RII on activated CD4+ T cells defines unique immunological features of cells.
Treg-depleted memory CD4+ T cells were stimulated for 48 hr with anti-CD3/28-coated microbeads to induce IL-1RI and IL-1RII expression. Cells were sorted into IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- cells and cultured for another 5 days with rhIL-1β (5 ng/ml) and rhIL-2 (50 IU/ml). (A) Representative flow cytometric plot and the frequencies of IL-17- and IFN-γ-producing cells in sorted IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- memory CD4+ T cells at day 7 post-stimulation (n = 6). (B) The amount of IL-17 and IFN-γ in the culture supernatant (A) by ELISA (n = 6). (C) Representative flow cytometric plot and the frequency of IFN-γ+ cells and Foxp3+ cells in the IL-17 producing IL-1RI+IL-1RII- or IL-1RI+IL-1RII+ cells (n = 5). (D) The frequencies of Treg-related marker- or Th17-related marker-expressing cells on sorted IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- memory CD4+ T cells at day 7 post-stimulation (n = 4). Bar graphs show the mean ± SEM. * = p<0.05, ** = p<0.01, and *** = p<0.001 by two-tailed paired t-test.
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Figure 6—source data 1
Figure 6A Frequency of IL-17 & IFN-γ producing sorted IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- cells.
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Figure 6—source data 2
Figure 6B Concentraion of IL-17 & IFN-γ in the culture supernatant of sorted IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- cells.
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Figure 6—source data 3
Figure 6C Frequency of Foxp3 & IFN-γ producing cells of IL-17 producing IL-1RI+IL-1RII- and IL-1RI+IL-1RII+ cells.
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Figure 6—source data 4
Figure 6D Expression of Treg related markers on sorted L-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- cells.
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Figure 6—figure supplement 1
IL-1β-mediated changes in pathogenic Th17-cell-associated gene signature of TCR-activated memory CD4+ T cells in humans.
Treg-depleted memory CD4+ T cells were stimulated for 48 hr to induce the expression of IL-1RI and IL-1RII. IL-1RI+IL-1RII- and IL-1RI+IL-1RII+ cells were purified by cell sorting and stimulated through their TCR for 5 days in the presence of IL-1β. Expression of several pathogenic Th17 cell-associated genes in IL-1RI+IL-1RII- cells was presented relative to their expression in IL-1RI+IL-1RII+ cells. Bar graphs show the mean ± SEM (n = 3).
Figure 6—figure supplement 2
Effect of IL-23 on Th17 production in IL-1RI+IL-1RII- cells and IL-1RI+IL-1RII+ memory CD4 T cells.
(A) Sorted IL-1RI+IL-1RII-, IL-1RI+IL-1RII+, and IL-1RI-IL-1RII- cells were stimulated with anti-CD3/28-coated microbeads and IL-2 (50 IU/ml) for 5 days under the indicated cytokine conditions (5 ng/ml of IL-1β and 25 ng/ml of IL-23). The amounts of IL-17 and IFN-γ in the culture supernatant were quantified by ELSIA (n = 3). (B) Time kinetics of IL-1RI, IL-1RII, and IL-23R expression on TCR-stimulated memory CD4+ T cells (n = 3). Bar graphs and line graphs show the mean ± SEM.
Figure 7 with 1 supplement
Aberrant expression of IL-1RI and IL-RII in synovial CD4 T cells in patients with rheumatoid arthritis (RA).
(A and B) Representative flow cytometric plot (A) and the frequencies (B) of IL-1RI and IL-1RII expression on ex vivo memory CD4+ T cells from peripheral blood (n = 23) and synovial fluid (n = 15) of RA patients and peripheral blood of HCs (n = 8). SFMC: synovial fluid mononuclear cells. (C and D) Representative flow cytometric plot (C) and the frequencies (D) of IL-1RI and IL-1RII expression on TCR-stimulated memory CD4+ T cells from peripheral blood (n = 13) and synovial fluid (n = 5) of RA patients and peripheral blood of HCs (n = 6) at day 2 post-stimulation. (E) The ratio of IL-1RI+IL-1RII+ to IL-1RI+IL-1RII- cells in (D). (F and G) The effect of anti-IL-1RII neutralizing Ab treatment on IL-1β-mediated IL-17 production by TCR-stimulated memory CD4+ T cells of HCs (n = 4). Fold change indicates the ratio of IL-1β-mediated IL-17 production between the anti-IL-1RI Ab-treated group and control isotype-treated group. Scatter plot and bar graphs show the mean ± SEM. * = p<0.05, ** = p<0.01, and *** = p<0.001 by two-tailed uppaired or paired t-test.
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Figure 7—source data 1
Figure 7B Ex vivo expression of IL-1RI and IL-1RII on CD4+ T cells between HC PBMC, RA PBMC, and RA SFMC.
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Figure 7—source data 2
Figure 7D Expression of IL-1RI and IL-1RII on stimulated memory CD4+ T cells between HC PBMC, RA PBMC, and RA SFMC.
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Figure 7—source data 3
Figure 7E Expression of IL-1RI and IL-1RII on stimulated memory CD4+ T cells between HC PBMC, RA PBMC, and RA SFMC (ratio).
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Figure 7—source data 4
Figure 7F and G IL-1β-mediated IL-17 & IFN-γ production in response to TCR stimulation compared with HC and RA.
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Figure 7—figure supplement 1
Foxp3 Tregs cells in peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA) and peripheral blood of HCs.
Representative flow cytometric plot and the frequencies of IL-1RI and Foxp3 expression on ex vivo memory CD4+ T cells from peripheral blood and synovial fluid of RA patients and peripheral blood of HCs. SFMC: synovial fluid mononuclear cells. Bar graphs show the mean ± SEM. *** = p<0.005 by two-tailed paired t-test. NS indicates not significant.
Author response image 1
IL-1β-mediated changes in pathogenic and nonpathogenic gene signature of TCR-activated memory CD4+ T cells in humans.
(A) Purified memory CD4+ T cells were stimulated with anti-CD3/28 coated microbeads in the presence of PBS (as vehicle) or IL-1β (5 ng/ml) for 7 days. Expression of pathogenic or nonpathogenic genes in IL-1β-treated CD4 T cells was presented relative to their expression in PBS-treated CD4 T cells. (B) Treg-depleted memory CD4+ T cells were stimulated for 48 h to induce the expression of IL-1RI and IL-1RII. IL-1RI+IL-1RII- and IL-1RI+IL-1RII+ cells were purified by cell sorting and stimulated through their TCR for 5 days in the presence of IL-1β. Expression of pathogenic or nonpathogenic genes in IL-1RI+IL-1RII- cells was presented relative to their expression in IL-1RI+IL-1RII+ cells. Bar graphs show the mean ± SEM.
Author response image 2
Efficiency of sorting-based Treg depletion from CD4+ T cells.
Six subsets of CD4+ T cells defined by the expression of CD45RA and CD25: (I), CD25++CD45RA+ cells → resting Treg cells; (II), CD25+++CD45RA− cells → activated Treg cells; (III), CD25++CD45RA− cells → non-suppressive cytokine-producing Foxp3low T cells; (IV), CD25+CD45RA− cells; (V), CD25−CD45RA− cells; (VI), CD25−CD45RA+ cells (Miyara et al., 2009).
Author response image 3
Foxp3 Tregs cells in peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA) and peripheral blood of HCs.
(A) Representative flow cytometric plot of IL-1RI, IL-1RII, and Foxp3 expression on TCR-stimulated memory CD4+ T cells from peripheral blood of RA patients and peripheral blood of HCs at day 2 post-stimulation. The frequency of Foxp3+, IL-1RI+IL-1RII+, IL-1RI+IL-1RII- cells and ratio of IL-1RI+IL-1RII+ to IL-1RI+IL-1RII- cells. (B) Representative flow cytometric plot and frequency of IL-1RI and IL-1RII expression on TCR-stimulated memory CD4+ T cells from HCs with plasma and synovial fluid of RA patients and plasma of HCs at day 2 post-stimulation. Bar graphs show the mean ± SEM. * = p < 0.05 by two-tailed paired or unpaired t-test. NS indicates not significant.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo-sapiens) | Jurkat (clone E6-1) | ATCC | Cat# TIB-152, RRID:CVCL_0367 | |
| Cell line (Homo-sapiens) | Foxp3+ Jurkat | This paper | Foxp3-expressing cell line | |
| Transfected construct (Homo-sapiens) | pLECE3 | Lee et al., 2020 | Lentiviral expression vector | |
| Biological sample (Homo-sapiens) | Rheumatoid Arthritis patient (Blood and Synovial fluid sample) | Chungnam National University Hospital | Department of Internal Medicine | |
| Antibody | Anti-human-NFATc2 (mouse monoclonal) | Santacruz biotech | Cat# Sc-7296, RRID:AB_628012 | ChIP (2 μg/test) |
| Antibody | Anti-human-GITR PE-Cy7 (mouse monoclonal) | BioLegend | Cat# 371223, RRID:AB_2687170 | FACS (1:50) |
| Antibody | Anti-human-CD73 APC-Cy7 (mouse monoclonal) | BioLegend | Cat# 344021, RRID:AB_2566755 | FACS (1:25) |
| Antibody | Anti-human-CD39 BV421 (mouse monoclonal) | BioLegend | Cat# 328213, RRID:AB_10933084 | FACS (1:50) |
| Antibody | Anti-human-CTLA-4 PerCP-Cy5.5 (mouse monoclonal) | BioLegend | Cat# 369607, RRID:AB_2629673 | FACS (1:25) |
| Antibody | Anti-human-CXCR3 PE-Cy7 (mouse monoclonal) | BioLegend | Cat# 353719, RRID:AB_11218804 | FACS (1:50) |
| Antibody | Anti-human-CD127 BV421 (mouse monoclonal) | BioLegend | Cat# 351309, RRID:AB_10898326 | FACS (1:50) |
| Antibody | Anti-human-Foxp3 Alexa Fluor 647 (mouse monoclonal) | BioLegend | Cat# 320014, RRID:AB_439750 | FACS (1:25) |
| Antibody | Anti-human-IL-10 PE (mouse monoclonal) | eBioscience | Cat# 12-7108-81, RRID:AB_466178 | FACS (1:25) |
| Antibody | Anti-human-IL-17 PE-Cy7 (mouse monoclonal) | eBioscience | Cat# 25-7179-41, RRID:AB_11042972 | FACS (1:25) |
| Antibody | Anti-human-IFN-γ Alexa Fluor 700 (mouse monoclonal) | eBioscience | Cat# 56-7319-42, RRID:AB_2574509 | FACS (1:200) |
| Antibody | Anti- Human IL-23R Biotinylated (Goat polyclonal) | R and D systems | Cat# BAF1400, RRID:AB_355982 | FACS (1:50) |
| Antibody | Anti-human IL-1 RI PE (Goat polyclonal) | R and D systems | Cat# FAB269P, RRID:AB_2124912 | FACS (1:10) |
| Antibody | Anti-Human IL-1 RII Fluorescein-conjugated (mouse monoclonal) | R and D systems | Cat# FAB663F, RRID:AB_1964612 | FACS (1:10) |
| Antibody | Anti- Human IL-1RII (mouse monoclonal) | R and D systems | Cat# MAB263, RRID:AB_2125174 | Neutralization (20 μg/ml) |
| Antibody | Mouse IgG2A Isotype control (mouse monoclonal) | R and D systems | Cat# MAB003, RRID:AB_357345 | Neutralization control (20 μg/ml) |
| Antibody | Anti-human-CD4 v500 (mouse monoclonal) | BD Bioscience | Cat# 560768, RRID:AB_1937323 | FACS (1:50) |
| Antibody | Anti-human-CD4 APC (mouse monoclonal) | BD Bioscience | Cat# 555349, RRID:AB_398593 | FACS (1:25) |
| Antibody | Anti-human-CD3 APC-Cy7 (mouse monoclonal) | BD Bioscience | Cat# 557832, RRID:AB_396890 | FACS (1:50) |
| Antibody | Anti-human-CD25 PE-Cy5 (mouse monoclonal) | BD Bioscience | Cat# 555433, RRID:AB_395827 | FACS (1:25) |
| Antibody | Anti-human-CD25 APC (mouse monoclonal) | BD Bioscience | Cat# 561399, RRID:AB_10643029 | FACS (1:25) |
| Antibody | Anti-human-CD45RA PE-Cy7 (mouse monoclonal) | BD Bioscience | Cat# 560675, RRID:AB_1727498 | FACS (1:50) |
| Antibody | Anti-human-CD161 BV421 (mouse monoclonal) | BD Bioscience | Cat# 562615, RRID:AB_2737678 | FACS (1:25) |
| Antibody | Anti-human-CCR6 APC (mouse monoclonal) | BD Bioscience | Cat# 560619, RRID:AB_1727439 | FACS (1:25) |
| Antibody | Streptavidin conjugated Pacific Blue | BD Bioscience | Cat# 560797, RRID:AB_2033992 | FACS (1:25) |
| Antibody | Streptavidin conjugated Alexa Fluor 488 | Life technologies | Cat# S11223 | FACS (1:500) |
| Antibody | anti-human CD3 functional grade purified(mouse monoclonal) | eBioscience | Cat# 16-0037-81, RRID:AB_468854 | Functional T cell assay (0.1 ~ 10 µg/ml) |
| Antibody | Anti-human CD28 Functional Grade (mouse monoclonal) | eBioscience | Cat# 16-0289-85, RRID:AB_468927 | Functional T cell assay (1.5 µg/ml) |
| Recombinant DNA reagent | pGL4/IL-1RII-a1414 (luciferase plasmid) | This paper | Containing the IL-1RII promoter region (−1314 to +100) | |
| Recombinant DNA reagent | pGL4/IL-1RII-a814 (luciferase plasmid) | This paper | Containing the IL-1RII promoter region (−714 to +100) | |
| Recombinant DNA reagent | pGL4/IL-1RII-a215 (luciferase plasmid) | This paper | Containing the IL-1RII promoter region (−115 to +100) | |
| Recombinant DNA reagent | pGL4.74 (Renilla control) | Promega | Cat# E6921 | |
| Recombinant DNA reagent | pGL4.10 (luciferase plasmid) | Promega | Cat# E6651 | |
| Recombinant DNA reagent | pLEF-puro-FoxP3 | This paper | Foxp3 overexpression plasmid | |
| Peptide, recombinant protein | Recombinant human IL-1β | R and D systems | Cat# 201-LB-005 | |
| Peptide, recombinant protein | Recombinant human IL-6 | R and D systems | Cat# 206-IL-010 | |
| Peptide, recombinant protein | Recombinant human IL-23 | R and D systems | Cat# 1290-IL | |
| Peptide, recombinant protein | Recombinant human TGF-β1 | R and D systems | Cat# 240-B | |
| Peptide, recombinant protein | Recombinant human IL-2 | PeproTech | Cat# AF-200–02 | |
| Peptide, recombinant protein | Foxp3 393–403 | Lozano et al., 2015 (PMID:26324768) | NFAT/Foxp3 interaction inhibition peptide | |
| Peptide, recombinant protein | Foxp3 399A | Lozano et al., 2015 (PMID:26324768) | Control peptide | |
| Peptide, recombinant protein | dNP2-VIVIT | Lee et al., 2019 (PMID:31737742) | NFAT inhibition peptide (1 μM) | |
| Peptide, recombinant protein | dNP2-VEET | Lee et al., 2019 (PMID:31737742) | Control peptide (1 μM) | |
| Commercial assay or kit | QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn | genomics agilent | Cat# 200523 | |
| Commercial assay or kit | EasySep Human memory CD4+ T cell enrichment kit | STEMCELL | Cat# 19157 | |
| Commercial assay or kit | EasySep Human Naïve CD4+ T Cell Isolation Kit | STEMCELL | Cat# 19555 | |
| Commercial assay or kit | CD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit II, human | Miltenyi Biotec | Cat# 130-094-775 | |
| Commercial assay or kit | CD3/CD28 activation Dynabeads | Thermo Fisher | Cat# 11161D | |
| Commercial assay or kit | EZ-ChIP | Merck Millipore | Cat# 17–371 | |
| Commercial assay or kit | NucleoSpin gDNA clean-up kit | MACHEREY-NAGEL | Cat# 740230.50 | |
| Commercial assay or kit | Dual-Luciferase Reporter Assay System | Promega | Cat# E1910 | |
| Commercial assay or kit | GoScript Reverse Transcriptase | Promega | Cat# A5001 | |
| Commercial assay or kit | Power SYBR Green Master Mix | Bio-Rad | Cat# 4367659 | |
| Commercial assay or kit | Human IL-17A ELISA Ready-set-go | eBioscience | Cat# 88-7176-88, RRID:AB_2575036 | |
| Commercial assay or kit | Human IL-10 ELISA Ready-SET-Go! | eBioscience | Cat# 88-7106-86, RRID:AB_2575004 | |
| Commercial assay or kit | Human IFN-γ ELISA MAX Deluxe | Biolegend | Cat# 430104 | |
| Commercial assay or kit | Foxp3 Fix/Perm buffer set | Biolegend | Cat# 421403 | |
| Chemical compound, drug | Cyclosprorin A | Sigma | Cat# 30024 | 1 μM |
| Chemical compound, drug | 1a,25-dihydroxyvitamin D3 | Sigma | Cat# D1530 | 10 μM |
| Chemical compound, drug | Phobol 12-myristate 13-acetate (PMA) | Sigma | Cat# p8139 | 50 ng/ml |
| Chemical compound, drug | Ionomycin calcium salt | Sigma | Cat# I0634 | 1 μg/ml |
| Sequence-based reagent | All sequences are listed in Materials and methods. | |||
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | Promo 3.0 | ALGGEN | RRID:SCR_016926 | |
| Software, algorithm | VISTA | Joint genome institute | RRID:SCR_011808 | |
| Software, algorithm | Primer designing tool | NCBI | ||
| Other | Neon Transfection System | Thermo Fisher |
Additional files
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A two-part list of links to download the article, or parts of the article, in various formats.
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Induction of the IL-1RII decoy receptor by NFAT/FOXP3 blocks IL-1β-dependent response of Th17 cells
eLife 10:e61841.
https://doi.org/10.7554/eLife.61841
