1. Immunology and Inflammation
Download icon

Induction of the IL-1RII decoy receptor by NFAT/FOXP3 blocks IL-1β-dependent response of Th17 cells

  1. Dong Hyun Kim
  2. Hee Young Kim
  3. Sunjung Cho
  4. Su-Jin Yoo
  5. Won-Ju Kim
  6. Hye Ran Yeon
  7. Kyungho Choi
  8. Je-Min Choi
  9. Seong Wook Kang
  10. Won-Woo Lee  Is a corresponding author
  1. Laboratory of Autoimmunity and Inflammation (LAI), Department of Biomedical Sciences, Seoul National University College of Medicine, Republic of Korea
  2. Department of Microbiology and Immunology, Seoul National University College of Medicine, Republic of Korea
  3. Cancer Research Institute and Institute of Infectious Diseases, Seoul National University College of Medicine, Republic of Korea
  4. Department of Internal Medicine, Chungnam National University School of Medicine, 282 Munhwa-ro, Jung-gu, Republic of Korea
  5. Department of Life Science, College of Natural Sciences and Research Institute for Natural Sciences, Hanyang University, Republic of Korea
  6. Department of Biochemistry and Molecular Biology, Department of Biomedical Sciences, and Cancer Research Institute, Seoul National University College of Medicine, Republic of Korea
  7. Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine; Seoul National University Hospital Biomedical Research Institute, Republic of Korea
Research Article
Cite this article as: eLife 2021;10:e61841 doi: 10.7554/eLife.61841
10 figures, 1 table and 1 additional file

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.

Figure 1—source data 1

Figure 1B IL-1β significantly enhance IL-17 & IFN-γ producing memory CD4+ T cells.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig1-data1-v2.xlsx
Figure 1—source data 2

Figure 1C IL-1β regulated IL-10 producing memory CD4+ T cells.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig1-data2-v2.xlsx
Figure 1—source data 3

Figure 1D Th17-polarizing cytokines intensify the Th17 response of memory CD4+ T cells.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig1-data3-v2.xlsx
Figure 1—source data 4

Figure 1E IL-1β is important for promoting Th17 differentiation form naïve CD4+ T cells.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig1-data4-v2.xlsx
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.

Figure 2—source data 1

Figure 2A Ex vivo expression of IL-1RI & IL-1RII on CD4+ T cells.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig2-data1-v2.xlsx
Figure 2—source data 2

Figure 2B Ex vivo expression of IL-1RI between naïve and memory CD4+ T cells.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig2-data2-v2.xlsx
Figure 2—source data 3

Figure 2B Ex vivo expression of IL-1RI between naïve and memory CD4+ T cells.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig2-data3-v2.xlsx
Figure 2—source data 4

Figure 2D Time kinetics of IL-1RI & IL-1RII.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig2-data4-v2.xlsx
Figure 2—source data 5

Figure 2E Effect of TCR signaling strength on expression of IL-1RI & IL-1RII.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig2-data5-v2.xlsx
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).

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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig3-data1-v2.xlsx
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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig3-data2-v2.xlsx
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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig3-data3-v2.xlsx
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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig3-data4-v2.xlsx
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.

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.

Figure 4—source data 1

Figure 4A NFAT inhibitor CsA selectivley repress the expression of IL-1RII.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig4-data1-v2.xlsx
Figure 4—source data 2

Figure 4B NFAT inhibiton peptide VIVIT repress the expression of IL-1RII.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig4-data2-v2.xlsx
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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig4-data3-v2.xlsx
Figure 4—source data 4

Figure 4H Ratio of IL-1RII+/IL-1RI+.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig4-data4-v2.xlsx
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.

Figure 5—source data 1

Figure 5A The inhibitory effect of FOXP3 393–403, a specific inhibitor of the NFAT/FOXP3 interaction.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig5-data1-v2.xlsx
Figure 5—source data 2

Figure 5C NFAT/Foxp3 interaction inhibitor significantly repress the IL-1RII expression.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig5-data2-v2.xlsx
Figure 5—source data 3

Figure 5D IL-1RII promoer activity measured via luciferase assay.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig5-data3-v2.xlsx
Figure 5—source data 4

Figure 5E and F Result of NFAT & Foxp3 ChIP-qPCR via IL-1RII promter.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig5-data4-v2.xlsx
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.

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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig6-data1-v2.xlsx
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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig6-data2-v2.xlsx
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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig6-data3-v2.xlsx
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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig6-data4-v2.xlsx
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.

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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig7-data1-v2.xlsx
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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig7-data2-v2.xlsx
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).

https://cdn.elifesciences.org/articles/61841/elife-61841-fig7-data3-v2.xlsx
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.

https://cdn.elifesciences.org/articles/61841/elife-61841-fig7-data4-v2.xlsx
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), CD25CD45RA cells; (VI), CD25CD45RA+ 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 resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo-sapiens)Jurkat (clone E6-1)ATCCCat# TIB-152, RRID:CVCL_0367
Cell line (Homo-sapiens)Foxp3+ JurkatThis paperFoxp3-expressing cell line
Transfected construct (Homo-sapiens)pLECE3Lee et al., 2020Lentiviral expression vector
Biological sample (Homo-sapiens)Rheumatoid Arthritis patient (Blood and Synovial fluid sample)Chungnam National University HospitalDepartment of Internal Medicine
AntibodyAnti-human-NFATc2 (mouse monoclonal)Santacruz biotechCat# Sc-7296,
RRID:AB_628012
ChIP
(2 μg/test)
AntibodyAnti-human-GITR PE-Cy7 (mouse monoclonal)BioLegendCat# 371223, RRID:AB_2687170FACS
(1:50)
AntibodyAnti-human-CD73 APC-Cy7 (mouse monoclonal)BioLegendCat# 344021, RRID:AB_2566755FACS
(1:25)
AntibodyAnti-human-CD39 BV421 (mouse monoclonal)BioLegendCat# 328213, RRID:AB_10933084FACS
(1:50)
AntibodyAnti-human-CTLA-4 PerCP-Cy5.5 (mouse monoclonal)BioLegendCat# 369607, RRID:AB_2629673FACS
(1:25)
AntibodyAnti-human-CXCR3 PE-Cy7 (mouse monoclonal)BioLegendCat# 353719, RRID:AB_11218804FACS
(1:50)
AntibodyAnti-human-CD127 BV421 (mouse monoclonal)BioLegendCat# 351309, RRID:AB_10898326FACS
(1:50)
AntibodyAnti-human-Foxp3 Alexa Fluor 647 (mouse monoclonal)BioLegendCat# 320014, RRID:AB_439750FACS
(1:25)
AntibodyAnti-human-IL-10 PE (mouse monoclonal)eBioscienceCat# 12-7108-81, RRID:AB_466178FACS
(1:25)
AntibodyAnti-human-IL-17 PE-Cy7 (mouse monoclonal)eBioscienceCat# 25-7179-41, RRID:AB_11042972FACS
(1:25)
AntibodyAnti-human-IFN-γ Alexa Fluor 700 (mouse monoclonal)eBioscienceCat# 56-7319-42, RRID:AB_2574509FACS
(1:200)
AntibodyAnti- Human IL-23R Biotinylated (Goat polyclonal)R and D systemsCat# BAF1400, RRID:AB_355982FACS
(1:50)
AntibodyAnti-human IL-1 RI PE (Goat polyclonal)R and D systemsCat# FAB269P, RRID:AB_2124912FACS
(1:10)
AntibodyAnti-Human IL-1 RII Fluorescein-conjugated (mouse monoclonal)R and D systemsCat# FAB663F, RRID:AB_1964612FACS
(1:10)
AntibodyAnti- Human IL-1RII (mouse monoclonal)R and D systemsCat# MAB263, RRID:AB_2125174Neutralization
(20 μg/ml)
AntibodyMouse IgG2A Isotype control (mouse monoclonal)R and D systemsCat# MAB003, RRID:AB_357345Neutralization control
(20 μg/ml)
AntibodyAnti-human-CD4 v500 (mouse monoclonal)BD BioscienceCat# 560768, RRID:AB_1937323FACS
(1:50)
AntibodyAnti-human-CD4 APC (mouse monoclonal)BD BioscienceCat# 555349, RRID:AB_398593FACS
(1:25)
AntibodyAnti-human-CD3 APC-Cy7 (mouse monoclonal)BD BioscienceCat# 557832, RRID:AB_396890FACS
(1:50)
AntibodyAnti-human-CD25 PE-Cy5 (mouse monoclonal)BD BioscienceCat# 555433, RRID:AB_395827FACS
(1:25)
AntibodyAnti-human-CD25 APC (mouse monoclonal)BD BioscienceCat# 561399, RRID:AB_10643029FACS
(1:25)
AntibodyAnti-human-CD45RA PE-Cy7 (mouse monoclonal)BD BioscienceCat# 560675, RRID:AB_1727498FACS
(1:50)
AntibodyAnti-human-CD161 BV421 (mouse monoclonal)BD BioscienceCat# 562615, RRID:AB_2737678FACS
(1:25)
AntibodyAnti-human-CCR6 APC (mouse monoclonal)BD BioscienceCat# 560619, RRID:AB_1727439FACS
(1:25)
AntibodyStreptavidin conjugated Pacific BlueBD BioscienceCat# 560797, RRID:AB_2033992FACS
(1:25)
AntibodyStreptavidin conjugated Alexa Fluor 488Life technologiesCat#
S11223
FACS
(1:500)
Antibodyanti-human CD3 functional grade purified(mouse monoclonal)eBioscienceCat# 16-0037-81, RRID:AB_468854Functional T cell assay
(0.1 ~ 10 µg/ml)
AntibodyAnti-human CD28 Functional Grade (mouse monoclonal)eBioscienceCat# 16-0289-85, RRID:AB_468927Functional T cell assay
(1.5 µg/ml)
Recombinant DNA reagentpGL4/IL-1RII-a1414 (luciferase plasmid)This paperContaining the IL-1RII promoter region (−1314 to +100)
Recombinant DNA reagentpGL4/IL-1RII-a814 (luciferase plasmid)This paperContaining the IL-1RII promoter region (−714 to +100)
Recombinant DNA reagentpGL4/IL-1RII-a215 (luciferase plasmid)This paperContaining the IL-1RII promoter region (−115 to +100)
Recombinant DNA reagentpGL4.74 (Renilla control)PromegaCat# E6921
Recombinant DNA reagentpGL4.10 (luciferase plasmid)PromegaCat# E6651
Recombinant DNA reagentpLEF-puro-FoxP3This paperFoxp3 overexpression plasmid
Peptide, recombinant proteinRecombinant human IL-1βR and D systemsCat# 201-LB-005
Peptide, recombinant proteinRecombinant human IL-6R and D systemsCat# 206-IL-010
Peptide, recombinant proteinRecombinant human IL-23R and D systemsCat# 1290-IL
Peptide, recombinant proteinRecombinant human TGF-β1R and D systemsCat# 240-B
Peptide, recombinant proteinRecombinant human IL-2PeproTechCat# AF-200–02
Peptide, recombinant proteinFoxp3 393–403Lozano et al., 2015
(PMID:26324768)
NFAT/Foxp3 interaction inhibition peptide
Peptide, recombinant proteinFoxp3 399ALozano et al., 2015
(PMID:26324768)
Control peptide
Peptide, recombinant proteindNP2-VIVIT
Lee et al., 2019
(PMID:31737742)
NFAT inhibition peptide
(1 μM)
Peptide, recombinant proteindNP2-VEETLee et al., 2019
(PMID:31737742)
Control peptide
(1 μM)
Commercial assay or kitQuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxngenomics agilentCat# 200523
Commercial assay or kitEasySep Human memory CD4+ T cell enrichment kitSTEMCELLCat# 19157
Commercial assay or kitEasySep Human Naïve CD4+ T Cell Isolation KitSTEMCELLCat# 19555
Commercial assay or kitCD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit II, humanMiltenyi BiotecCat# 130-094-775
Commercial assay or kitCD3/CD28 activation DynabeadsThermo FisherCat# 11161D
Commercial assay or kitEZ-ChIPMerck MilliporeCat# 17–371
Commercial assay or kitNucleoSpin gDNA clean-up kitMACHEREY-NAGELCat# 740230.50
Commercial assay or kitDual-Luciferase Reporter Assay SystemPromegaCat# E1910
Commercial assay or kitGoScript Reverse TranscriptasePromegaCat# A5001
Commercial assay or kitPower SYBR Green Master MixBio-RadCat# 4367659
Commercial assay or kitHuman IL-17A ELISA Ready-set-goeBioscienceCat# 88-7176-88, RRID:AB_2575036
Commercial assay or kitHuman IL-10 ELISA Ready-SET-Go!eBioscienceCat# 88-7106-86, RRID:AB_2575004
Commercial assay or kitHuman IFN-γ ELISA MAX DeluxeBiolegendCat# 430104
Commercial assay or kitFoxp3 Fix/Perm buffer setBiolegendCat# 421403
Chemical compound, drugCyclosprorin ASigmaCat# 300241 μM
Chemical compound, drug1a,25-dihydroxyvitamin D3SigmaCat# D153010 μM
Chemical compound, drugPhobol 12-myristate 13-acetate (PMA)SigmaCat# p813950 ng/ml
Chemical compound, drugIonomycin calcium saltSigmaCat# I06341 μg/ml
Sequence-based reagentAll sequences are listed in Materials and methods.
Software, algorithmPrismGraphPadRRID:SCR_002798
Software, algorithmPromo 3.0ALGGENRRID:SCR_016926
Software, algorithmVISTAJoint genome instituteRRID:SCR_011808
Software, algorithmPrimer designing toolNCBI
OtherNeon Transfection SystemThermo Fisher

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)