IL-1R1 is expressed on both Helios+ and Helios−FoxP3+CD4+ T cells in the rheumatic joint
Corresponding Author
M. Müller
Rheumatology Unit, Department of Medicine, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden
Correspondence: M. Müller, Rheumatology Research Unit, CMM L8:04, Karolinska University Hospital, 171 76 Stockholm, Sweden. E-mail: [email protected]Search for more papers by this authorJ. Herrath
Rheumatology Unit, Department of Medicine, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden
Search for more papers by this authorV. Malmström
Rheumatology Unit, Department of Medicine, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden
Search for more papers by this authorCorresponding Author
M. Müller
Rheumatology Unit, Department of Medicine, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden
Correspondence: M. Müller, Rheumatology Research Unit, CMM L8:04, Karolinska University Hospital, 171 76 Stockholm, Sweden. E-mail: [email protected]Search for more papers by this authorJ. Herrath
Rheumatology Unit, Department of Medicine, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden
Search for more papers by this authorV. Malmström
Rheumatology Unit, Department of Medicine, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden
Search for more papers by this authorSummary
Synovial fluid from rheumatic joints displays a well-documented enrichment of forkhead box protein 3 (FoxP3)+ regulatory T cells (tissue Tregs). However, we have previously demonstrated that the mere frequency of FoxP3 expressing cells cannot predict suppressive function. Instead, extrinsic factors and the functional heterogeneity of FoxP3+ Tregs complicate the picture. Here, we investigated FoxP3+ Tregs from blood and synovial fluid of patients with rheumatic disease in relation to Helios expression by assessing phenotypes, proliferative potential and cytokine production by flow cytometry. Our aim was to investigate the discriminatory potential of Helios when studying FoxP3+ Tregs in an inflammatory setting. We demonstrate that the majority of the synovial FoxP3+CD4+ T cells in patients with inflammatory arthritis expressed Helios. Helios+FoxP3+ Tregs displayed a classical Treg phenotype with regard to CD25 and cytotoxic T lymphocyte-associated antigen (CTLA)-4 expression and a demethylated Treg-specific demethylated region (TSDR). Furthermore, Helios+FoxP3+ T cells were poor producers of the effector cytokines interferon (IFN)-γ and tumour necrosis factor (TNF), as well as of the anti-inflammatory cytokine interleukin (IL)-10. The less abundant Helios–FoxP3+ T cell subset was also enriched significantly in the joint, displayed a overlapping phenotype to the double-positive Treg cells with regard to CTLA-4 expression, but differed by their ability to secrete IL-10, IFN-γ and TNF upon T cell receptor (TCR) cross-linking. We also demonstrate a striking enrichment of IL-1R1 expression in synovial CD4+ T cells that was restricted to the CD25-expressing FoxP3 population, but independent of Helios. IL-1R1 expression appears to define a tissue Treg cell phenotype together with the expression of CD25, glucocorticoid-induced TNF receptor family-related gene (GITR) and CTLA-4.
Supporting Information
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| cei12668-sup-0001-suppinfo01.pptx349.6 KB |
Fig. S1. Synovial Helios+ and Helios− forkhead box protein 3 (FoxP3)+ T cells show the highest degree of CCR4 expression. The dot-plots show representative stainings of CXCR3, CCR6, CCR4 and CCR7 expression on CD4 T cells plotted against FoxP3 in paired synovial fluid (SF) (a) and peripheral blood (PB) (b) samples. The summary graphs display the frequency of CXCR3-, CCR6-, CCR4- and CCR7-positive Helios+/−FoxP3+ T cells as well as Helios+/−FoxP3– T cells in paired synovial fluid (SF) (a) and peripheral blood (PB) (b) samples [n = 9 diagnosis: ankylosing spondylitis (SpA)]. The data were compared using the Kruskal–Wallis test together with Dunn's multiple comparison post-test, significance: ***P < 0·001, **P < 0·005 and *P < 0·05. |
| cei12668-sup-0002-suppinfo02.pptx446.2 KB |
Fig. S2. Co-expression of interleukin (IL)-1R1 and CCR7 enriches for Helios−forkhead box protein 3 (FoxP3)+ cells in peripheral blood (PB) but not in synovial fluid (SF). The summary graphs show the frequencies of Helios−FoxP3+ in PB (a) and SF (b) and the frequencies of Helios+FoxP3+ T cells in PB (c) and SF (d) within CD4+CD25++ cells or in the different CCR7/IL-1R1 cell subpopulations within the CD4+CD25++ cells. Representative fluorescence activated cell sorter (FACS) plots of paired cell samples of PB (e) and SF (f) show the application of the CCR7/IL-1R1 marker combination in distinguishing Helios subsets within CD4+CD25+ cells [n = 4, diagnosis ankylosing spondylitis (SpA)]. The summary graphs (g,h) depict the frequencies of Helios–FoxP3+ cells gained when gating on CD25++IL-1R1+ cells compared to using a CD25++ gate alone, in PB (g) and SF (h) [n = 13, eight SpA and five rheumatoid arthritis (RA)]. For data analysis, the Kruskal–Wallis test together with Dunn's multiple comparison post-test was used when comparing several groups and grouped data were compared using the Wilcoxon's signed-rank test, **P < 0·001 and ***P < 0·0001. |
| cei12668-sup-0003-suppinfo03.pptx157.8 KB |
Fig. S3. Interleukin (IL)-1R1 displays similar expression patterns in peripheral blood (PB) and synovial fluid (SF) joints of both rheumatoid arthritis (RA) and ankylosing spondylitis (SpA). The graphs summarize flow cytometric analysis of the frequencies of IL-R1+forkhead box protein 3 (FoxP3)+ cells in paired PB and SF divided into patients diagnosed with SpA and RA (a). Frequencies of IL-1R1 expressing Helios+ and Helios−FoxP3+ cells of each subpopulation is shown for SpA (b) and RA (c) and the frequencies of CD25++ cells in the IL-1R1+FoxP3+ cell population in PB and SF from patients diagnosed with SpA and RA, respectively (d) [n = 14, nine SpA and five RA]. Data were compared using Wilcoxon's signed-rank test, **P < 0·001 and ***P < 0·0001. |
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