Fig 1: Changes of genes and function in Treg cells before and after anti-IL6R treatment.(A) UMAP clustering defined by single-cell RNAseq of bead-enriched Treg cells of blood+SF samples of ICI-IA before and after anti-IL6R treatment (n = 3). (B) Distribution of AtpTreg cell cluster across treatment conditions (Tregpre and Tregpost) in blood and SF sample Tregs. (C and D) Differentially expressed genes across treatment conditions (Tregpre and Tregpost) in blood (C) and SF (D) samples. (E–H) Canonical Treg/Th17 Signature scores by clusters in blood (E and F) and in SF (G and H) Tregpre and Tregpost. ****P < 0.0001, calculated using paired Wilcoxon test. (I–K) Treg suppression assay with Tregs for Tresp from healthy controls. CFSE labelled T responder (Tresp) cells were cocultured with Tregs isolated from pretreatment and posttreatment blood samples. FlowJo figures are shown for CFSE negative cells at a Tresp‑to‑Treg ratio of 1:4 (I and J) and suppression analysis plot shown (K). (L and M) ELISA test of MMP-3 (L) and MMP-10 (M) in MH7A in coculture system, grouped by blood Tregpre and Tregpost, as compared with controls. (N) CCK-8 test of cell counts of MH7A in each group of coculture system, grouped by Tregpre and Tregpost in blood, as compared with controls. *P < 0.05, calculated using paired Wilcoxon test.
Fig 2: An atypical Treg cell type as signature of inflammatory arthritis after checkpoint blockade.(A) Research flowchart of single-cell surface proteomics of peripheral blood mononuclear cells from ICI-IA (17,323 cells, n = 5), RA-CA (17,541 cells, n = 4) and ICI-NC (18,619 cells, n = 5). (B) Annotated UMAP clustering defined by single-cell surface proteomics of blood samples of ICI-IA, RA-CA, and ICI-NC. (C) Distribution of cell clusters in each group. (D) Distribution of Treg cells per patient in each group. (E) UMAP subclustering of Treg cells (3,744 cells) from the original single-cell surface proteomics samples. (F) Signature proteins of the 2 Treg subclusters depicted as the bubble plot. (G) Distribution of Treg subclusters in each group, showing ICI-IA group has high percentage cluster # 0. (H) Research flowchart of single-cell RNA-seq (11,864 cells) of bead-enriched Treg cells. (I) UMAP clustering defined by single-cell RNA-seq of blood Treg cells of all samples. (J) Signature proteins. (K) Distribution of single-cell RNA-seq–defined cell clusters in each group. (L) GSEA of differential genes in cluster # 2. (M) Th17 scoring in each cluster. (N) Heatmap of average expression of each marker of single-cell secreting proteomics of AtpTreg and other Tregs per sample. (O and P) Treg suppression assay with Tregs for Tresp from healthy controls. FlowJo figures are shown for CFSE negative cells at a Tresp‑to‑Treg ratio of 1:4. (O) and suppression analysis plot shown (P). (Q) OLINK proteomics of human synovial lines (MH7A) cocultured with blood-derived Treg cells of patients with ICI-IA, as compared with human synovial lines (MH7A) without coculture system. (R and S) ELISA test of stromelysin MMP-10 (R) and stromelysin MMP-3 (S) in MH7A in coculture system. (T) CCK-8 test of cell counts of MH7A in coculture system. **P < 0.01, ***P < 0.001, calculated using an independent Wilcoxon test. *P < 0.05, **P < 0.01, ***P < 0.001, calculated with independent Wilcoxon signed-rank test.
Supplier Page from Abcam for Human MMP10 ELISA Kit