Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: Single-cell profiling of PBMCs from RA patients and HC revealed distinct clustering patterns of CD20 + T cell populations. a t-SNE plot of CD20 + T cells from PBMCs of RA patients ( n = 9) and HC ( n = 3), colored by group. b tSNE plot (left panel) and subgroup proportion pie chart (right panel) of CD20 + T cells from all donors. Subgroups were divided based on the expression levels of marker genes CD4 or CD8A . c tSNE plot of CD20 + T cells from PBMCs of all donors, showing 3 clusters based on the unsupervised graph-based clustering. d Dot plot for expression of marker genes for three CD20 + T cell subtypes. The color of the dots shows the relative expression levels of genes, and the size of the dots shows the percentage of expressed cells. e Heatmap showing expression levels of discriminative genes for each cluster. Cluster 0 prominently features high expression of CCL5 , NKG7 and GNLY ; Cluster 1 is defined by combined enrichment of CCR7 , IL6ST and TRABD2A ; Cluster 2 shows distinct expression of GATA3 , ITGB1 and S100A11 . f Feature plots of single-cell expression for identified genes across three CD20 + T cell clusters

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Expressing, Marker

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: The proportion of CCL5 + T cells in CD20 + CD8 + T cells was significantly elevated in naïve RA patients. a Schematic representation of gating strategy for flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) derived from HC and RA patients. b The frequencies of CD20 + CD8 + T cells were analyzed in PBMCs samples collected from HC ( n = 63) and naïve RA patients ( n = 69). Data shown as median with interquartile range (IQR), Mann-Whitney U test, **** P < 0.0001. c The frequencies of CCL5 + cells within CD20 + CD8 + T cells were shown for HC ( n = 32) and naïve RA patients ( n = 35). Data shown as median with interquartile range (IQR), Mann-Whitney U test, **** P < 0.0001. d The proportion of CCL5 + cells was significantly higher in CD20 + CD8 + T cells than in CD20 – CD8 + T cells in both HC and RA patients. Data shown as median with interquartile range (IQR), Wilcoxon matched-pairs signed-rank test, **** P < 0.0001. e The proportion of CCL5 + cells within CD20 + CD8 + T cells positively correlated with inflammation and disease activity of RA. Data shown as individual scatter points, Spearman’s rank correlation, P < 0.05. f KEGG pathway analysis revealed that the top five most significantly enriched pathways for DEGs between CD20 + CD8 + T and CD20 – CD8 + T cells included the chemokine signaling pathway, and CXCR3 and CCR5 were identified as genes significantly enriched in this pathway. g Violin plots (upper panel) demonstrated that the expression levels of CXCR3 and CCR5 genes were significantly upregulated in CD20 + CD8 + T cells compared to CD20 – CD8 + T cells. **** P < 0.0001; * P < 0.05. UMAP plot (lower panel) visualized the distribution of CXCR3 + or CCR5 + cells among CD8 + T cells. ( h Flow cytometric analysis demonstrated significantly higher proportions of CXCR3 + or CCR5 + cells among CD20 + CD8 + T cells compared to CD20 – CD8 + T cells in PBMCs from RA patients ( n = 27 in each group). Wilcoxon matched-pairs signed-rank test, **** P < 0.0001

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Derivative Assay, MANN-WHITNEY, Activity Assay, Expressing

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: CD20 + CD8 + T cells may promote the expansion of invasive FAPα + FLS through CCL5-mediated activation of the JAK-STAT signaling pathway. a Gating strategy for identifying FLS involved sequential selection of CD45-negative (CD45 – ) cells followed by positive selection for PDPN (podoplanin) expression. The frequencies of FAPα + cells in the PDPN + CD45 – CD31 – population were quantified and compared between normal human controls (NH, n = 9) and RA patients ( n = 9). (mean ± SEM, unpaired two-tailed t -test, *** P < 0.001). b Representative flow cytometry plots depict FAPα + cells (gated on PDPN + CD45 – CD31 – populations) with corresponding quantitative analysis following 48-hour treatment under various experimental conditions. (mean ± SEM, n = 7 in each group, one-way ANOVA with Tukey’s post hoc test, * P < 0.05, **** P < 0.0001). c Representative images from the Transwell assay demonstrate the migratory capacity of FLS under the experimental conditions corresponding to panel (b). Scale bar: 50 μm. (mean ± SEM, n = 3 in each group, one-way ANOVA with Tukey’s post hoc test, * P < 0.05; *** P < 0.001). d The frequency of FAPα-expressing FLS was assessed by flow cytometry following 48-hour incubation with CCR1 antagonist BX471 (5 µM) or CCR5 antagonist maraviroc (5 µM) in IL-1β (5 ng/mL) + CD20 + CD8 + T-CM cultures. (mean ± SEM, n = 6 in each group, one-way ANOVA with Tukey’s post hoc test, * P < 0.05; ** P < 0.01). e Representative histogram plots demonstrate FAPα expression in FLS following stimulation with IL-1β (5 ng/mL) in combination with either CD20 + CD8 + T-CM alone, or T-CM + tofacitinib (JAK inhibitor). (mean ± SEM, n = 6 in each group, paired t- test, *** P < 0.001). ( f ) ELISA was performed to quantify CCL5 concentrations in culture supernatants under experimental conditions corresponding to panels (B) and (C). (mean ± SEM, n = 6 in each group, one-way ANOVA with Welch’s correction, followed by Dunnett’s T3 post hoc test, ** P < 0.01). g Heatmap depicting transcriptomic profiling results, showing expression levels of differentially expressed genes across experimental groups. h GO (upper panel) and KEGG pathway (lower panel) analyses of upregulated genes in FLS treated with CD20 – CD8 + T-CM + IL-1β (left), and FLS treated with CD20 + CD8 + T-CM + IL-1β (right), both compared to IL-1β alone control (CTL). q -value < 0.05

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Activation Assay, Selection, Expressing, Two Tailed Test, Flow Cytometry, Transwell Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: Schematic of CCL5 + CD20 + CD8 + T cells driving synoviocyte transformation. In RA, circulating CD20 + CD8 + T cells with high co-expression of chemokine receptors CXCR3 and CCR5 are chemoattracted from peripheral blood into synovial tissues by cognate chemokines (such as CXCL9 and CCL5). CD20 + CD8 + T cells further engage in crosstalk with fibroblast-like synoviocytes (FLS) through CCL5 secretion. This chemokine-dependent signaling engages cognate receptors (i.e., CCR1, CCR5) on FLS, subsequently activating the JAK/STAT signaling cascade, inducing phenotypic transformation of FLS (PDPN + CD45 – CD31 – ) into pathogenic FAPα + subpopulation. The FAPα + subpopulation demonstrates enhanced proliferative capacity, increased invasive migration, and robust secretion of synovial inflammatory cytokines, chemokines, and pro-angiogenic factors, collectively driving pannus formation and progressive joint destruction. ECM: extracellular matrix; MMPs: matrix metalloproteinases; TNFα: tumor necrosis factor α.

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Transformation Assay, Expressing, Migration