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: For mechanistic validation, parallel cultures of 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL) with FLS were supplemented with either maraviroc (5 μM; CCR5 antagonist, MCE, Cat#HY-13004) or BX471 (5 μM; CCR1 antagonist, MCE, Cat#HY-12080) to assess chemokine receptor involvement; Separately, identical cultures of 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL) with FLS were treated with tofacitinib (1 μM; JAK inhibitor, MCE, Cat#HY-40354 A) to evaluate JAK/STAT pathway activation.
Techniques: Activation Assay, Selection, Expressing, Two Tailed Test, Flow Cytometry, Transwell Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control
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