Fig. 2

Frequencies of SIGIRR-positive memory CD4 T cells in PBMCs of patients with RA and the healthy population. Related to Additional file 1: Figure S2. A, B Gating strategies used to analyze SIGIRR expression in memory CD4 T cells in human peripheral blood mononuclear cells (PBMCs). Memory CD4 T cells were identified as CD4 ⁺ CD45RO ⁺ CD45RA ⁻ previously gated on single live (FVS780 ⁻) lymphocytes (FSC ^low SSC ^low). Representative flow cytometry histogram plots showing SIGIRR expression by memory CD4 T cells in human PBMCs in healthy and RA cohorts (n = 50 for healthy and 78 for RA individuals), with quantification of results as frequency (B). C Linear regression analysis of the proportion of SIGIRR in memory CD4⁺ T cells and disease activity score 28 (DAS28) of 78 RA patients (r =  − 0.8928, P < 0.0001, n = 78). D, E SIGIRR⁻ memory CD4 T cells in RA PBMCs (n = 4) were flow-sorted and transduced with retroviral empty vectors (empty) or vectors encoding SIGIRR (SIGIRR OE). Then, SIGIRR⁻ memory CD4 T cells (1 × 10⁶ cells/mL) were treated with IL-1β in the presence of α-CD3/α-CD28 antibodies for 48 h. TNF-α production in the supernatants of the SIGIRR⁻ memory CD4⁺ T cell cultures stimulated with was measured by ELISA (D), and the relative expression of TNF-α was monitored by quantitative real-time RT‒PCR (E). Each symbol B, C represents an individual human; small horizontal lines indicate the median ± interquartile (B). *P < 0.05 and ****P < 0.0001, Mann‒Whitney test (B), Spearman’s correlation (C) or paired t test (D, E)