Fig. 3

Regulation of T-cell proliferation by lymph node stromal cells (LNSCs). a Flow cytometry gating strategy used to identify CD4⁺ and CD8⁺ T-cell subsets according to CD45, CD4 and CD8 expression. Numbers adjacent to the outlined areas indicate percentages of cells in the gated population. A representative carboxyfluorescein succinimidyl ester (CSFE) dilution plot is shown for 2 of 15 donors. b Proliferation of CSFE-labelled CD4⁺ and CD8⁺ T cells out of 50,000 peripheral blood mononuclear cell (PBMCs) (all from one donor) activated with αCD3 and αCD28 for 96 h without LNSCs (passages 4 to 8) or co-cultured with 1250 (1:40), 5000 (1:10), 10,000 (1:5) or 25,000 LNSCs (1:2). LNSCs were cultured from healthy donors, individuals with RA risk and patients with RA and pre-treated with interferon-γ (IFN-γ) or not. Data are presented as the percentage of total cells found in the respective cell division (mean and SD of n = 5 donors per group; donor characteristics listed in Table 3). Upper panels show CD4⁺ T-cell data; lower panels show data of CD8⁺ T cells displayed co-cultured together with LNSCs of respective donor groups. Differences between T cell with or without LNSCs were assessed using two-way analysis of variance ANOVA. * P < 0.050, ** P < 0.010, *** P < 0.001, **** P < 0.0001 c Nitric oxide (NO) production in co-culture supernatants was measured after 96 h of co-culture using Griess reagent. Mean and SD of 10 donors (healthy, n = 5; individuals with RA risk, n = 3; patients with RA, n = 2) are shown. Differences between conditions were assessed using two-way ANOVA. * P < 0.050, ** P < 0.010. FSC-A Forward scatter area, FSC-W Forward scatter width, SSC-A Side scatter area, SSC-W Side scatter width