Fig. 3. Immunofibroblast ICOSL contributes to TLS establishment.

a Histogram showing flow cytometry staining for ICOSL expression on dendritic cells (CD45+ CD11c+ cells) and immunofibroblasts (CD45-EpCAM-CD31-pdpn+) in wt mice at day 0 (blue line) and day 5 p.c. (green line), and isotype control (red line). b icosl mRNA expression in FACS sorted CD45-EpCAM-CD31-pdpn+ immunofibroblasts (black bars), CD45-EpCAM-CD31-pdpn− fibroblasts (grey bars) at day 0, 5 and 8 p.c. and sorted activated dendritic cells (DCs) cells at day 8 p.c. (white bar). mRNA transcripts were assessed by qPCR and normalized to housekeeping gene β-actin. RQ values were calculated with calibrator day 0 pdpn- fibroblasts. c, d Absolute numbers of T cells (c) and LTα-producing T cells (d) in wt (black bars), Icosl^−/− mice reconstituted with wt bone marrow (grey bars) and wt mice reconstituted with Icosl^−/− bone-marrow (bm) (white bars) at day 5 p.c were determined by flow cytometry. e, f Quantitative real time PCR analysis of mRNA transcripts of ltα (e) and ccl19 (f) in wt (black bars), Icosl^−/− mice reconstituted with wt bone-marrow (grey bars) and wt mice reconstituted with Icosl^−/− bone marrow (white bars). mRNA transcripts were normalized to housekeeping gene β-actin. g, h FACS sorted wt T cells were either co-cultured directly (g) or in trans-wells (h) with either wt or Icosl^−/− CD45-EpCAM-CD31-pdpn+ fibroblasts and dendritic cells (DCs) isolated at day 0 p.c. additionally stimulated with adenovirus. Graphs qPCR analysis of ltα mRNA transcripts normalized to the housekeeping gene β-actin are presented as relative quantitation (RQ) values calibrated to T cells cultured alone and the asterisks show significance as compared to T cells cultured alone. Data are mean ± s.e.m from two independent experiments with three to six mice analyzed per group. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. non-significant, one-way ANOVA with Dunnett’s multiple comparison test (b, g, h) and one-way ANOVA with Tukey’s multiple comparison test (c–f).