CD4 T cells isolated from IL-1R1^ΔT mice do not respond to IL-1β administration <i>in vitro</i>.

<p>(A) <i>In vitro</i> proliferation assay of CD4 T cells labeled with violet cell tracer (VCT) and activated with the indicated stimuli (FACS histograms, gated on VD^-/CD4⁺ cells). (B) Proliferation index of cultures supplemented with indicated stimuli as shown in (A). (C) Total numbers of live CD4 T cells harvested from cultures supplemented with indicated stimuli as shown in (A). MACS-purified CD4 T cells from control and IL-1R1^ΔT mice (n = 4 of each genotype) were cultured 4 days under different indicated conditions. Data are (A) representative FACS histograms of control (black) and IL-1R1 deficient (blue) cultures and (B, C) individual values with mean. Experiments were performed twice with the similar results. *p < 0.05, **p < 0.01, ***p < 0.001, N. S.–not significant; two-tailed unpaired t-test.</p

CD4 T cells isolated from IL-1R1^ΔT mice display non-impaired differentiation <i>in vitro</i>.

<p><i>In vitro</i> polarization assay of MACS-purified CD4 T cells activated under Th0, Th17 and Th1 conditions (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161505#sec002" target="_blank">materials and methods</a> section) for 4 days. Data are shown as representative FACS plots gated on VD^-/CD4⁺ cells with average frequencies per group and as mean +SEM of n = 3 of each genotype. Experiments were performed twice with similar results.</p

T cell specific deletion of IL-1R1 results in impaired Th17 cell expansion in anti-CD3 treatment model.

<p>(A) Analysis of activation status of CD4 T cells isolated from the spleen after anti-CD3 treatment at indicated time points. Data are shown as representative FACS plots gated on VD^-/CD90.2⁺/CD4⁺ cells with mean frequencies per group ± average deviation; and as mean +SEM of n = 4 of each genotype. (B-C) Analysis of cytokine expression by CD4 T cells isolated from the (B) spleen and (C) IEL compartment of the small intestine after anti-CD3 treatment at indicated time points. Data (B, C) are shown as representative FACS plots gated on VD^-/CD90.2⁺/CD4⁺ cells with mean frequencies per group and as mean +SEM of n = 4 control and n = 5 IL-1R1^ΔT mice analyzed at 48 h, as well as of n = 3 control and n = 4 IL1R1^ΔT mice analyzed at 100 h. Experiments were performed twice with similar results. *p < 0.05; two-tailed unpaired t-test.</p

Conditional deletion of IL-1R1 in T cells.

<p>(A) Schematic representation of targeted murine <i>Il1r1</i> allele with exon 5 flanked by loxP sites. Cre mediated recombination results in excision of exon 5 and leads to the deletion of <i>Il1r1</i> gene. Open squares—numbered exons, triangles—loxP sites. All components are out of scale. (B) FACS analysis of IL-1R1 expression by CD4 T cells (upper row, gated on VD^-/TCR-β⁺/CD4⁺ cells) and γδ-TCR⁺ T cells (lower row, gated on VD^-/γδ-TCR⁺ cells) isolated from the draining lymph nodes of mice immunized with CFA. (C) Mean Fluorescence Intensity (MFI) of IL-1R1 staining by cell populations shown in (B). Data are (B) representative FACS plots and (C) mean +SEM of n = 3 of each genotype, and are representative of three independent experiments. **p < 0.01, ***p < 0.001, N. S.–not significant; two-tailed unpaired t-test.</p