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

IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells

Interleukin‐1 (IL‐1) is implicated in numerous pathologies, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which IL‐1 is involved in the generation of pathogenic T cells and in disease development remains largely unknown. We found that following EAE induction, pertussis toxin administration leads to IL‐1 receptor type 1 (IL‐1R1)‐dependent IL‐1β expression by myeloid cells in the draining lymph nodes. This myeloid‐derived IL‐1β did not vitally contribute to the generation and plasticity of Th17 cells, but rather promoted the expansion of a GM‐CSF (+) Th17 cell subset, thereby enhancing its encephalitogenic potential. Lack of expansion of GM‐CSF‐producing Th17 cells led to ameliorated disease in mice deficient for IL‐1R1 specifically in T cells. Importantly, pathogenicity of IL‐1R1‐deficient T cells was fully restored by IL‐23 polarization and expansion in vitro. Therefore, our data demonstrate that IL‐1 functions as a mitogenic mediator of encephalitogenic Th17 cells rather than qualitative inducer of their generation

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

Generation of a Novel T Cell Specific Interleukin-1 Receptor Type 1 Conditional Knock Out Mouse Reveals Intrinsic Defects in Survival, Expansion and Cytokine Production of CD4 T Cells

Interleukin-1 (IL-1) plays a crucial role in numerous inflammatory diseases via action on its only known signaling IL-1 receptor type 1 (IL-1R1). To investigate the role of IL-1 signaling in selected cell types, we generated a new mouse strain in which exon 5 of the Il1r1 gene is flanked by loxP sites. Crossing of these mice with CD4-Cre transgenic mice resulted in IL-1R1 loss of function specifically in T cells. These mice, termed IL-1R1ΔT, displayed normal development under steady state conditions. Importantly, isolated CD4 positive T cells retained their capacity to differentiate toward Th1 or Th17 cell lineages in vitro, and strongly proliferated in cultures supplemented with either anti-CD3/CD28 or Concanavalin A, but, as predicted, were completely unresponsive to IL-1β administration. Furthermore, IL-1R1ΔT mice were protected from gut inflammation in the anti-CD3 treatment model, due to dramatically reduced frequencies and absolute numbers of IL-17A and interferon (IFN)-γ producing cells. Taken together, our data shows the necessity of intact IL-1 signaling for survival and expansion of CD4 T cells that were developed in an otherwise IL-1 sufficient environment