Fig 2: Effector CD4 T Cells constitutively express IL-1R and require IL-1R signaling for cytokine production. a, b Splenic CD4 T cells were gated on naive (Tnaive) and memory (Tem) T cell markers and stained for a IL-6Ra, IL-4Ra and IL-12Rb1 b IL-1R1, IL-18Ra and IL-33R (ST2). c Relative expression of Il1r1 transcripts in Tnaive and Tem cells. Data is normalized to 18s. d-f Tem cells were stimulated with splenic DCs and soluble aCD3 (30 ng ml-1) in the presence or absence of IL-1R antagonist (IL-1Ra) for 48 h d IL-17A, IL-17F, IL-22, e IL-13, IL-4, IL-5, and f IFN? were measured. g CD4 T cells from SI-LP were stained for IL-1R1. h CD4 T cells from SI-LP were stimulated using CD11C + DCs and soluble aCD3 (30 ng ml-1) in the presence or absence of IL-1Ra for 48 h followed by cytokine ELISA. i IL-17A, IL-13 and IFN? levels in supernatants obtained from Tem cell co-cultures with WT or Il1r-/-CD11c + splenic DCs in the presence of aCD3 (30 ng ml-1) after 48 h of stimulation. Control in receptor staining refers to fluorescence minus one control. IL-17A, IL-13 and IFN? in the culture supernatants were measured using paired-antibody ELISA. Error bars indicate SEM; paired t test, c–f, h, i 4–8 mice were pooled per experiment

Fig 3: Both frequency of responding cells and per-cell activation levels increase with increasing signal strength.(A) Purified AND T cells and CD11c+ APCs were co-cultured in the presence of indicated peptides at the indicated concentrations for 24 hr. Flow cytometry was then used to phenotype the CD4+ T cells. The histograms show CD69 expression of CD4+ T cells. The annotated bar indicates the gate used to identify CD69+ CD4 cells in subsequent figures. (B) Gating on CD4+ cells as in 1A, there is a bimodal distribution of CD25 expression resulting from activating levels of high-affinity (PCC) or low-affinity (K99A) peptides. The annotated bar indicates the gate used to identify CD25+ cells. (C) The percent of CD4+ cells that are CD69+ (using gate shown in 1A) varies with the peptide presented and concentration of the indicated peptide. (D) The percent of CD4+ cells that are positive for the activation marker CD25 varies with both peptide and dose. (E) Gating on CD4+ CD69+ cells (as shown in 1A), the geometric mean fluorescence intensity (MFI) of CD69 per cell in each condition varies. (F) The geometric MFI of CD25, gated on CD4+ CD25+ cells (as shown in 1C), varies with peptide and dose. (P-values based on Student’s t test; *p<0.05, **p<0.01, ***p<0.001.)DOI: http://dx.doi.org/10.7554/eLife.10134.003

Fig 4: IL-1R signaling stabilizes Th17 and Th2 cytokine transcripts. a CD44hi CD62Llo Tem cells were stimulated by splenic DCs and soluble aCD3 (30 ng ml-1) in the presence or absence of IL-1Ra for 48 h. Respective cytokine transcripts were quantified in DC-T cell co-culture cell lysate. All data is normalized to 18 s and untreated control. b CD4 T cells were stimulated with CD11c + DCs and soluble aCD3. c CD4 T cells were stimulated using immobilized aCD3 (0.5 µg ml-1) and aCD28 (0.5 µg ml-1). b, c Actinomycin D was added 18 h post stimulation and lysates were collected at indicated time points post-actinomycin D treatment. RNA was isolated post-stimulation and cDNA was synthesized for qRT PCR. All data are normalized to 18 S rRNA. Error bars indicate SEM; paired t test. b, c Data are representative of three to four independent experiments

Fig 5: Dendritic cells provide key signals necessary for optimal functioning of effector CD4 T cells. a CD44hi CD62Llo CD4 T cells (Tem) were stimulated with soluble αCD3 (30 ng ml−1) in the presence of either live splenic CD11C + DCs or αCD3 (0.5 μg ml−1) and αCD28 (0.5 μg ml−1) antibodies immobilized on a plate. b CD44hi CD62Llo CD4 T cells (Tem) were stimulated with soluble αCD3 (30 ng ml−1) in the presence of either live DCs, naive and activated (CpG-stimulated) CD19 + B cells. Cytokine levels in the culture supernatants were determined after 48 h of activation using paired-antibody ELISAs. Error bars indicate SEM; paired t test