IL-1R signaling modulates CD4⁺ T cell apoptosis in vivo and in vitro. (A) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT or Il1r1^−/− mice. At sacrifice, cLPLs were isolated and Ki67 expression by cLPLs CD4⁺ T cells was assessed by intracellular staining. Data represent mean ± SEM (n = 20–21 from 3 pooled independent experiments). Representative FACS plots (gated on CD4⁺ T cells) and quantitation are shown. (B) C57BL/6 Rag1^−/− mice were transferred with 2 × 10⁶ CD4⁺ CD25⁻ CD45Rb^Hi T cells from C57BL/6 WT or Il1r1^−/− mice and sacrificed 2 wk after transfer. Total number of CD4⁺ T cells and Th17 cells from the colonic lamina propria were assessed by FACS. Data are shown as mean ± SEM (n = 5 mice/group). (C and D) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT or Il1r1^−/− mice. Mice were sacrificed when recipients of WT naive CD4⁺ T cells developed clinical signs of colitis, and CD4⁺ T cells were FACS sorted from cLPL preparations. (C) Expression levels of indicated chemokine receptors as revealed by qRT-PCR analysis. (D) Bcl-2 and Bcl-XL expression by WT or Il1r1^−/− CD4⁺ T cells upon ex vivo restimulation with 0.1 µg/ml PMA and 1 µg/ml ionomycin overnight in complete medium (n = 5 mice/group, 1 representative experiment out of 2 is shown). (E) Purified CD4⁺ T cells from WT or Il1r1^−/− mice were cultured for 3 d in the presence of plate-bound αCD3 and αCD28 mAb at the indicated concentrations. After staining with the phosphatidylserine-binding protein Annexin V and the vital dye 7AAD, Annexin V⁺ 7AAD⁻ cells were identified as early apoptotic events. The average frequency of Annexin V⁺ 7AAD⁻ cells in WT cells was set as a baseline (100%), and data were expressed as relative increase over baseline (n = 10 from 4 pooled independent experiments). Representative FACS plots (gated on CD4⁺ T cells) and quantitation are shown. (F) 129SvEv Rag2^−/− mice were infected with H. hepaticus for 8 wk. CD45⁺lin⁻ Sca1⁺Thy1.2^Hi ILCs were FACS sorted from the colon and cultured overnight in complete medium alone (n/a) or in the presence of IL-1β (10ng/ml). Bcl2 expression was evaluated by qRT-PCR and normalized on Hprt expression. Data are shown as mean ± SEM from two pooled independent experiments. In each experiment, 10–18 mice were pooled.