Extended Data Fig. 5: Effect of IL-18 on T cell development in vitro.

a–c, 50,000 lineage depleted bone marrow cells were co-cultured with OP9-DLL1^GFP fibroblasts confluent in a 6-well dish with 5 ng/mL Flt3L plus 1 ng/mL IL-7 and 0, 1 or 10 ng/mL rIL-18 (n = 1/group). a, (Top) CD45⁺ Thy1⁺ CD4⁻ CD8⁻ DN1-4 thymocyte differentiation measured according to CD44 and CD25 expression 2 weeks post co-culture and (Bottom) CD45⁺Thy1⁺ thymocyte differentiation into CD4⁺ CD8⁺ Double Positive population 3 weeks post co-culture. (Right) Ratio of DN2:DN1 thymocyte differentiation 2 weeks post co-culture. b, Thymocyte expansion measured by non-adherent cell count quantified 2 and 3 weeks post co-culture with OP9-DLL1^GFP adherent cells. c, 50,000 bone marrow CD45+ Lineage- cKit+ Sca-1+ LSKs were FACS purified and co-cultured with OP9-DLL1^GFP fibroblasts confluent in a 6-well dish with 5 ng/mL Ftl3L plus 1 ng/mL IL-7 and 0, 1 or 10 ng/mL rIL-18 (n = 4/group) and 10 days later, thymocyte expansion was quantified by measuring non-adherent cell expansion. d, Female 1-2 mo C57BL/6 mice were administered 200μg αNK1.1 monoclonal antibody or control PBS (i.p) at days -1, 1 and 3 days post SL-TBI and thymuses assessed at day 7. Thymus NK1.1⁺IL-18R⁺ (Left) and NKG2D⁺IL-18R⁺ (Right) cells (parent gated on viable CD45⁺CD4⁻CD8⁻ cells) (n = 9/group). e, Female 1-2 mo C57BL/6 WT (Cd1d ^+/+) and Cd1d^−/− mice were given SL-TBI and thymus cellularity was measured 35 days later (n = 5/group). f, Female 1-2 mo Il18r1^fl/fl:Ncr1-Cre⁻ (Il18r1^WT, n = 5) and Il18r1^fl/fl:Ncr1-Cre⁺ (Il18r1^ΔNK/ILC1, n = 6) mice were given SL-TBI and thymus cellularity measured on day 7 post SL-TBI. g, Female 1-2 mo l18r1^WT and Il18r1^ΔNK/ILC1 thymus CD45⁺ NK1.1⁺ TCRβ^—CD49b⁺ NK cells at baseline (n = 5/group). Graphs represent mean ± SEM; each dot represents an individual biological replicate; ns = not significant. Statistics were generated for e, f using unpaired two-tailed t-tests.

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