Extended Data Fig. 6: Increased NK cells is a function of both extrathymic recruitment and intrathymic expansion.
From: Damage-induced IL-18 stimulates thymic NK cells limiting endogenous tissue regeneration
a, Taken from scRNAseq described in from Fig. 2a: integrated UMAP showing undamaged, baseline thymus major clusters and annotations (left) and heatmap expression Ncr1, Itga1 and Itga1. b, Number of NK cells at day 3 in WT or Il18r1−/− mice (n = 5/group). c, d, Female C57BL/6 CD45.1+ and CD45.2+ mice were surgically conjoined to establish parabiotic pairs when both members of the pair received sublethal total body irradiation (SL-TBI; 550 cGy). Intrathymic/extrathymic chimerism was calculated on days 0, 1, 4, or 7 days after SL-TBI using congenic markers (CD45.1 and CD45.2) to determine mouse of origin (that is, cells expressing the same CD45 isoform as assessed mouse pair thymus = “intrathymic”; cells expressing the alternate isoform = “extrathymic”-derived). Absolute chimerism (c) (n = 8/group) and fold-change in NK cell number (d) across timepoints (d1, n = 6; d4, n = 6; d7, n = 8). e, Concatenated flow cytometry plots shown of NKG2D expression on CD45+ NK1.1+ TCRβ—CD49b+ NK cells and gMFI of NKG2D expression of IL-18R− and IL-18R+ NK cells at day 3 post SL-TBI (n = 3/group). f–h, Female 1-2 mo C57BL/6 WT (-rIL-18, n = 6; +rIL-18, n = 4) or Ifng-reporter (-rIL-18, n = 5; +rIL-18, n = 3) mice were given rIL-18 (s.c., 2.5 mg/kg) or PBS. f, Total thymus cellularity assessed on day 3. g–h, Expression of Ifng-GFP (g) or Perforin (h) at day 3. i, Female 1-2mo C57BL/6 mice were given SL-TBI and 3 days later administered with rIL-18 (s.c., 2.5 mg/kg, n = 3) or PBS (n = 4) as in Fig. 4k. On day 7 NK1.1+IL-18R+TCRβ−CD49b+ NK cells were then FACS purified and cocultured with cell-dye labeled RMA-S target cells at a 5:1 Effector to Target ratio. RMA-S target cell Annexin V expression was measured 5 h post co-culture. Graphs represent mean ± SEM; each dot represents an individual biological replicate; ns = not significant. Statistics were generated for b–d and f–i using unpaired two-tailed t-tests, e using paired two-tailed t-tests, and for c using one-way ANOVA with Dunnet’s correction for multiple comparisons.
