Fig 2: Co-culture with gut and lung organoids (LOs) drive tissue-specific ILC2 imprinting(A) Representative bright-field and confocal image of murine primary distal lung epithelial organoid showing cystic or saccular structures (scale bars: 25 µm).(B) Fold change expansion of Live, EpCAM-, CD45+ ILCPs after 7-day co-culture with SIOs or LOs (N = 6 animals, two independent experiments).(C) Relative proportion of mature ILC groups, showing Live, EpCAM-, CD45+ group 1 ILCs (magenta, Live EpCAM-CD45+Lineage-ROR?t-NKp46+NK1.1+Gata3-), ILC2 (green, gated as Live EpCAM-CD45+Lineage-ROR?t-NKp46-NK1.1-Gata3+), and ILC3 (blue, EpCAM-CD45+Lineage-ROR?t+NKp46+/-NK1.1+/-Gata3-) derived from 7-day co-culture of ILCPs with SIOs or LOs.(D) Quantification of absolute ILC2 frequency in (C) as a proportion of all LIN- cells (N = 5 animals).(E) Representative flow plots of Klrg1, ST2, ICOS, CD25, IL-13, and IL-5 expression in EpCAM-CD45+NKp46-NK1.1-ROR?t- putative ILC2s after co-culture with SIOs or with LOs (FMOs, magenta and blue), with the GeoMFI of IL-5 and IL-13 cytokine expression in ILC2 and IL-22 expression in ROR?t+ ILC3 quantified in (F) after 4-h unbiased PMA/Ionomycin stimulation (N = 4, two experiments).(G) Schematic of SIO-to-LO experimental design.(H) Frequency of Klrg1+, ST2+, and CD25+ ILC2s after 14-day co-culture (N = 3–5 animals).(I) Relative gene expression of Il33 in whole primary small intestine and lung tissue (N = 5 animals), as well as in SIOs and LOs (n = 3 wells of organoids) in (J).(K) Frequency of Klrg1+, ST2+, and CD25+ ILC2s in SIO-to-LO co-cultures with and without neutralizing dose of rmIL-33 blocking antibody (50 ng/mL, N = 3 animals).Error bars represent SEM; p values are from unpaired Student’s t tests.

Fig 3: CCR2 and CCR4 serve as tissue specific markers of ILC2s.a Violin plots showed ILC2 signature genes in the lung and the intestine. Ccr2 was highly expressed in lung ILC2s and Ccr4 is restricted to intestine ILC2s. Box plots indicate median (middle line), 25th, 75th percentile (box) of the data and the maximum and minimum values as endpoints for the whiskers. b Maturation trajectory of lung and intestine ILC2s were analyzed by Monocle pseudotime analysis. c, d Expression of CCR2 and CCR4 on ILC2s. CCR2-mNeonGreen reporter mice (c) and CCR4-mNeonGreen reporter mice (d) were generated as described in “Methods” and analyzed for the expression of CCR2 and CCR4 on ILC2s by flow cytometry. Lin-CD45+CD127+ lymphocytes were gated for analysis of KLRG1 and ST2. (Lin=CD3e,CD8a,CD19,CD11b,CD11c,Gr1,F4/80,Ter119). e, f CCR2-mNeonGreen and CCR4-mNeonGreen mice were intraperitoneally injected with 200 ng/mouse/day IL-25 for three constitutive days. Expression of CCR2-mNeonGreen (e) and CCR4-mNeonGreen (f) on ILC2s from respective mice were analyzed by flow cytometry. g, h CCR2-mNeonGreen mice (g) and CCR4-mNeonGreen mice (h) were analyzed for the expression of CCR2-mNeonGreen and CCR4-mNeonGreen on ILC2s after i.p. injection of 400 ng/mouse/day IL-33 for three constitutive days. (Gate strategies for ILC2s are: Lin-CD127+ST2+KLRG1+ for lung ILC2s, Lin-CD127+ST2-KLRG1+ for iILC2, Lin-CD127+KLRG1+ for gut ILC2s, Lin=CD3e,CD19,CD11b,CD11c,Gr1,F4/80,NK1.1). Data are representative at least three independent experiments. Source data are provided as a Source Data file.

Fig 4: HIOs promote proliferation and maturation of systemic human ILCPs(A) Gating strategy for PBMC-derived ILCPs, pregated on live, single, CD45+ cells (FMOs, blue and magenta).(B) Schematic of HIO + ILCP co-cultures, indicating the presence of mesenchymal cells and CD45+ ILCPs.(C) Representative plot overlays of EpCAM+ IECs, double-negative mesenchymal cells (M, magenta), and CD45+ ILCs (blue) after 14-day co-cultures, highlighting Matrigel debris in gray, and quantified in (D) as count of EpCAM-, CD45+, LINn- ILCs and (E) fold change expansion of ILCs after 14-day co-culture relative to the number of ILCPs seeded on day 1 (N = 3–15 across seven experiments).(F) Representative image of CD45+ ILCPs co-cultured with mesenchyme-depleted, EpCAM+ HIOs (scale bar: 25 µm). Error bars represent SEM; p values are from unpaired Student’s t tests.(G) Count of Live, EpCAM-CD45+LIN-ROR?t+ ILCs after 14-day co-culture with SD-HIOs or epithelial-depleted HIO-STROs expressing markers CCR6, NKp44, and/or T-bet (ILCPs from N = 3 PBMC donors).(H) Relative group 1 (Live, CD45+Lin-CRTH2-c-kit- [CD127+CD161+ in primary gut ILC only] and [EpCAM-ROR?t-GATA3- in SD-HIO and HIO-STRO only]), group 2 (Live, CD45+Lin-CRTH2+c-kit+/-, [CD127+CD161+/- in primary gut ILC only] and [EpCAM-ROR?t-GATA3+ in SD-HIO and HIO-STRO only]), group 3 (Live, CD45+Lin-CRTH2-c-kit+, NKp44+/- and [CD127+ CD161+ in primary gut ILC only] and [EpCAM-ROR?t+GATA3- in SD-HIO and HIO-STRO only]), and other LIN- ILCs (e.g., undifferentiated precursors; Live, CD45+, Lin-, CRTH2-, c-kit- and [CD127+ CD161+ in primary gut ILC only] and [EpCAM-, ROR?t-, GATA3- in SD-HIO and HIO-STRO only]) in unstimulated, primary human intestine (N = 13, adapted from Krämer et al., 2017), SD-HIOs (N = 13 from six experiments), and HIO-STROs (N = 7 from three experiments).

Fig 5: Murine ILCPs yield group 1, 2, and 3 ILCs after co-culture with SIOs(A) Confocal image of SIOs with apical ZO-1 and Lyzozyme-1+ Paneth cells (scale bar: 50 µm).(B) Diagram of co-culture system and representative confocal image of SIO-ILCP co-culture (scale bar: 50 µm).(C) Schematic of experimental design.(D) Representative flow plot of DAPI-Lineage- (hematopoeitic stem cell [HSC] cocktail [CD3, CD45R, CD11b, TER-119] Ly-G6, CD5, CD19, and NK1.1), CD127+a4ß7+Flt3-, PD-1+ ILCP, and CD25+ ILC2P, gated based on PD-1 fluorescence minus one (FMO) controls (overlaid in red).(E) Quantification of ILCP count yielded per animal (N = 15 animals, five experiments).(F) Representative flow plots of DAPI-EpCAM+CD45- IECs and EpCAM-CD45+ ILCs from 7-day cultures of PD-1+ ILCPs only, ILCP + SIO, and SIO-only cultures. Arrows indicate corresponding lack of Lineage markers.(G) Fold change of CD45+Lineage- cells yielded after culture relative to the number of seeded precursors (N = 15 animals, five independent experiments).(H) Contour plot overlay representing expression of NKp46, ROR?t, NKp46, NK1.1, Klrg1, and Sca-1 in Lineage- populations derived from (F) in populations pregated as putative group 1 (magenta; Live, EpCAM-, CD45+, Lin-, ROR?t-, Klrg1-, NK1.1+, NKp46+), group 2 (green; Live, EpCAM-, CD45+, Lin-, ROR?t-, NKp46-, Klrg1+, Sca-1+), NKp46+ group 3 (lavender; Live, EpCAM-, CD45+, Lin-, Klrg1-, ROR?t+, NKp46+), NKp46- group 3 (blue; Live, EpCAM-, CD45+, Lin-, Klrg1-, ROR?t+, NKp46-), and a putative immature “other” population (gray; Live, EpCAM-, CD45+, Lin-, ROR?t-, Klrg1-, NK1.1-, NKp46-) in cultures derived from ILCPs only or ILCP co-culture with SIOs.(I) Quantification of pooled ILC groups depicted in (H) (ILCPs from N = 4 animals).Error bars represent SEM; p values are from unpaired Student’s t tests.