Fig 1: 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.
Fig 2: SIOs provide an essentially GF model of gut-specific ILCs(A) Representative flow plots of NKp46 expression in ILCP + SIO co-culture-derived ILCs or SI-LP-derived CD127+ ILCs, with the frequency of NKp46+ ILC3s (co-culture: Live, EpCAM-, Lin-, CD45+, ROR?t+; primary tissue: Live, CD45+, Lin-, CD127+, Klrg1-, NK1.1+/-, ROR?t+) additionally quantified for ILCPs cultured without SIOs or with GF SIOs in (B) (N = 2–5 animals, pooled from two experiments).(C) Relative frequency of mature ILC subsets excluding immature or other cells, depicting group 1 (magenta; Live, EpCAM-, CD45+, Lin-, ROR?t-, ST2-, Klrg1-, NK1.1+, NKp46+), group 2 (green; Live, EpCAM-, CD45+, Lin-, ROR?t-, NK1.1-, ST2+, Klrg1+, Sca-1+), NKp46+ group 3 (lavender; Live, EpCAM-, CD45+, Lin-, ST2-, Klrg1-, ROR?t+, NKp46+), and NKp46- group 3 ILCs (blue; Live, EpCAM-, CD45+, Lin-, ST2-, Klrg1-, ROR?t+, NKp46-) in live, unstimulated co-cultures derived from SPF-SIOs or GF-SIOs compared with primary SPF ileum (no Peyer’s patches).(D) Diagram of transwell culture strategy.(E) Relative frequency of group 1, 2, and 3 ILCs derived from PD-1+ ILCP + SIO +/- transwell insert (TW) separation (N = 3, two experiments).(F and G) Count of putative LIN-, ROR?t-, NKp46-, Klrg1+, Sca-1+ ILC2 (F) and geometric mean fluorescence intensity (GeoMFI) of Klrg1 in LIN- ILCs after co-culture of CD25+ ILC2Ps with SIOs, TW separation, or without SIOs (N = 4, two experiments) (G).(H–K) Representative flow plots indicating expression of Gata3, IL-25R, IL-13, and IL-5 in ILC2P-derived and SI-LP-derived ILC2 after 4-h stimulation with PMA/Ionomycin (H), quantified in (I)–(K) (FMO, cyan and magenta; error bars represent SD; N = 3).(L) Gene expression heatmap (magenta = high, cyan = low, white = not detected) of genes of interest derived from bulk RNA sequencing of EpCAM+, CD45- IECs after 7-day co-culture with precursor-derived lymphocytes, without immune cells but with IL-2, IL-7, and Flt3-ligand supplementation, or in basal SIO media.(M) Schematic of metabolite microinjection strategy.(N) SIOs microinjected with 20-kDa FITC-dextran and 5 mM succinate 16 h after injection.(O) Representative confocal images of SIOs microinjected with PBS or with 5 mM succinate stained for Tuft cell marker Dclk1 (green) and crypt marker CD44 (magenta) (scale bars, 50 µm).(P) Expression of Il25/Il17E normalized to Hprt1 in SIOs injected with PBS or 5 mM succinate (n = 3 wells of SIOs in one experiment).(Q) Frequency of Klrg1+ ILC2s after co-culture of ILC2Ps with SIOs injected with PBS or with 5 mM succinate (ILC2Ps split between conditions from N = 4 animals in one experiment).Error bars represent SEM; p values are from unpaired Student’s t tests.
Fig 3: Gut-matured ILC2 upregulates ST2 on transfer to HLO culture(A) Representative image of SD-HIOs and SD-HLOs showing E-cadherin+ (E-CAD) epithelium, CD45+ ILCs, and nuclei (Hoechst) after 14-day co-culture (scale bars: 50 µm).(B) Count of EpCAM-, CD45+, LIN- ILCs after 14-day co-culture with SD-HIOs or SD-HLOs, with corresponding count of EpCAM-, CD45- mesenchyme.(C) Representative flow plots of ROR?t, CCR6, IL-22, IL-17A, IFN-?, and NKp44 after 14-day co-culture with SD-HIOs or SD-HLOs after 4-h PMA/Ionomycin stimulation (pregated population in gray, representative of N = 3).(D) IL-5 and IL-13 expression in putative GATA3+ ILC2s after 14-day co-culture and 4-h PMA/Ionomycin stimulation (FMOs, magenta and cyan).(E) Expression of CD25 and ST2 in putative GATA3+ ILC2s after 14-day co-culture with SD-HIOs or SD-HLOs.(F and G) Relative frequency of CD25+ and ST2+ putative ILC2s (F) and GeoMFI of ST2 in the same population with corresponding histogram overlay of ST2 (PE) after 14-day co-culture with SD-HIOs or SD-HLOs, followed SD-HIO-to-HIO, SD-HIO-to-HLO, or SD-HIO-to-HLO with 50 ng/mL hIL-33 neutralizing antibody (G). All experiments were performed with ILCPs from N = 3 donors; unpaired two-tailed Student’s t tests were used; and error bars represent SEM.
Fig 4: SIOs promote ILCP stemness and maturation(A) Representative flow contour plots of ILCs yielded from ILCP + SIO or ILCP only in Matrigel (“NEG” black = ILCP + SIO; “NEG” magenta = ILCP without SIO).(B) Relative frequency of a4ß7+PD-1+CD25+c-KIT+ of all Lineage- cells.(C) Relative frequency of ROR?t-CCR6-NK1.1-NKp46-Klrg1-ST2- (NEG) within the Lineage- population, count of NEG, % of CD25+, c-KIT+, a4ß7+, and PD-1+ within the negative population (frequency of parent, SD).(D) Frequency and count of ILC1 (Live, EpCAM-, CD45+, Lin-, ROR?t-, Klrg1-, NK1.1+, NKp46+, Eomes-, T-bet+) and NK cells (Live, EpCAM-, CD45+, Lin-, ROR?t-, NK1.1+, NKp46+, T-bet+, Eomes+) depicted in (E) derived from PD-1+ ILCP co-cultures +/- SIO and compared with putative ILC3 (Live EpCAM-CD45+Lin-ROR?t+Klrg1-NK1.1+/-NKp46+/-Eomes-T-bet+/-) and ILC2 (Live EpCAM-CD45+Lin-ROR?t-Klrg1+NK1.1-NKp46-Eomes-T-bet-) (N = 7 animals, two experiments).(F) Frequency of ILC1 and NK cells expressing IFN-? (FMO, blue) in (E) and granzyme B after 4-h stimulation with PMA/Ionomycin and IL-18 compared with SI-LP ILC1s and NK cells depicted in (G).(H) Frequency and count of ILC3 (Live EpCAM-CD45+Lin-NK1.1+/-NKp46+/-Klrg1-ST2-ROR?t+CCR6-) and CCR6+ LTi-like cells (Live EpCAM-CD45+Lin-NK1.1-NKp46-Klrg1-ST2-ROR?t+CCR6+) depicted in (I) derived from PD-1+ ILCP co-cultures +/- SIOs and compared with primary ILC3s (N = 5 animals, two experiments).(J) Frequency of ILC3 and CCR6+ LTi-like cells expressing IL-22 (FMO, blue) and IL-17A (FMO, magenta) in (I) after 4-h stimulation with PMA/Ionomycin and IL-23 when compared with SI-LP ILC3 and Lti depicted in (K).Error bars represent SEM; p values are from unpaired Student’s t tests.
Fig 5: 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.
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