Figure 3.
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+, RORγt+; primary tissue: Live, CD45+, Lin−, CD127+, Klrg1−, NK1.1+/−, RORγt+) 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−, RORγt-, ST2−, Klrg1−, NK1.1+, NKp46+), group 2 (green; Live, EpCAM−, CD45+, Lin−, RORγt−, NK1.1−, ST2+, Klrg1+, Sca-1+), NKp46+ group 3 (lavender; Live, EpCAM−, CD45+, Lin−, ST2−, Klrg1−, RORγt+, NKp46+), and NKp46− group 3 ILCs (blue; Live, EpCAM−, CD45+, Lin−, ST2−, Klrg1−, RORγt+, 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−, RORγt−, 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.