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: WNT11 knockdown in tumor cells stimulates CD8+ T-cell proliferation and CD8+ T-cell-mediated tumor cell killing via AFF3.a–d Flow cytometry analysis of the proliferation and apoptosis of CD8+ T-cells in MC38 (a, b) and Panc02 (c, d) liver metastases between scramble and shWnt11 group. e, f FACS analysis of CD8+ T-cell proliferation (CFSE low) in cocultures with MC38 (e) or Panc02 (f) scramble and shWnt11 cancer cells. g. Schematic representation of an in vitro T-cell killing assay. OVA-expressing tumor cells were cocultured in different ratios with CD8+ T-cells isolated from OVA-specific T-cell receptor transgenic (OT1) mice that had been preactivated using an OVA peptide and IL-2 plus IL-7 treatment. h Titration assay involving coculture at different effector-to-target ratios assessing the sensitivity to cytotoxic T-cell killing of MC38-OVA scramble, shWnt11, and shWnt11-shAff3 cells. i Alterations in AFF3 and β-catenin protein levels in MC38 cells following treatment with WNT11 and inhibitors of CAMKII, JNK, and PKC as indicated. j Knockdown of β-catenin abrogated the AFF3-induced increase in expression following Wnt11 knockdown. k, l Knockdown of WNT11 increased the nuclear translocation of β-catenin. m FACS analysis of CD8+ T-cell proliferation (CFSE low) in cultured with conditioned media derived from MC38 cells treated with WNT11 and CAMKII, JNK, and PKC inhibitors as indicated. n qPCR analysis of the expression of Camk2b in MC38 cells following CAMKII knockdown. o Aff3 mRNA levels were detected using qPCR in MC38 cells following CAMKII knockdown and WNT11 treatment as indicated. p FACS analysis of CD8+ T-cell proliferation (CFSE low) in cultured with conditioned media derived from MC38 cells with CAMKII knockdown and WNT11 treatment as indicated. q Titration assay involving coculture at different effector-to-target ratios assessing the sensitivity to cytotoxic T-cell killing of MC38-OVA scramble and shCamk2b cells with or without WNT11 treatment. b, d P-values were calculated using a Student’s t test (two-tailed), n = 5 biological replicates. e, f, m–p P-values were calculated using a one-way ANOVA followed by a Tukey’s multiple comparison test, n = 3 biological replicates. h, q P-values were calculated using a two-way ANOVA, n = 3 biological replicates. Data are presented as the mean ± SD. Source data are provided as a Source Data file.