Figure 5.

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⁻RORγt⁺ 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⁻RORγt⁻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⁻RORγt⁻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⁻RORγt⁺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⁻, RORγt⁻, 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).