Fig. 1. Intestine-derived TRM cells integrate homeostatically into autologous organoids to form IIOs.
a, Schematic overview of establishment of autologous IIOs (adapted with permission from ref. 60). b,c, Flow cytometry-based, t-distributed stochastic neighbour-embedding (t-SNE) analysis of donor-matched gut TRM cell and circulating PBMC T cell subgroups based on surface marker expression, derived from one donor and representative of six biological replicates. t-SNE plot coloured according to original source of T cells (b; light grey for PBMCs and black for TRM cells), and expression of ten individual markers of naivety, memory and tissue residency (c). d, Fluorescent live image 24 h following IIO co-culture set-up (nuclei, teal; T cells, pink). Similar images were observed with four biological replicates. e–g, mIF staining of cultures 24 h following co-culture with autologous TRM cells (e,f) or PBMCs (g). Intestinal organoids highlighted by E-cadherin+ (ECAD) epithelium (red). CD4+ (green) and CD8+ (turquoise) TRM cells integrated within larger (e) or smaller (f) organoids whereas blood-derived CD4+ and CD8+ T cells surrounded the organoid (g). Box i and box ii highlight the presence (f) or absence (g) of immune cell integration into two representative regions (i and ii) of the epithelium. e–g, miF images representative of three independent biological replicates. IO, intestinal organoid. h, Immune cell count detected per organoid, each data point representing an individual organoid; n = 18 for PBMCs and n = 54 for TRM cells, two-tailed Mann–Whitney test. i, Ratio of epithelial to immune cells within each organoid (n = 35), 24 h following organoid supplementation with autologous TRM cells. h,i, Data represent the collation of two independent IIO cultures. Similar results were observed in three biological replicates. j, Elongated flossing T cell inserting between basal and lateral epithelial cell junctions. Scale bars, 25 µm (d–g, j).