Extended Data Fig. 6: uLIPSTIC to study epithelial cell – immune cell interactions in the gut.

(A–C) Flow cytometry strategy for intraepithelial immune cells. (A) Representative gating strategy for γδ TCR and αβ TCR (Cd8αα⁺, CD8αβ⁺, and CD4⁺) IEL subsets. (B) Top, expression of SrtA (FLAG) and capture of LIPSTIC substrate by IEC donor cells and bottom, transfer of substrate onto CD45⁺ acceptor cells in SrtA-expressing and control mice. (C) Sorting strategy for the scRNA-seq experiment. Samples were enriched for rarer (e.g., B cell, CD4⁺ IEL) populations by first sorting 12,500 total cells then an additional 12,500 cells depleted of the dominant γδ, CD8αα, and CD8αβ IEL populations. Three independent samples were sorted and stained with different hashtag oligos for downstream identification. (D–L) Clustering analysis of the immune interactome of IECs in the small intestine. (D) UMAP colored by Leiden clustering of the entire scRNA-seq/uLIPSTIC dataset (n = 3,677 cells) used as an intermediate step in cell type annotations. (E) Left, UMAP colored by biological replicate. Right, bar plot indicating cluster composition by biological replicate, cluster size indicated at the right of each bar. (F-G) Further analysis of cluster 10 shows that is a composite comprising proliferating T and B cells. This co-clustering of B and T cells held true for varying number of PCs between 20 and 100 (not shown). (F) Left, Leiden cluster 10 was isolated and sub-clustered, yielding two separate clusters (UMAP). Right, normalized expression of Cd79a and Cd8a for these two sub-clusters of cluster 10 determines their annotation as either B or T cells. (G) UMAP showing the S and G2M phase cell cycle gene list scores (obtained using the ‘score_genes_cell_cycle()‘ function with lists from the Seurat package⁷²), characterizing Leiden cluster 10 as proliferating cells, thus explaining their co-clustering. (H) UMAP showing final clustering of the entire data, with Leiden cluster 10 subdivided into clusters 10a and 10b. (I) Dendrogram representing transcriptional similarities among clusters. Differentially expressed genes were identified for each cluster (log2FC > 1, FDR < 0.05, see Methods), and normalized expression of all such genes (5,956 genes total), averaged per cluster, was used for the hierarchical clustering analysis that produced the dendrogram. Final annotation clusters shown in Fig. 4 are indicated below the Leiden cluster numbers. (J) Dot plot of marker genes indicating their level of expression in each cluster. Dot size indicates the fraction of cells in the cluster with Pearson residual normalized expression greater than 0, dot color represents level of expression. (K) Violin plot showing levels of normalized uLIPSTIC signal for each Leiden cluster. (L) UMAP showing presence of rearranged TCRα and β in each cell.