Figure 5. Kras-mutant epithelial states participate in a feedback loop with immune populations.

(A) A feedback loop identified by Calligraphy in the pre-malignant pancreas. Arrows depict cognate R-L interactions. (B) tSNEs of immune and epithelial scRNA-seq data from pre-malignant stages (K1–K3, n = 6 mice), displaying imputed expression (69) of key genes from the loop in (A). Arrows between plots indicate sequential steps of the loop. (C) Co-immunofluorescence (co-IF) images showing co-expression of IL-33 and E-cadherin (epithelial marker), and apposition of FOXP3-expressing Tregs (arrows) and IL-33-expressing epithelial cells. Scale bar, 10 μm. (D) Distance in pixels (0.325 μm per pixel) of IL-33⁺ epithelial cells to Tregs against a null model of spatial distribution in co-IF data pooled across all biological replicates from K2 tissue. Distances are calculated between each IL-33⁺ epithelial (E-cadherin⁺) cell and its closest Treg (CD3⁺ FOXP3⁺). Asterisks, significant difference (one-tailed, un-paired t-test, p value < 0.0001) compared to random distances calculated by permuting epithelial cells. (E) IL-33-centric crosstalk paths originating from epithelial Gastric module E6 (central circle, magenta), with each outward concentric circle illustrating possible communication paths from inner to outer modules based on links inferred by Calligraphy. Arc length is proportional to the number of inner-module ligands that can bind to cognate receptors in the outer module. (F) tSNE as in (B), colored according to the step in which communication events from the IL-33-centric path in (E) reach the module expressed by that cell. Cells are assigned to their highest-expressed module, and each module is scored by the earliest step in which it appears along any paths through the Calligraphy network emanating from E6-derived IL-33. Cells expressing modules which are not downstream of IL-33 are colored in gray.