Fig. 9

Functional validation of P3 and P4 cells using the newly identified phenotype by scRNAseq. a Feature plots of key discriminating genes consisting of the t-SNE plot from Figure 6B on which is overlaid the gene expression level of selected markers genes (TREM1, FCAR, MCR1, CD209, and MERTK) for cluster E and F. Color scheme is according to log2(TPM + 1) expression value, with color gradient from purple (low) to yellow (high) expression. b TREM1 (n = 4) and CD89 (n = 3), CD206 (n = 4), CD209 (n = 6), and MERTK (n = 4) protein expression in the two CD14⁺CD64^hiCD163⁻, subsets after gating on HLADR⁺SIRPα⁺ cells. c TREM1⁺CD209⁻MERTK- cells display P3 (CD64^hiCD163⁻) phenotype and TREM1⁻CD209⁺MERTK⁺ cells show P4 (CD64^hiCD163^hi) markers. Shown is one representative experiment among four. d Gating strategy for isolation of CD4⁺ T cells and MNPs: strategy b (TREM1⁺CD209⁻MERTK⁻, P3/b and TREM1⁻CD209⁺MERTK⁺, P4/b) in comparison to strategy a, as depicted in Fig. 4. e Representative dot plots of CD4⁺ T cells co-cultured with autologous colonic P3/b (n = 6) and P4/b (n = 2) cells for 6 days (left panels); IL-17/IFN-γ expression measured via intracytoplasmic staining after gating on CD3⁺ T cells (right panels). f Mean fluorescence intensity of IL-17 in colonic CD4⁺ T cells co-cultured for 6 days with autologous P3/b (n = 6), P3 (n = 16) or in the presence of IL-1β (n = 6). b, e, f, Wilcoxon signed test