Supplementary MaterialsResearch summary. to identify a module of BIBW2992 manufacturer co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, Tim-3, Lag-3, and TIGIT), but also a number of novel surface receptors. We functionally validated two novel co-inhibitory receptors, Activated protein C receptor (Procr) and Podoplanin (Pdpn). The module of co-inhibitory receptors is usually co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in multiple physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors Prdm1 and c-Maf as cooperative regulators of the co-inhibitory module, which we validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies novel regulators of T cell function with the potential to regulate autoimmunity and tumor immunity. We used single-cell RNA-seq (scRNA-Seq) to analyze co-inhibitory and co-stimulatory receptor expression in 588 CD8+ and 316 CD4+ tumor-infiltrating lymphocytes (TILs) from B16F10 melanoma3. We found that PD-1, Tim-3, Lag-3, CTLA-4, 4C1BB, and TIGIT strongly co-vary in CD8+ TILs. CD4+ TILs showed a similar pattern with the additional co-expression of ICOS, GITR, and OX40 (Fig. 1a, top). Single-cell mass cytometry (CyTOF) confirmed the surface co-expression of these receptors (Fig. 1a, bottom, Supplementary Table Information 1). Expression of PD-1, Lag-3, Tim-3, and TIGIT was tightly correlated on both CD8+ and CD4+ TILs (Fig. 1a, bottom). Clustering analysis (t-SNE4, Methods) showed two groups of CD8+ TILs (clusters 1 and 2) (Fig. 1b, Extended Data Fig. 1a,c) where PD-1, Lag-3, Tim-3, and TIGIT were mainly expressed in cluster 1 cells (Fig. 1b, Extended Data Fig. 1c) as were LILRB4 (Extended Data Fig. 1a), and co-stimulatory receptors of the TNF-receptor family, 4C1BB, OX-40, and GITR. In contrast, ICOS and CD226 were less restricted to cluster 1 (Extended Data Fig. 1a). We further observed two discrete clusters of CD4+ TILs (clusters 3 and 4) wherein PD-1, Tim-3, Lag-3, and TIGIT co-expression was restricted to cluster 3 (Fig. 1b, Extended Data Fig. 1c). Open in a separate window BIBW2992 manufacturer Physique 1. Multiple co-inhibitory receptors are expressed as a module on CD4+ and CD8+ T cellsa) CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) were harvested from WT mice bearing B16F10 melanoma tumors. Top panels, co-expression analysis of co-inhibitory and co-stimulatory receptor mRNA expression as BIBW2992 manufacturer determined by single-cell RNA-seq for 316 CD4+ and 588 CD8+ TILs. Bottom panels, protein expression by CyTOF for 23,656 CD4+ and 36,486 CD8+ TILs. Spearman correlation, followed by dendrogram ordering of the matrix using Euclidian distance is shown. Data are from biologically impartial experiments. b) TILs from WT mice bearing B16F10 melanoma were analyzed using CyTOF with a custom panel of antibodies against co-inhibitory and co-stimulatory cell surface receptors2,24 (Supplementary Information Table 1). Data were analyzed using vi-SNE. Polygons indicating clusters 1, 2 (in CD8+ T cells), 3 and 4 (in CD4+ T cells) are shown. Individual panels show expression of the indicated markers. c) Na?ve T cells from either wild type (WT) or IL-27ra deficient (IL27ra KO) mice were stimulated with anti-CD3/CD28 in the presence or absence of IL-27. Indicated co-inhibitory receptors expression was examined by real-time PCR (qPCR) at 96hr (CD4) and 72hr (CD8). Data are from biologically impartial animals. mean + s.e.m LEP is shown. d) vi-SNE plot showing WT (red) and IL27ra KO (blue) cells. e) ScRNA-seq of TILs from mice bearing B16F10 melanoma. Data were analyzed using t-SNE. Polygons indicating cluster 4 (in CD4+ T cells, orange) and cluster 5 (in CD8+ T cells, blue) are shown. Individual panels show expression of the indicated markers. Bar graphs show the mean signal intensity for indicated co-inhibitory receptors from WT (CD4+ (n=849); CD8+ (n=1752)) and IL27ra KO (CD4+ (n=628); CD8+ (n=541)) TILs for CyTOF (d) or WT (CD4+ (n=707); CD8+ (n=825)) and IL27ra KO (CD4+ (n=376); CD8+ (n=394)) TILs for ScRNA-seq (e). Error bars indicate s.e.m. and *p 0.05, **p 0.01, ***p 0.001; two-sided t-test. The co-expression of co-inhibitory receptors on CD8+ and CD4+ T cells suggests a common trigger. One candidate is usually IL-27, a heterodimeric member of the IL-12 cytokine family that suppresses autoimmunity5, induces IL-10-secreting Type 1 regulatory (Tr1) cells6,7, and induces expression of Tim-3 and PD-L1 on CD4+ and CD8+ T cells8,9. Activation of CD4+ and CD8+ T cells in the presence of IL-27 induced Tim-3 (Havcr2), Lag-3, and TIGIT at mRNA (Fig. 1c) and protein levels (Extended Data Fig. 2a). Expression of Tim-3, Lag-3, and TIGIT was reduced in IL-27R-deficient T cells, whereas PD-1 (Pdcd1) expression was unaffected by IL-27 (Fig. 1c, Extended Data Fig. 2a). CyTOF analysis showed.