Additional file 2 of PD-1 signaling uncovers a pathogenic subset of T cells in inflammatory arthritis
Creators: name: Straube, Johanna
name: Bukhari, Shoiab
name: Lerrer, Shalom
name: Winchester, Robert J.
name: Gartshteyn, Yevgeniya
name: Henick, Brian S.
name: Dragovich, Matthew A.
name: Mor, Adam
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 2: Supplement Figure 1. Flow cytometry histogram.  A flow cytometry histogram shows the IL-2 expression levels of the cells sorted in the final stage of the Jurkat T cells CRISPR screen. Supplement Figure 2. PTPN2 is required for PD-1 functions. The percent of inhibition of secreted IL-2 from shirt hairpin (sh) scramble and sh PTPN2 Jurkat T cells occulted overnight with Raji B cell vs. Raji B cell that overexpressed PD-L1 at different concentrations of SEE (A). p&lt;0.05, n=5. ELISA measured IL-2 levels.  Flow cytometry quantifies CD69 expression inhibition from sh Scramble and sh PTPN2 Jurkat T cells occulted overnight with Raji B cell vs. Raji B cell that overexpressed PD-L1 in the presence of SEE (B). Two different shRPTN2 clones are shown at different concentrations of SEE (A). p&lt;0.05, n=5. Supplement Figure 3. FUT6 signaling downstream of the T cell receptor.  Growth assay of the different clones of short hairpin stably expressing Jurkat T cells stimulated with anti-CD3 and anti-CD28 over three days (A). Quantification of western blots showing the total ERK and phosphorylated ERK levels in the indicated sh Jurkat T cell lines stimulated with beads coated with antiCD3 for the appropriate time, as indicated in Figure (B). Fold change in secreted IL-2 levels from anti-CD3 stimulated shirt hairpin different Jurkat T cell lines measured by ELISA after overnight stimulation (C). n=3-5, *p&lt;0.05. Cartoon showing the glycosylation pattern on FUT6 protein (D). Flow cytometry quantification of cell surface CD69 from Jurkat T cell line stably expressing the indicated sh and stimulated as indicated in Figure (E). Supplement Figure 4. The genes used to validate the dysfunctional T cell scores.  Heatmaps showing differential expression levels of genes that we discovered to be involved in PD-1 downstream signaling and that are also differentially expressed in the other scRNA sequencing datasets (GSE120575, SCP1288, and GSE164237), as reported in Fig. 2D. The green columns represent non-responders. The brown columns represent responders (A). The top gene list in each square is shown on the right side of the heat map. Volcano plot based on data from scRNA sequencing datasets (GSE120575, SCP1288, and GSE164237) displaying fold change in the expression levels of genes that were either up or down-regulated in the cancer patients that failed to respond to PD-1 blockade (B). Supplement Figure 5. Top-ranked genes are downregulated in T cells of other autoimmune diseases. Expression levels on top-ranked genes that we discovered as needed for PD-1 signaling were upregulated in cancer patients that responded to PD-1 inhibition in other autoimmune T cell bult RNA sequencing (A). Both the T cell and non-T cell datasets were analyzed. Heatmap showing genes that have high T cell dysfunctional score (upper right box) and their expression levels in ICI datasets immunosuppress datasets and in our CRISPR screen (lower right box) (B). The list of top genes in each square is shown on the right side of the heat map. Representative genes plotted as box plots showing the mean RNA expression levels in SF RA vs. PB RA (Ci) or PB RA vs. PB HC (Cii). Supplement Figure 6. PD1+ CD4 and CD8 EM T cells are functionally and transcriptionally different. Density ridge plots reveal elevated activation scores (HLA-DRB1, CD69, PCDC1, LAG3)   in CD8 EM T cells gated on PD1+ T cells in synovial fluid (SF) compared to peripheral blood (PB). Conversely, the activation score for CD4 EM T cells appears comparable between SF and PB. This analysis indicates a downregulation of KLRG1 expression in CD8 EM cells from SF, while no significant change is observed in the CD4 EM subset (A). Volcano plot displaying differentially expressed genes in CD4 and CD8 EM T cells gated on PD1+ cells (B). UMAP plots show cells in the CD8 EM cluster representing high EOMES, NKG7, and CCL4 levels compared to the CD4 EM cluster (C). Supplement Figure 7. Enrichment analysis of transcription factors. Heatmaps showing the predicted expression levels of transcription factors (TF) in different activated T cell subsets (CD4+ PRF1+, CD8+ HLA-DRHIGH, and CD8+ HLA-DRLOW T cells) based on top-ranked genes that we discovered in the screen to be involved in PD-1 signaling and the context of PD-1, LAG3, and CTLA-4 (shown in purple) (A). Genes we discovered to be involved in PD-1 signaling are shown in pink. Expression levels in both PB and SF are shown. TF expression in PB, SF, and other T cell subsets (B). Spider plot depicting the enrichment of TF that are associated with the genes of CD8+ PD-1+ KLRG1LOW T cells in SF (B). Supplement Figure 8.CD8+PD1+KLRG1low synovial T cells are functionally more activated than in peripheral blood T cells. Gating strategy for flow cytometry performed on T cells isolated from SF and matched PB T cells from RA patients (RA1, RA2), SF T cells from RA patient (RA3), and PB from healthy controls (HC1, HC2) (A). Flow cytometry analysis is represented in a dual Y-axis horizontal bar plot showing the percentage of positive CD69 and HLA-DR T cells in SF and PB gated on CD8+PD1+KLRG1low T cells (B). Supplement Figure 9. Genes associated with PD-1 signaling exhibit downregulation in activated CD8 T cell subsets in inflammatory arthritis. Density ridge plots showing the downregulation of RNA expression in SF compared to PB of KLRG1 gene and PD-1 associated genes (combined average expression of KLRG1, CRTAM, SLAMF7, and PTPN2) in CD8 T cells gated on high, moderate, and low levels of PRF1 (A), HLA-DRB1 (B), CD69 (C). All CD8 T cells were equally activated, as shown by the levels of CD69 expression between SF and PB. Data is plotted on non-zero entries.