Fig 1: Clustering of TCGA dataset based on angiogenesis, IL6, and PD-1 pathway expression. A, RNAscope and IHC for IL6 and IL6R in HGSOC patient biopsies. B, Heatmap illustrates GSVA enrichment scores for Lu tumor angiogenesis up, Kegg JAK-STAT signaling pathway, and Hallmark IL6-JAK-STAT signaling from MSigDB calculated for each sample of the TCGA ovarian dataset (n = 571). C, Boxplots illustrate GSVA scores for Lu tumor angiogenesis up, Kegg JAK-STAT signaling pathway, and Hallmark IL6-JAK-STAT across clusters 6, 7, 8, and 9 representing four major patterns of pathways’ expression (n = 57, 65, 38, 79 respectively). D, ConsensusTME applied on sample clusters identified in (B). Boxplots illustrate GSVA scores for macrophages and endothelial cells across the four clusters of interest. E, Heatmap illustrates GSVA enrichment scores for Lu tumor angiogenesis up and Reactome PD-1 signaling calculated for each sample of TCGA ovarian dataset. F, Boxplots illustrate GSVA scores for Lu tumor angiogenesis up and Reactome PD-1 signaling across clusters 5, 1, 10, and 9 representing the four major expression patterns of these pathways (n = 46, 65, 76, 43 respectively). G, ConsensusTME was applied on the sample clusters identified in (E). Boxplots illustrate GSVA scores for CD4 and CD8 T cells across four clusters of interest.
Fig 2: PD1 expression of T cells in WT and OT1/OT2 mice in peripheral blood, before and after CLL tumor transplantation. (A) Transplantation scheme. CLL tumors were derived from the Tcl1 mouse model. Splenocytes containing CLL cells were transferred intraperitoneally into congenic recipient mice (created with BioRender.com, 11 June 2021). (B) Percentage of PD1 positivity in the CD8/CD4 T cell compartments of WT mice (all Vbpos) and of transgenic Vb5pos CD8 (OT1 mice) and Vb5pos CD4 (OT2 mice) T cells in peripheral blood before and post-transplantation (last measurement before sacrifice day) analyzed by flow cytometry. (C) Representative flow cytometry data showing PD1pos T cell fractions in peripheral blood samples of WT and OT1/OT2 mice post-transplantation. Left panel shows PD1 isotype control, right panel shows actual PD1 signal.
Fig 3: Non-cancer-specific T cells upregulate PD1 after CLL transplantation. (A) Representative flow cytometry data of splenocytes of transplanted OT1/OT2 mice stained with a FITC-conjugated antibody cocktail targeting various TCR Vb chains (VbX) and a PE-conjugated antibody recognizing the OVA-specific TCR (Vb5). The VbX antibody cocktail recognizes around 71% of all endogenous TCR Vb chains of a WT mouse. Thus, Vb5neg VbXneg populations represent endogenous T cells expressing single TCR Vb chains not covered by the VbX antibody cocktail. The left panel represents an isotype control for VbX, the right panel shows the actual VbX signal. (B) PD1pos T cell fractions of OT1/OT2 mice (n = 3), of the flow cytometry quadrants depicted in A. Populations consisting of <100 events were not analyzed. P values are only shown when significant. Error bars represent SD. (C) Correlation of PD1pos T cell fraction in Vb5pos and Vb5neg T cell populations in OT1/OT2 mice. n.d.: not detected, SD: standard deviation.
Fig 4: PD1 expression of T cells in OT1 Rag and OT2 Rag mice. (A) Transplantation scheme of OT1/OT2, OT1 Rag/OT2 Rag, and WT mice. Spleens derived from transplanted or non-transplanted WT mice were homogenized and T cells were depleted via a pan-B/CLL cell MACS isolation in order to inject CLL or healthy B cells without large amounts of endogenous T cells (created with BioRender.com, 11th June 2021). (B) Flow cytometry data of splenic PD1pos CD8pos T cells of WT (all Vbpos), OT1 (Vb5pos), and OT1 Rag (Vb5pos) mice either transplanted with CLL cells (CLL Tx), B cells (B cells Tx), or non-transplanted (no Tx). Bars represent mean. (C) Splenic CD4pos PD1pos T cells from either CLL-transplanted (CLL Tx) or non-transplanted (no Tx) WT (all Vbpos), OT2 (Vb5pos), or OT2 Rag (Vb5pos) mice. Bars represent mean. (D) Percentage of non-CLL-specific Vb5pos CD8pos PD1pos T cells in OT1 Rag mice in peripheral blood before and after tumor transplantation. (E) Percentage of CD4pos Vb5pos PD1pos T cells of OT2 Rag mice before and after tumor injection in peripheral blood. (F) Correlation of the amount of endogenous T cells (Vb5neg) (x-axis) with PD1 levels of CD8pos T cells (y-axis) in WT, OT1, and OT1 Rag mice. CD8pos T cells of y-axis correspond to endogenous T cells (all Vbpos) in WT mice and Vb5pos T cells in OT1 and OT1 Rag mice. (G) Correlation of the amount of endogenous T cells (Vb5neg) (x-axis) with PD1 levels of CD4pos T cells (y-axis) in WT, OT2, and OT2 Rag mice. CD4pos T cells of y-axis correspond to endogenous T cells (all Vbpos) in WT mice and Vb5pos T cells in OT2 and OT2 Rag mice. (H) Representative flow cytometry data of PD1pos spleen T cell (Vb5pos) fractions in OT1 Rag and OT2 Rag mice. Left panel shows isotype control of PD1, right panel shows actual PD1 levels.
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