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Figure S1 from VISTA Targeting of T-cell Quiescence and Myeloid Suppression Overcomes Adaptive Resistance

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posted on 2023-04-04, 02:04 authored by Evelien Schaafsma, Walburga Croteau, Mohamed ElTanbouly, Elizabeth C. Nowak, Nicole C. Smits, Jie Deng, Aurelien Sarde, Cecilia A. Webber, Dina Rabadi, Chao Cheng, Randolph Noelle, J. Louise Lines

Quality control and cluster identification of CD45-enriched scRNAseq data. In the I/V model, BALB/c mice were administered 100k CT26 cells and treated when tumors reached a size of 40mm3 with isotype (I) or anti-VISTA (V). In the CPV model, BALB/c mice were administered 100k CT26 cells and treated with anti-CTLA-4 and anti-PD-1 plus isotype (CP) or plus anti-VISTA (CPV) when tumors reached a size of 600mm3. In both models, CD45+ cells were isolated from the TME and analyzed by scRNAseq. (A) Histogram of the number of reads (left) and the number of genes (right) detected in all cells within the I/V dataset. (B) Histogram of the number of reads (left) and the number of genes (right) detected in all cells within the CP/CPV dataset. (C) Visual representation of lymphoid and myeloid cell distributions between experimental replicates using UMAP plots. (D) Expected and observed Local Inverse Simpson’s Index (LISI) measuring the degree of mixing among datasets. The LISI score ranges from 1 in an unmixed space to 2 in a perfectly mixed space with equal numbers of cells in each dataset. (E) AUCs calculated based on fivefold cross-validation where cells from each cluster are split into training and test datasets, a logistic regression classifier is trained on the training dataset and applied to the test dataset to measure model accuracy for each cluster. Higher AUC values correspond to better cluster discrimination. (F) Comparison of the number of cells detected per cluster in the I/V and CP/CPV datasets. (G) Comparison of the number of genes detected per cluster. (H) UMAP of all CD45+ cells sorted from I/V- and CP/CPV-treated tumor infiltrates, colored by cell cluster based on Louvain clustering. (I) Dot plots of cluster-defining marker genes for cell type annotations (as in H). Dot size represents fraction of cells expressing a gene in each cluster. Dot color represents scaled average expression by gene column.

Funding

National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)

United States Department of Health and Human Services

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Division of Cancer Epidemiology and Genetics, National Cancer Institute (DCEG)

Center for Biomedical Informatics and Information Technology, National Cancer Institute (CBIIT)

Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases (DMID)

Cancer Prevention and Research Institute of Texas (CPRIT)

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ARTICLE ABSTRACT

V domain immunoglobulin suppressor of T-cell activation (VISTA) is a premier target for cancer treatment due to its broad expression in many cancer types and enhanced expression upon development of adaptive immune checkpoint resistance. In the CT26 colorectal cancer model, monotherapy of small tumors with anti-VISTA resulted in slowed tumor growth. In a combination therapy setting, large CT26 tumors showed complete adaptive resistance to anti–PD-1/CTLA-4, but inclusion of anti-VISTA led to rejection of half the tumors. Mechanisms of enhanced antitumor immunity were investigated using single-cell RNA sequencing (scRNA-seq), multiplex image analysis, and flow cytometry of the tumor immune infiltrate. In both treatment models, anti-VISTA upregulated stimulated antigen presentation pathways and reduced myeloid-mediated suppression. Imaging revealed an anti-VISTA stimulated increase in contacts between T cells and myeloid cells, further supporting the notion of increased antigen presentation. scRNA-seq of tumor-specific CD8+ T cells revealed that anti-VISTA therapy induced T-cell pathways highly distinct from and complementary to those induced by anti–PD-1 therapy. Whereas anti–CTLA-4/PD-1 expanded progenitor exhausted CD8+ T-cell subsets, anti-VISTA promoted costimulatory genes and reduced regulators of T-cell quiescence. Notably, this is the first report of a checkpoint regulator impacting CD8+ T-cell quiescence, and the first indication that quiescence may be a target in the context of T-cell exhaustion and in cancer. This study builds a foundation for all future studies on the role of anti-VISTA in the development of antitumor immunity and provides important mechanistic insights that strongly support use of anti-VISTA to overcome the adaptive resistance seen in contemporary treatments involving PD-1 and/or CTLA-4.See related Spotlight by Wei, p. 3

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