Introduction

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) have revolutionized cancer treatment in the recent years, affording long-term survival benefit in a broad range of cancer patients. PD-1 is an inhibitory cell surface receptor that is upregulated upon T cell activation. Upon binding to its ligands PD-L1 and PD-L2, that are expressed on antigen presenting cells and/or tumor cells [1], PD-1 recruits the phosphatases SHP1 and SHP2 which in turn suppresses T cell activation and function [2]. While this mechanism of regulating T cell immune response is necessary in maintaining immune tolerance to autoantigens, several tumors over-express PD-L1 in response to inflammatory mediators and downregulate anti-tumor function of T cells leading to tumor immune evasion [64]. Plots were generated with ggplot2 version 3.4 [65], https://cloud.r-project.org/web/packages/ggplot2/index.html) within R-4.0.2 (https://cran.r-project.org). Due to the analysis of DTC samples generated in two groups, batch correction was performed with combat-seq (Zhang et al., NAR Genomics and Bioinformatics, 2020: 2(3):lqaa078) after iterative testing of gene and sample filtering parameters using principal components analysis with the preprocessCore version 1.56 R package (Bolstad B 2023, https://github.com/bmbolstad/preprocessCore).

Pathway analysis

The logFC values from edgeR differential analysis were used for gene set enrichment analysis (GSEA). GSEA was performed with these gene sets via the clusterProfiler R version 4 package (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html, [66]. The Gene Ontology Biological Process (GOBP) and Hallmark gene sets were obtained from the Broad Institute’s msig.db website (http://www.gsea-msigdb.org/gsea/msigdb). Immune gene sets were extracted from the LM22 matrix [30] by sorting the coefficients for each of the 22 immune cell types and retaining the top 30 genes for each. Immune exhaustion gene sets were derived from the seminal paper on immune exhaustion in the LCMV model system [29].

Statistics

Statistical tests were carried out using GraphPad Prism Software version 9. Statistical tests were used as indicated in the figure legends. p values are reported as follows: *p < 0.05, ** p< 0.01, ***p < 0.001 and ****p < 0.0001.