Single-cell transcriptomics reveals the effect of PD-L1/TGF-β blockade on the tumor microenvironment

Background: The anti-tumor activity of anti-PD-1/PD-L1 therapies correlates with T cell infiltration in tumors. Thus, a major goal in oncology is to find strategies that enhance T cell infiltration and efficacy of anti-PD-1/PD-L1 therapy. TGF-beta has been shown to contribute to T cell exclusion, and anti-TGF-beta improves anti-PD-L1 efficacy in vivo. However, TGF-beta inhibition has frequently been shown to induce toxicity in the clinic, and the clinical efficacy of combination PD-L1 and TGF-beta blockade has not yet been proven. To identify strategies to overcome resistance to PD-L1 blockade, the transcriptional programs associated with PD-L1 and/or TGF-beta blockade in the tumor microenvironment should be further elucidated. Results: We used single-cell RNA sequencing in a mouse model to characterize the transcriptomic effects of PD-L1 and/or TGF-beta blockade on nearly 30,000 single cells in the tumor and surrounding microenvironment. Combination treatment led to upregulation of immune response genes, including multiple chemokine genes such as CCL5, in macrophages, and downregulation of extracellular matrix genes in fibroblasts. Analysis of publicly available tumor transcriptome profiles showed that the chemokine CCL5 was strongly associated with immune cell infiltration in various human cancers. Further investigation with in vivo models showed that intratumorally administered CCL5 enhanced cytotoxic lymphocytes and the anti-tumor activity of anti-PD-L1. Conclusions: Taken together, our data could be leveraged translationally to complement or find alternatives to anti-PD-L1 plus anti-TGF-beta combination therapy, for example through companion biomarkers, and/or to identify novel targets that could be modulated to overcome resistance.