Immune cell dynamics induced by a single dose of pembrolizumab as revealed by single-cell RNA profiling

Abstract

Background: To improve our understanding of the molecular mechanisms underlying response to immune checkpoint inhibition, we monitored dynamic changes within the tumour microenvironment exposed to immune checkpoint inhibition by single-cell RNA sequencing (scRNA-seq). Method(s): In a window-of-opportunity setting, fresh tumor tissue was collected from breast cancer patients before and 10 days after a single dose of 200 mg Pembrolizumab monotherapy. Hematoxylin-eosin (H&E) staining to determine tumor infiltrating lymphocytes (TILs) and scRNA-seq was performed on fixed and fresh tumor tissue, respectively. Single cell suspensions were subjected to 10X Genomics scRNA- and T-cell receptor-sequencing using the Chromium Single Cell 5' kit on an Illumina NovaSeq. Expression matrices were generated using CellRanger and analyzed by Seurat. Dimensionality reduction was applied by PCA. Result(s): Currently, 7 paired tumor samples have been analyzed. All were ER/PRnegative IDA, of which 3 were pre-treated with neo-adjuvant chemotherapy. On H&E slide, there were no notable differences in TILs comparing biopsies before versus after Pembro. By scRNA-seq, we did observe several prominent changes. First, there was a decrease in cancer cells versus an increase in immune cells after Pembro. Second, the increase in T-cells was due to an increased prevalence of exhausted CD4+and CD8+ cytotoxic cells, as well as regulatory T-cells, which were all positive for PD1 expression. Cytolytic activity in these T-cell subpopulations also increased significantly, and this was accompanied by a specific increase in TCR clonotypes in these. Finally, in 3 out of 7 pairs such changes in T-cells were not detectable. In these post-treatment samples, a decrease in expression ofMHC class 1 genes was observed specifically in cancer cells. Conclusion(s): Our data suggest that scRNA-seq creates accurate maps of the tumor micro-environment and is capable of monitoring dynamic changes already induced by a single dose of immunotherapy. Particularly, in 4/7 patients changes in T-cells reactivity reminiscent of a clinical response to Pembrolizumab were noted. Other patients failed to display such reactivity presumably due to a failure in presenting neo-epitopes byMHCclass 1 genes.