High Dimensional Analysis Delineates Myeloid and Lymphoid Compartment Remodeling during Successful Immune Checkpoint Cancer Therapy

Using complementary forms of high dimensional profiling we define differences in CD45+ cells from syngeneic mouse tumors that either grow progressively or eventually reject following immune checkpoint therapy (ICT). Unbiased assessment of gene expression of tumor infiltrating cells by single cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages distinguishable by the combinatorial presence or absence of CD206, CX3CR1, CD1d and iNOS, markers of different macrophage activation states that change over time during ICT in a manner partially dependent on IFNγ. Both the CyTOF data and additional analysis of scRNAseq data support the hypothesis that macrophage polarization/activation results from effects on circulatory monocytes/early macrophages entering tumors rather than on pre-polarized mature intratumoral macrophages. Thus, ICT induces transcriptional and functional remodeling of both myeloid and lymphoid compartments. Droplet-based 3′ end massively parallel single-cell RNA sequencing was performed by encapsulating sorted live CD45+ tumor infiltrating cells into droplets and libraries were prepared using Chromium Single Cell 3′ Reagent Kits v1 according to manufacturer’s protocol (10x Genomics). The generated scRNAseq libraries were sequenced using an Illumina HiSeq2500.