Tumor PD-L1 engages myeloid PD-1 to suppress type I interferon to impair cytotoxic T lymphocyte recruitment (single cells)

A hallmark of PD-1/L1 blockade is long-term, sustained remission of metastatic disease. How the immune system coordinates the destruction of macro- and micro-metastases following checkpoint blockade, however, remains unclear. Here, we show that tumor-expressed PD-L1 (tPD-L1) enhanced metastasis in a mechanism distinct from and independent of its role in primary tumor growth. This difference in metastatic growth was mediated by cytotoxic T lymphocytes (CTLs), however, tPD-L1 did not promote effector CTL exhaustion or suppress lytic activity in vivo. Instead, single cell RNA sequencing revealed that tPD-L1 engaged macrophage-expressed PD-1 to antagonize type I interferon production and signaling, creating an immunologically ‘cold’ microenvironment. Loss of tPD-L1 eliminated metastases by driving interferon-mediated sensitization of tumor cells to CTL lysis and CTL recruitment. Balb/c mice were injected i.v. with 2.5*105 4T1 WT or PDL1-KO cells. After fourteen days, mice were sacrificed, lungs were perfused with PBS and harvested. Following collagenase digestion, leukocytes were positively selected using CD45 magnetic nanobeads (Biolegend). Cells were suspended in PBS (without Ca or Mg) with 0.04% BSA. Cell viability was assessed using trypan blue and assured of ≥ 80% viable cells. Cells were loaded at a concentration to capture approximately 2-3 x 103 targeted cells on a Chromium Chip B (10x Genomics). scRNA-seq libraries were generated using the Chromium Single Cell 3’ Reagent Kit v3 (10x Genomics) according to the manufacturer’s instructions. The libraries were sequenced on Illumina NextSeq 500 platform under the following sequencing protocol: 28 bp (Read 1), 8 bp (indexing Run), and 91 bp (RNA Read 2). Reads from the raw fastq files were mapped to mm10 Mouse Genome reference by STAR aligner in Cell Ranger 3.1.0 pipeline. Alignment generated 30-39K reads/cell with 75-102 million reads per sample at a target of 2.3-2.7 x 103 cells per sample identified at 88% in Q30 Bases in RNA read as well as greater than 91% genome-mapping rates along with approximately 1600 median genes per cell.