DRG2 is required for cell surface localization of PD-L1 in melanoma cells and optimum response to anti-PD-1 immune checkpoint blockade [scRNA-seq]

Tumors expressing high level of programmed cell death-1 (PD-1) ligand 1 (PD-L1) are more likely to respond to immune checkpoint blockers (ICBs) targeting PD-1 or PD-L1. However, more than half of tumor patients with high PD-L1 expression does not respond to ICBs and the underlying mechanisms are yet to be clarified. Here we show that depletion of developmentally regulated GTP-binding protein 2 (DRG2) inhibited recycling of endosomal PD-L1 and reduced surface PD-L1 level in melanoma cells. DRG2-depleted cells showed decreased binding with recombinant PD-1. Although DRG2-depleted cells expressed high levels of PD-L1, anti-PD-1 ICB did not activate T cells within DRG2-depleted tumors and failed to improve the survival of DRG2-depleted tumor-bearing mice. Cohort analysis of melanoma patients under anti-PD-1 treatment revealed that patients bearing tumors with high DRG2 protein level were more sensitive to PD-1 anti-PD-1 ICBs. These findings identify DRG2 as a regulator of recycling of endosomal PD-L1 and a key determinant for response to anti-PD-1 ICB and provide insights into how to increase the correlation between PD-L1 expression and response to ICB. B16F10 tumors were collected at 3 days after 2nd administration of anti-PD-1 antibody. The tumor samples were first mechanically dissociated using a scalpel, then enzymatically dissociated in digestion medium (2mg/ml Collagenase P (Sigma Aldrich) and 0.2mg/ml DNAse I (Roche) in DMEM (Thermo Fisher Scientific)). Red blood cells were removed from the cell suspension using red blood cell lysis buffer (Roche), and the cells were filtered using a 40-µm Flowmi tip strainer (VWR). The number of living cells was determined using a LUNA automated cell counter (Logos Biosystems). We performed single-cell RNA-seq on the single-cell suspension using the Chromium Single Cell 3’ Solution from 10x Genomics according to the manufacturer’s instructions. Up to 5,000 cells were loaded onto a 10x Genomics cartridge for each sample. Cell-barcoded 5′ gene expression libraries were sequenced on an Illumina NextSeq and/or NovaSeq6000 system.