UC San Diego

Programmed Death Ligand 1 (PD-L1) overexpressed on cancer cells can help tumors evade the immune system by triggering the coinhibitory receptor Programmed Death 1 (PD-1) on T-cells, subsequently inhibiting T-cell activity. PD-L1 binding to PD-1 is reciprocally regulated by two proteins, CD80 and CKLF-like MARVEL-containing Transmembrane 6 (CMTM6). CD80 is a key immunostimulatory ligand that binds PD-L1 in cis on the same membrane, and blocks PD-L1:PD-1 inhibitory signaling. Conversely, CMTM6 is a multi-pass transmembrane protein that prevents the degradation of PD-L1, and thereby promotes PD-L1:PD-1 inhibitory signaling. However, the biochemical nature of CMTM6:PD-L1 interaction, and how it may be influenced by CD80, is poorly defined. Here, I established an intact cell-based, quantitative, and functional assay of CMTM6 to determine the molecular requirements for PD-L1 and CMTM6 interaction, and its potential regulation by CD80. Using domain-swapping and cellular reconstitution, I found that PD-L1 chimeras with the transmembrane (TM), extracellular (EC), or intracellular (IC) domains of PD-L1 swapped with that of the CMTM6 non-client PD-L2, were all upregulated by CMTM6, but to a lesser extent than wild-type PD-L1. The TM replacement showed the greatest impairment in CMTM6-mediated upregulation, suggesting that CMTM6 primarily interacts with the TM of PD-L1. Finally, co-expression of CD80 did not affect the magnitude of the CMTM6 upregulation of PD-L1, suggesting that cis-CD80:PD-L1 interaction does not perturb CMTM6:PD-L1 interaction. These data provide a better understanding of the trimolecular relationship between PD-L1, CMTM6, and CD80.