Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

Mechanistic understanding of the immune checkpoint receptor PD1 is largely based on mouse models, but human and mouse PD1 orthologs exhibit only 59.6% identity in amino acid sequences, raising the question of potential cross-species functional differences. Based on our biochemical evidence that human PD1 is more inhibitory than mouse PD1, we addressed the functional consequence of PD1 humanization in a mouse melanoma model with adoptively transferred T cells. Using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), we found that humanization of PD1 intracellular domain (ICD) in CD8 T cells decreases the numbers, effector functions, and differentiation of intratumoral T cells. Specifically, PD1 humanization reduced the number of effector-like exhausted T cell subset while increasing the precursor-exhausted T cell subset, the latter of which is known to respond to anti-PD(L)1 therapy. Adoptively transferred P14 TCR-transgenic CD8 T cells expressing either wild-type or ICD-humanized mouse PD1 were isolated from the B16.gp33 tumor in each mice, hash-tagged with ssDNA-conjugated anti-MHCI total-seq antibodies, and then analyzed using CITE-seq.