Tyrosinase in melanoma inhibits anti-tumor activity of PD-1 deficient T cells
Melanoma is one of the most commonly diagnosed malignancies and serves as a model for studying immunotherapy. The B16 melanoma model is characterized by low T cell infiltration in the tumor, and blocking the PD-1 pathway shows no significant anti-tumor activity, mirroring melanoma patients with a cold tumor immunophenotype. Therefore, understanding the molecular basis that prevents T cell-mediated anti-tumor activity in B16 melanoma is of great significance. In this study, we generated Tyr knockout B16 melanoma cells using CRISPR/Cas9 and discovered that tyrosinase in melanoma significantly inhibits the anti-tumor activity of T cells. Tyrosinase deficiency significantly increases T-cell infiltration and activation within the tumor. Single-cell RNA sequencing reveals an altered cold tumor immunophenotype in tyrosinase-deficient B16 melanoma. In wild-type mice, T cells in tyrosinase-deficient tumors express elevated levels of PD-1 and Foxp3. However, strikingly, in PD-1 deficient mice, the loss of tyrosinase in B16 melanoma unleashes the anti-tumor activity of PD-1 deficient T cells. This enhanced anti-tumor activity is explained by significantly increased tumor T cell infiltration accompanied by reduced frequencies of Tregs in PD-1 knockout mice. Targeting tyrosinase may enhance the anti-tumor efficacy of PD-1 blockade in cold tumors, offering a novel strategy to render cold tumors responsive to immunotherapy. Clinically, higher levels of tyrosinase expression in human melanoma are associated with a poorer prognosis, indicating that our findings could potentially improve the efficacy of immunotherapy in melanoma patients. Overall design: For scRNA-seq analysis of tumor-infiltrating immune cells (TIICs), wildtype (WT) , oca2 knockout and Tyr knockout B16F10 melanoma cells were implanted in the right flank of female C57BL/6 mice (n=3). At the endpoint, the tumors were dissected from the surrounding fascia, and single cell suspensions were prepared from both the Tyr knockout and the WT melanoma cells. TIICs were isolated using the FACSAria ? cell sorter (BD Biosciences, USA) and stained with anti-mouse CD45.2 PE-cy7 (eBioscience). CD45+ TIICs were labeled with the BD Single-Cell Multiplexing Kit (BD Biosciences) according to the manufacturer's protocol. Cells from each group were labeled with sample tags, washed, and pooled. The pooled sample was then counted, resuspended, and subjected to single-cell capture and cDNA Synthesis using the BD Rhapsody Express Single-Cell Analysis System (BD Biosciences). cDNA libraries were prepared using the mRNA Targeted and Sample Tag Library preparation method along with the BD Rhapsody Targeted mRNA Amplification and BD Single-Cell Multiplexing Kits and protocols from BD Biosciences. The libraries were sequenced on the Illumina Novaseq platform (Novogene), and clustering analysis and high-dimensional visualization were performed using the SeqGeqTM software.