Tyrosinase in melanoma inhibits anti-tumor activity of PD-1 deficient T cells

Abstract

Background: Melanoma is one of the most commonly diagnosed malignancies and serves as a model for studying immunotherapy. The B16 melanoma model, resembling human cold tumors that lack T cell infiltration and show minimal response to PD-1 blockade, is widely used for studying melanoma and its resistance to immunotherapy. Therefore, understanding the molecular basis that prevents T cell-mediated anti-tumor activity in B16 melanoma is of great significance.

Results: In this study, we generated tyrosinase knockout B16 melanoma cells using CRISPR/Cas9 and discovered that tyrosinase in melanoma significantly inhibits the anti-tumor activity of T cells. Tyrosinase deficiency leads to a 3.80-fold increase in T-cell infiltration and enhances T-cell 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 regulatory T cells in PD-1 knockout mice.

Conclusions: These findings suggest that targeting tyrosinase could convert cold tumors into an immune-responsive state in vivo using murine models. Inhibiting tyrosinase could enhance the effectiveness of PD-1 blockade, offering a new approach for melanoma patients who fail in current PD-1 inhibitor treatment.

Keywords: Melanoma; PD-1; Tumor infiltrating T cells; Tyrosinase.

Fig. 1

Generation of Tyr knockout B16 melanoma cells by CRISPR/Cas9. A Elevated Tyr expression in SKCM tumor tissues compared to normal tissues. The data were obtained from the GEPIA2 database (http://gepia2.cancer-pku.cn). T: SKCMtumor tissues, N: Normal tissues. (*p < 0.05). B Analysis of the correlation between Tyr expression and clinical prognosis in SKCM patients, indicated by overall survival rates from the GEPIA2 database. C Illustration of sgRNA sequences targeting Tyr gene in B16-F10 cells. A visual depiction of the three guide RNA sequences designed to target exon 1 of the Tyr gene in B16-F10 cells is presented in this schematic. The protospacer adjacent motifs (PAMs) are highlighted in red. D Identification of Tyr knockout clones in B16-F10 cells through PCR screening. The three deletion clones are identified as C1, C2, and C3. M referred to the DNA size marker, WT referred to the wild-type B16-F10 control, and H₂O was used as a negative control for PCR amplification. E Visual comparison of pigmentation in wild-type (WT) and Tyr knockout (KO) B16-F10 cells

Fig. 2

Characterization of tumor-infiltrating immune cells in WT and Tyr^−/− B16-F10 melanoma model. A Representative images of WT and Tyr^−/− B16-F10 tumors from female C57BL/6 mice on day 14. B The weight of WT and Tyr^−/− B16-F10 tumors on day 14. C The immune infiltration profiling in WT and Tyr^−/− B16-F10 tumors through scRNA-Seq. CD45⁺ immune cells from WT and Tyr^−/− B16-F10 tumors were sorted and analyzed. Pooled samples from three mice per group were used. Dimensionality reduction was done with the t-SNE algorithm and cell clustering using Seurat. Each cluster was denoted a unique color. t-SNE, t-distributed stochastic neighbor embedding. D Proportions of different immune cell types in WT and Tyr^−/− B16-F10 tumors. E Gating strategy for tumor-infiltrating immune cells, including CD11b⁺ cells and T cells, in WT and Tyr^−/− B16-F10 tumors. Cells were gated based on CD45⁺, CD11b⁺, CD19⁺, CD3⁺ TCR-β⁺, CD4⁺, and CD8⁺ markers. F Quantification of tumor-infiltrating CD45⁺ cells in WT and Tyr^−/− B16-F10 tumors. G The proportion of T cells, CD4⁺ T cells and CD8⁺ T cells among tumor-infiltrating CD45⁺ cells in WT and Tyr^−/− B16-F10 tumors. H Proportion of CD11b⁺ cells among tumor-infiltrating CD45⁺ cells in WT and Tyr^−/− B16-F10 tumors. I-J Cell counts of T cells, CD4⁺ T cells, CD8⁺ T cells (I), and CD11b⁺ cells (J) per gram of tissue in WT and Tyr^−/− B16-F10 tumors. Data are presented as mean ± SEM (WT tumors, n = 9; Tyr^−/− tumors, n = 8 or 9). All data are pooled from two independent experiments. Statistical significances were calculated using Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant)

Fig. 3

Enhanced PD-1 and Foxp3 expression in tyrosinase-deficient tumors T Cells. A-B Flow cytometric analysis of PD-1 expression in WT and Tyr^−/− tumors. Cells were firstly gated on CD45⁺ and Lin⁻ (CD19, CD11b, NK1.1, Ly6G), then further gated on CD5⁺ TCR-β⁺, CD4⁺, and CD8⁺ cells. C Percentage of PD-1⁺ cells within CD4⁺ and CD8⁺ T cells in WT and Tyr^−/− B16-F10 tumors. D-E Flow cytometric analysis of PD-1 expression in WT and Tyr^−/− tumors. Proportion of CTLA-4⁺ cells within CD4⁺ and CD8⁺ T cells in WT and Tyr^−/− B16-F10 tumors (E). F Flow cytometric analysis of Foxp3 expression in WT and Tyr^−/− tumors. Histograms on the right show the proportion of Foxp3 in CD4⁺ T cells in WT and Tyr^−/− tumors. Data are presented as mean ± SEM (WT tumors, n = 6; Tyr^−/− tumors, n = 5). All data are representative of two independent experiments. Statistical significances were calculated using Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001)

Fig. 4

Altered cold-tumor immunophenotype revealed by single-cell RNA sequencing in tyrosinase-deficient B16 melanoma. A q-PCR analyses of Tbx21, Nr4a1, TNF-α and IFN-γ mRNAs in WT and Tyr^−/− B16-F10 tumors (WT tumors, n = 5; Tyr^−/− tumors, n = 5). Each point represents the mean value obtained from two replicates for an individual mouse. Statistical significances were calculated using Student’s t-test (* p < 0.05; ** p < 0.01). B The expression levels and distribution of CD3 in WT and Tyr^−/− tumors demonstrated by t-SNE. C t-SNE visualization of the expression of IL-2, Tbx21, Nr4a1, TNF-α, and IFN-γ in CD4 cells (cluster 5). D t-SNE visualization of the expression of IL-2, Tbx21, Nr4a1, TNF-α, and IFN-γ in CD8 cells (cluster 10)

Fig. 5

The effects of PD-1 blockade on tumor growth and T cells in Tyr^+/+ B16-F10 tumors. A The tumor weight of Tyr^+/+ B16-F10 cell grafted Pdcd1^−/− and B6 mice. B Quantification of tumor-infiltrating T cells by flow cytometry in Tyr^+/+ B16-F10 tumors from Pdcd1^−/− and B6 mice. C Representative flow cytometry analysis of CTLA-4⁺ cells in Tyr^+/+ B16-F10 tumors from Pdcd1^−/− and B6 mice. Cells were identified based on CD45⁺, TCR-β⁺, CD4⁺, and CD8⁺ markers. D Quantification of the frequency of CTLA-4⁺ cells within CD4⁺ and CD8⁺ T cells in Tyr^+/+ B16-F10 tumors from Pdcd1^−/− and B6 mice. E–F Flow cytometric analysis of Foxp3 expression in Tyr^+/+ B16-F10 tumors from Pdcd1^−/− and B6 mice. Proportion of Foxp3 in CD4⁺ T cells of Tyr^+/+ B16-F10 tumors from Pdcd1^−/− and B6 mice (F). Data are presented as mean ± SEM (B6 mice, n = 6; Pdcd1^−/− mice, n = 8). Statistical significances were calculated using Student’s t-test (ns, not significant)

Fig. 6

Loss of tyrosinase in B16 melanoma unleashes T cell-mediated tumor immunity of PD-1 deficient mice. A The tumor weight of Tyr^−/− B16-F10 cell grafted Pdcd1^−/− and B6 mice. B Quantification of tumor-infiltrating T cells by flow cytometry in Tyr^−/− B16-F10 tumors from Pdcd1^−/− and B6 mice. C Representative flow cytometry analysis of CTLA-4⁺ cells in Tyr^−/− B16-F10 tumors from Pdcd1^−/− and B6 mice. Cells were identified based on CD45⁺, TCR-β⁺, CD4⁺, and CD8⁺ markers. D Quantification of the frequency of CTLA-4⁺ cells within CD4⁺ and CD8⁺ T cells in Tyr^−/− B16-F10 tumors from Pdcd1^−/− and B6 mice. E Flow cytometric analysis of Foxp3 expression in Tyr^−/− B16-F10 tumors from Pdcd1^−/− and B6 mice. Proportion of Foxp3 in CD4⁺ T cells of Tyr^−/− B16-F10 tumors from Pdcd1^−/− and B6 mice. F q-PCR analyses of Tbx21, Nr4a1, TNF-α and IFN-γ mRNAs in in Tyr^−/− B16-F10 tumors from Pdcd1^−/− and B6 mice (WT tumors, n = 4; Tyr^−/− tumors, n = 4). Each point represents the mean value obtained from two replicates for an individual mouse. Data in 6 A-E are presented as mean ± SEM (B6 mice, n = 7; Pdcd1^−/− mice, n = 7), representative of two independent experiments. Statistical significances were calculated using Student’s t -test (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant)

Fig. 7

Schematic diagram. In tyrosinase-sufficient Tyr^+/+ tumors, Pdcd1^−/− mice exhibit a tumor weight comparable to that of B6 WT mice. There are no significant changes in T-cell infiltration or Tregs expression in the tumors of either B6 WT or Pdcd1^−/− mice. The absence of tyrosinase in B16 melanoma enhances the anti-tumor activity of PD-1 deficient T cells. In tyrosinase-deficient Tyr^−/− tumor, the tumor weight is significantly reduced in Pdcd1^−/− mice compared to the B6 WT controls. This reduction is associated with a significant increase in T-cell infiltration and a decrease in Treg frequencies in the Pdcd1^−/− mice