PD-1, PD-L1, and PD-L2 Gene Expression and Tumor Infiltrating Lymphocytes in Canine Melanoma
Valentina B. Stevenson, Samantha N. Perry, S. Michelle Todd, William R. Huckle, Tanya LeRoith
Abstract
Melanoma in humans and dogs is considered highly immunogenic; however, the function of tumor-infiltrating lymphocytes (TILs) is often suppressed in the tumor microenvironment. In humans, current immunotherapies target checkpoint molecules (such as PD-L1, expressed by tumor cells), inhibiting their suppressive effect over TILs. The role of PD-L2, an alternative PD-1 ligand also overexpressed in malignant tumors and in patients with anti-PD-L1 resistance, remains poorly understood. In the current study, we evaluated the expression of checkpoint molecule mRNAs in canine melanoma and TILs. Analysis of checkpoint molecule gene expression was performed by RT-qPCR (real-time quantitative polymerase chain reaction) using total RNA isolated from formalin-fixed and paraffin-embedded melanomas ( n = 22) and melanocytomas ( n = 9) from the Virginia Tech Animal Laboratory Services archives. Analysis of checkpoint molecule expression revealed significantly higher levels of PDCD1 ( PD-1) and CD274 ( PD-L1) mRNAs and an upward trend in PDCD1LG2 ( PD-L2) mRNA in melanomas relative to melanocytomas. Immunohistochemistry revealed markedly increased numbers of CD3 + T cells in the highest PD-1-expressing subgroup of melanomas compared to the lowest PD-1 expressors, whereas densities of IBA1 + cells (macrophages) were similar in both groups. CD79a + cell numbers were low for both groups. As in human melanoma, overexpression of the PD-1/PD-L1/PD-L2 axis is a common feature of canine melanoma. High expression of PD-1 and PD-L1 correlates with increased numbers of CD3 + cells. Additionally, the high level of IBA1 + cells in melanomas with low PD-1 expression and low CD3 + cells levels suggest that the expression of checkpoint molecules is modulated by interactions between T cells and cancer cells rather than histiocytes.