CD4 and CD8 T-cell Lymphocytes from Penile Squamous Cell Carcinoma Tumors are more Differentiated with higher PD-1 Expression compared to Lymphocytes in Peripheral Circulation.

Abstract

Background: Penile squamous cell carcinoma (PSCC) is the commonest malignancy of the penis, with a higher incidence and poor treatment outcomes in developing countries. T-cell phenotypes have been shown to identify patients who may respond favorably to immune therapy, and also associate with treatment outcomes. This study aimed to determine and compare the tumor and peripheral blood T-cell phenotypes of individuals with PSCC, and whether factors such as smoking and presence of HPV associate with these T-cell phenotypes.

Methods: We conducted a prospective cross-sectional study at the University Teaching Hospital, Lusaka, Zambia. Participants with a histologically-confirmed PSCC were recruited into the study. Socio-demographic information was obtained, and whole blood was collected and subjected to peripheral blood mononuclear cells (PBMCs) isolation. Fresh penile tumors were mechanically and enzymatically digested. CD4 and CD8 cells were sorted from PBMCs and tumor, stained using antibodies against CD3, CD45RO, CCR7, PD-1, CD103 and CD69, and subjected to flow cytometry. Parts of the tumor were subjected to HPV detection, and histological grading and staging.

Results: Twenty-four participants were recruited into the study. The median age was 55.5 years, 45.8% were smokers, 87.5% were HIV positive, 62.5% had high-risk HPV detected in the tumors, and 25% had advanced-stage disease. There was a significantly higher proportion of naïve cells among CD4 T-cells from PBMCs than tumor (40.2% vs 3.8%; p=0.01). CD4 T cells from the tumor demonstrated a significantly higher proportion of cells expressing CD69 (3.2% vs 95.9%; p=0.0001), CD103 (0.7% vs 7.3%; p=0.0001), and PD-1 (35.5% vs 92%; p=0.0001) than the ones from PBMCs. Tumoral CD8 T-cells had a significantly lower proportion of terminally-differentiated effector cells but higher proportion of central memory cells compared to PBMCs, (7.9% vs 15.1%, p=0.04) and (55% vs 14.4%; p=0.01) respectively. Tumoral CD8 T-cells also had a significantly higher proportion of cells expressing CD69 (96.7% vs 8.5%; p=0.0001), CD103 (22.2% vs 1.2%; p=0.0001), and PD-1 (79.3% vs 18.8%; p=0.0001) when compared to the PBMCs. Early-stage disease was associated with a significantly higher proportion of central memory CD4 T-cells among the PBMCs when compared with advanced stage disease (46.7% vs 30%; p=0.01), while smoking was associated with a significantly higher proportion of tumoral CD8 T-cells expressing the homing marker CD103 (28.2% vs 17.8%; p=0.01).

Conclusion: PSCC tumors demonstrate a higher proportion of primed T-cells with a memory phenotype compared to T-cells in the circulation. T-cells from PSCC tumors also have a higher proportion of cells expressing the immune checkpoint PD-1 and homing markers than those from the circulation.