Human papillomavirus infection affects the immune microenvironment and antigen presentation in penile cancer

Abstract

Penile squamous cell carcinoma (PSCC) is a largely neglected condition, predominantly affecting underdeveloped regions, and is associated with risk factors such as low socioeconomic status, phimosis, and human papillomavirus (HPV) infection. Unlike other urogenital cancers, its pathophysiology and therapeutic targets remain poorly understood, particularly regarding the immune response to the tumor microenvironment. This study aims to investigate immune cell infiltration profiles, dendritic cell maturation, and lymphocyte apoptosis in both HPV-positive and HPV-negative PSCC. Clinical and histopathological data, along with peripheral blood and tumor tissue samples, were collected from 30 patients (66.6% were HPV-positive and 33.3% HPV-negative), with an additional 19 healthy donors serving as controls. Tumor-infiltrating immune cells were analyzed following enzymatic digestion of tumor tissue, enabling detailed phenotypic characterization. A simulated tumor microenvironment was created using supernatants derived from primary cultures of HPV-positive PSCC tumors. Peripheral blood mononuclear cells were isolated and differentiated into dendritic cells (Mo-DCs) for further phenotyping and lymphoproliferation assays. Lymphocytes from healthy donors and patients were exposed to tumor culture supernatants to evaluate apoptosis induced by the tumor microenvironment. Results showed that HPV-positive tumors exhibited lower T lymphocyte frequencies compared to HPV-negative tumors. Additionally, patients infected with high-risk HPV demonstrated reduced maturation rates of Mo-DCs and decreased expression of co-stimulatory molecules on these cells compared to healthy donors. Furthermore, Mo-DCs from hrHPV-positive patients showed impaired lymphoproliferation capacity relative to controls, while HPV-negative patients exhibited a trend towards reduced lymphoproliferative ability. Regarding the simulated tumor microenvironment, lymphocytes from healthy donors underwent apoptosis, contrasting with patients' lymphocytes, which showed increased viability when cultured with tumor supernatants. These results underscore the impact of HPV infection on T lymphocyte infiltration, Mo-DC maturation, and lymphocyte survival in PSCC, offering critical insights for advancing our understanding of the tumor microenvironment and guiding the development of immunotherapy strategies.

Keywords: HPV-related cancer; cancer immunomodulation; costimulatory molecules; dendritic cells; urological carcinoma.

Figure 1

Frequency of cells positive for immune cell markers infiltrated in tumor tissue of hrHPV-positive and HPV-negative PSCC patients. Tumor tissue samples were enzymatically digested and labeled with surface markers CD3+, CD19+, CD56+, and CD14+, to identify T lymphocytes, B lymphocytes, Natural Killer (NK) cells, and Monocytes/Macrophages, respectively. Data were presented as mean ± standard error of the mean (SEM), and an unpaired t-test was performed for each marker, with statistical significance set at *p<0.05, comparing HPV-positive (n=8) and HPV-negative (n=8) carcinoma samples.

Figure 2

Comparison of the phenotypic and functional profile of Mo-DCs obtained from patients with hrHPV-positive and HPV-negative PSCC patients, compared to healthy donors. (A) Frequency of Mo-DCs expressing the co-stimulatory molecule CD86 in patients with hrHPV-positive (n=6) and HPV-negative (n=6) PSCC, compared to healthy donors (n=9). (B) Mean Fluorescence Intensity (MFI) of CD86 on positive cells from patients with hrHPV-positive (n=6) and HPV-negative (n=6) PSCC, compared to healthy donors (n=9). (C) Proliferation index of healthy allogeneic lymphocytes co-cultured with Mo-DCs derived from hrHPV-positive (n=6) and HPV-negative (n=6) PSCC patients, compared to healthy donors (n=6). Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparison post hoc test, with significance set at p<0.05. Significance levels are denoted as *p < 0.05 and **p < 0.01.

Figure 3

Viability of lymphocytes obtained from PSCC patients with hrHPV-positive and HPV-negative carcinoma and healthy donors, treated with supernatant obtained from ex vivo culture of tumor cells from penile carcinoma. (A) Frequency of lymphocytes in early apoptosis from patients with hrHPV-positive (n=6) and HPV-negative (n=6) PSCC, compared to healthy donors (n=5). (B) Frequency of lymphocytes in late apoptosis from hrHPV-positive (n=6) and HPV-negative (n=6) PSCC patients, compared to healthy donors (n=5). (C) Frequency of viable lymphocytes from hrHPV-positive (n=6) and HPV-negative (n=6) PSCC, compared to healthy donors (n=5). Data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was conducted using One-way ANOVA followed by Tukey’s multiple comparison post hoc test and an unpaired t-test, with significance set at p<0.05. Statistical significance is denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.