Local T-Cell Dysregulation and Immune Checkpoint Expression in Human Papillomavirus-Mediated Recurrent Respiratory Papillomatosis

Abstract

Human papillomavirus-mediated recurrent respiratory papillomatosis (RRP) is a premalignant neoplasia of the upper airway characterized by significant dysphonia and respiratory obstruction. Immune checkpoint blockade has emerged as a potential alternative to repeated surgical interventions in RRP. Here, we investigated the intralesional T-cell composition and expression of the immune checkpoints programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) in RRP. We analyzed tissue samples from 30 patients treated at a tertiary care center between 2009 and 2021, including paired samples from individual patients collected at different time points. Immunohistochemical staining was performed for CD4, CD8, CTLA-4, FoxP3, and PD-L1 and correlated with disease severity and previous adjuvant therapies. Overall disease burden and intervention-free survival were not associated with the abundance of CD4⁺, CD8⁺, or FoxP3⁺ T cells, nor with immune checkpoint expression. However, patients with aggressive disease exhibited a higher intralesional FoxP3/CD4 T-cell ratio. Prior intralesional cidofovir treatment was associated with reduced CD4⁺ T-cell infiltration. These findings suggest that a locally immunosuppressive microenvironment, reflected by an elevated FoxP3/CD4 ratio, contributes to disease severity in RRP. Consistent CTLA-4 expression across all evaluated samples supports further investigation of anti-CTLA-4 therapy, either alone or in combination with other checkpoint inhibitors.

Keywords: CTLA4; human papillomavirus; lymphocytes; recurrent respiratory papillomatosis.

Figure 1

CD4-, CD8-, FoxP3-, CTLA4-, and PD-L1-positive immune cells infiltrate recurrent respiratory papillomatosis (RRP) tissue. Tissue samples of patients with RRP were stained for the indicated markers by immunohistochemistry. (a) Representative images of tissue samples with high and low expression of the indicated molecules. (b) Number of positively stained cells/mm² displayed as individual values, mean, and standard deviation (n = 30). (c) CD4-, CD8-, FoxP3-, CTLA-4- and PD-L1-positive cells/mm² in consecutive surgical specimens (n = 7); ns, not significant; mm, millimeter. Significant differences were calculated by Mann–Whitney test.

Figure 2

Correlation of T-cell abundance and immune checkpoint expression with clinical severity reveals more previous surgeries in patients with a high ratio of FoxP3⁺/CD4⁺ cells. Patients are divided into three groups—low, intermediate, and high—according to expression of the respective target molecules (n = 10 per group). (a) Number of previous lifetime surgeries of patients with respect to immune cell infiltration and immune checkpoint expression. (b) Intervention-free survival in relation to the expression of CD4, CD8, FoxP3, CTLA4, and PD-L1. (c) Lifetime surgeries in relation to the intralesional ratio of CD8⁺/CD4⁺ cells, FoxP3⁺/CD4⁺ cells, PD-L1⁺/CD8⁺ cells, PD-L1⁺/CD4⁺ cells, and CTLA4⁺/CD4⁺ cells. IFS, intervention-free survival. Significant differences were calculated by Mann–Whitney test (comparing low vs. high groups) and log-rank test.

Figure 3

Immune cell infiltration is more abundant in stromal tissue than in epithelial tissue. (a) Workflow for the classification of recurrent respiratory papillomatosis tissue stroma and epithelium, as well as positive cell detection. (b) Investigation of the distribution of CD4-, CD8-, FoxP3-, CTLA4-, and PD-L1-positive cells within the stromal and epithelial compartments of RRP tissue; mm, millimeter. Statistical differences were calculated by Mann–Whitney test.

Figure 4

Cidofovir treatment influences CD4⁺ T-cell infiltration in RRP. Tissue samples of RRP patients were analyzed by immunohistochemistry for the expression of the indicated markers. (a) Samples without previous adjuvant therapy (n = 15) were compared to samples with either previous bevacizumab (n = 8) or cidofovir (n = 7); statistical differences were calculated by Mann–Whitney test (none vs. bevacizumab or cidofovir). (b) Prophylactic HPV vaccination (Gardasil^® or Gardasil 9 ^®) (n = 6) compared to unvaccinated (n = 24) patients; significant differences were calculated by Mann–Whitney test.