Profiling of VEGF Receptors and Immune Checkpoints in Recurrent Respiratory Papillomatosis

Abstract

Objectives: Recurrent respiratory papillomatosis (RRP) is caused by human papilloma virus (HPV) infection of the aerodigestive tract that significantly impacts quality-of-life including the ability to communicate and breathe. Treatment was traditionally limited to serial ablative procedures in the O.R. with possible local adjuvant therapy, but new systemic therapies, such as Vascular endothelial growth factor (VEGF) inhibitors, are showing significant promise. This study aims to determine whether rationale exists for combination therapeutic approaches using VEGF inhibitors and/or immune checkpoint blockade.

Methods: Using fresh specimens from the O.R., we performed flow cytometry on papilloma, normal adjacent tissue, and blood. Papilloma and surrounding tissue were examined for expression of PD-L1, PD-L2, Galectin-9, VEGFR2, and VEGFR3. CD8+ and CD4+ T cells were assayed for expression of PD-1, TIGIT, LAG3, and TIM3.

Results: Our data shows that papilloma tissue exhibits significantly higher levels of PD-L1 and PD-L2 compared to adjacent tissue. Elevated levels of the VEGF receptor VEGFR3 were also observed in papilloma tissue. When examining T cells within the papilloma, elevated PD-1 and TIGIT expression was observed on CD8+ T cells, while levels of PD-1, TIGIT, and TIM3 were elevated on CD4+ T cells compared to PBMCs. Heterogenous marker expression was observed between individuals.

Conclusions: Our analysis shows that RRP tissue shows elevated levels of multiple immune check point targets and VEGFR3, with varied patterns unique to each papilloma patient. Some of these immune checkpoint markers already have novel immunotherapies available or in development, providing molecular rationale to offer these systemic treatments to selected patients affected by RRP alongside VEGF inhibitors. Laryngoscope, 134:2819-2825, 2024.

Keywords: flow cytometry; human papillomavirus; immunotherapy; larynx; recurrent respiratory papillomatosis.

Figure 1.. Papilloma tissue expresses elevated levels of VEGFR3 and B7 family members PD-L1 and PD-L2.

To evaluate the levels of VEGF receptors and immune checkpoint ligands on papilloma tissue, single cells were prepared. Samples were stained with an antibody cocktail including CD45 for immune cell dumping and a viability dye and analyzed by flow cytometry. Representative histogram of expression normalized to mode in papilloma tissue (red) or normal adjacent tissue (purple) of PD-L1 (a), PD-L2 (c), Galectin-9 (e), VEGFR2 (g), and VEGFR3 (i). Bar graph showing expression across patients (individual points) as well as median expression of PD-L1 (b), PD-L2 (d), Galectin-9 (f), VEGFR2 (h), and VEGFR3 (j). tSNE plots showing the heterogeneity in expression of PD-L1 (k) and PD-L2 (l) in normal adjacent (top row) and papilloma tissue (bottom row) in participants (rows).

Figure 2.. Expression of immune checkpoint markers on papilloma-infiltrating CD8+ T cells.

After single cell suspensions were prepared and appropriate gating was performed to identify CD8+ T cells, expression of immune exhaustion markers and CD69 was evaluated. Representative histogram of expression normalized to mode in papilloma-infiltrating CD8+ T cells (red) or CD8+ T cells from PBMCs (purple) of PD-1 (a), TIGIT (c), LAG3 (e), TIM3 (g), and CD69 (i). Bar graph showing expression (individual points) as well as median expression across patients of PD-1 (b), TIGIT (d), LAG3 (f), TIM3 (h), and CD69 (j).

Figure 3.. Immune checkpoint marker expression on CD4+ T cells.

As with CD8+ T cells, CD4+ T cells were identified by appropriate gating and expression of immune exhaustion markers and CD69 was evaluated. Representative histogram of expression normalized to mode in papilloma-infiltrating CD4+ T cells (red) or CD4+ T cells from PBMCs (purple) of PD-1 (a), TIGIT (c), LAG3 (e), TIM3 (g), and CD69 (i). Bar graph showing expression (individual points) as well as median expression across patients of PD-1 (b), TIGIT (d), LAG3 (f), TIM3 (h), and CD69 (j).