Th2 type inflammation promotes the gradual progression of HPV-infected cervical cells to cervical carcinoma

Abstract

Objectives: To investigate the role of immunological parameters in tumorigenesis of cervical cancer in women infected with high risk human papillomavirus (hr-HPV), and determine whether key findings with human material can be recapitulated in the mouse TC1 carcinoma model which expresses hr-HPV epitopes.

Methods: Epithelial and lymphoid cells in cervical tissues were analyzed by immunohistochemistry and serum IL10 levels were determined by ELISA. Tumor draining lymph nodes were analyzed in the mouse TC1 model by flow cytometry.

Results: The mucosa was infiltrated by CD20+ and CD138+ cells already at cervical intraepithelial neoplasia 1 (CIN1) and infiltration increased in cervical intraepithelial neoplasia 3 (CIN3)/carcinoma in situ (CIS) and invasive cervical cancer (ICC), where it strongly correlated with infiltration by CD32B+ and FoxP3+ lymphocytes. GATA3+ and T-bet+ lymphoid cells were increased in ICC compared to normal, and expression in epithelial cells of the Th2 inflammation-promoting cytokine TSLP and of IDO1 was higher in CIN3/CIS and ICC. As a corollary, serum levels of IL10 were higher in women with CIN3/CIS or ICC than in normals. Finally we demonstrated in the mouse TC1 carcinoma, which expresses hr-HPV epitopes, an increase of cells expressing B cell or plasma cell markers or Fc receptors in tumor-draining than distal lymph nodes or spleen.

Conclusions: hr-HPV initiates a local Th2 inflammation at an early stage, involving antibody forming cells, and fosters an immunosuppressive microenvironment that aids tumor progression.

Figure 1. Th2 type lymphoid cells are significantly associated with CIN3/CIS and ICC

The presence of stromal infiltrating CD20+ (A), CD138+ (B) and CD32B+ (C) lymphoid cells was determined on cervical tissue blocks by immunohistochemistry analysis. Wilcoxon Rank Sum Tests were used to compare IHC marker expression in cervical tissues with various histological diagnosis.

Figure 2. Stromal infiltrating CD20+, CD138+ and CD32B+ positive lymphoid cells are increased in CIN3/CIS and ICC

Figure 3. Cervical lesions are associated with a local immunosuppressive environment

The epithelial expression of Th2 type cytokine TSLP (A) and IDO1 (B), as well as stromal infiltrating FoxP3+ cells were determined on cervical tissue blocks by immunohistochemistry analysis. Wilcoxon Rank Sum Tests were used to compare IHC marker expression in cervical tissues with various histological diagnoses.

Figure 4. Correlation between Th2 inflammation and immunosuppression in cervical epithelium

Spearman correlation coefficients were used to determine correlation between stromal CD32B+ cells and FoxP3+ cells (A), between stromal CD32B+ cells and epithelial IDO1 expression (B), between stromal infiltrating Foxp3+ cells and epithelial IDO1 expression (C), and between epithelial TSLP and IDO1 expression (D).

Figure 5. IL10 was elevated in serum samples collected from women with CIN3/CIS and ICC

Serum level IL10 was determined by the ELISA assay (A), and significantly correlated with epithelial CD32B+ cells (B) by Spearman correlation coefficients test.

Figure 6. Tumor draining lymph nodes from TC1 mice have increased CD19+, CD138+, CD32+ lymphocytes

Lymphocytes from spleens of TC1 bearing mice and naïve mice were analyzed for CD19+, CD138+, CD32+ cells by flow cytometry analysis. Similar analysis was performed on lymphocytes collected from tumor-draining lymph nodes (TDLNs) and distal lymph nodes (DLNs) from TC1 bearing mice as well as normal lymph nodes (NLNs) from naïve mice (3 mice/group). Each type of cells was presented as the percentage of CD45+ leukocytes. Data are presented as mean±SEM (A). Representative dotspots show CD19+, CD138+ and CD32+ cells in NLNs, DLNs and TDLNs (B).