Cervical (pre)neoplastic microenvironment promotes the emergence of tolerogenic dendritic cells via RANKL secretion

Abstract

The progression of genital human papillomavirus (HPV) infections into preneoplastic lesions suggests that infected/malignant cells are not adequately recognized by the immune system. In this study, we demonstrated that cervical/vulvar cancer cells secrete factor(s) that affect both the maturation and function of dendritic cells (DC) leading to a tolerogenic profile. Indeed, DC cocultured with cancer cell lines display both a partially mature phenotype after lipopolysaccharide (LPS) maturation and an altered secretory profile (IL-10^high and IL-12p70^low). In addition, tumor-converted DC acquire the ability to alter T-cell proliferation and to induce FoxP3⁺ suppressive T cells from naive CD4⁺ T cells. Among the immunosuppressive factors implicated in DC alterations in genital (pre)neoplastic microenvironment, we identified receptor activator of nuclear factor kappa-B ligand (RANKL), a TNF family member, as a potential candidate. For the first time, we showed that RANKL expression strongly increases during cervical progression. We also confirmed that RANKL is directly secreted by cancer cells and this expression is not related to HPV viral oncoprotein induction. Interestingly, the addition of osteoprotegerin (OPG) in coculture experiments reduces significantly the inhibition of DC maturation, the release of a tolerogenic cytokine profile (IL-12^low IL-10^high) and the induction of regulatory T (Treg) cells. Our findings suggest that the use of inhibitory molecules directed against RANKL in cervical/vulvar (pre)neoplastic lesions might prevent alterations of DC functionality and represent an attractive strategy to overcome immune tolerance in such cancers.

Keywords: LC, Langerhans cells; LPS, lipopolysaccharide; APC, antigen presenting cells; DC, dendritic cells; GILZ, glucocorticoid-induced leucine zipper; HPV, human papillomavirus; HSIL, high grade intraepithelial lesions; IHC, immunohistochemistry; ILT3, Immunoglobulin-like transcript 3; KN, normal keratinocytes; LSIL, low grade intraepithelial lesion; MFI, mean fluorescence intensity; OPG, osteoprotegerin; PBMC, peripheral blood mononuclear cells; pDC, plasmacytoid dendritic cells; RANKL; RANKL, Receptor activator of nuclear factor kappa-B ligand; SCC, squamous cell carcinoma; SIL, squamous intraepithelial neoplasia; Treg cells; Treg cells, regulatory T cells; VIN, vulvar intraepithelial neoplasia; cervical cancers; dendritic cells; tolerogenicity.

Figure 1.

DC cultured in a cervical/vulvar tumor microenvironment display a semi-mature phenotype and functional defects. (A) DC were either cultured alone (control DC) or in the presence of normal (KN) or malignant cells (CaSki, SiHa, C4II and A431) in a Transwell Chamber assay. At day 6, epithelial cells were removed and DC were stimulated with LPS for 24 h. DC phenotype was then assessed by flow cytometry (maturation markers: CD80, CD83, CD86, HLA-DR, and CCR7). Data were normalized to control DC (= 100%). Data are from 21 different experiments and mean values are shown as percentages of positive cells ± standard deviation. Statistical differences were determined by performing the one-way ANOVA test between control DC or KN and the other cell culture conditions (CaSki, SiHa, C4II, A431) (*P < 0.05; **P < 0.01; ***P < 0.001, ns: not significant). (B) DC differentiated in the presence of SCC cell lines produce high levels of IL-10 and low amount of IL-12. Data are from 20 different experiments and mean values are shown as cytokine concentration (pg/mL) ± standard deviation (**P < 0.01; ***P < 0.001). (C) DC cocultured with SCC cell lines inhibit T-cell proliferation and induce suppressive T cells in MLR assays. MLR of DC cocultured with genital CaSki cell line, KN or alone (Control) and then cultured with allogeneic T lymphocytes for 7 d. Responder cells: lymphocytes T; effector cells: DC. Data represent mean ± standard deviation of ³H-Tdr incorporation (**P < 0.01; ***P < 0.001). (D) Suppressor activity of T cells originally primed by DC cocultured with CaSki, SiHa, C4II, KN or alone (control DC). Allogeneic CD4⁺ T cells were purified and cocultured during 7 d with irradiated control DC or DC cocultured with CaSki, SiHa, C4II or KN. Cell mixture was than mixed with freshly isolated T cells and with other allogeneic DC. ³H-Tdr incorporation was measured after 5 d of culture. Data represent mean ± standard deviation of ³H-Tdr incorporation from six experiments (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 2.

Squamous carcinoma cells express RANKL in vitro and in situ. (A) RANKL expression in cervical biopsy specimens. (a) Normal exocervix, (b) squamous epithelial metaplasia, (c) low-grade squamous intraepithelial lesions, (d) high-grade squamous intraepithelial lesions, (e) cervical SCC. Original magnification: X100. The RANKL immunoreactivity is observed in the epithelial compartment. (f) Semi-quantitative evaluation of RANKL expression in normal exocervix (n = 22), epithelial metaplasia (n = 9), LSIL (n = 18), HSIL (n = 27) and SCC (n = 12). Asterisks indicate statistically significant differences (*P < 0.05; ***P < 0.001). (B) RANKL secretion by SCC cell lines (SiHa, CaSki, A431, C4II) and normal keratinocytes (KN) was determined by an ELISA assay. Data represent mean ± standard deviation of RANKL concentration (pg/mL/10⁶ cells) from five independent experiments (*P < 0.05; **P < 0.01). (C) RANKL mRNA expression by SCC cell lines (CaSki, SiHa, C4II, A431) and KN was determined by classical PCR. Densitometric analysis (ratio) shows that RANKL mRNA level is higher in SCC cell lines compared to KN.

Figure 3.

RANKL induces a semi-mature phenotype in DC and modifies their IL-10/IL-12 secretion. (A) DC were cultured in the presence of human recombinant RANKL (0.1 or 0.5 μg/mL). At day 6, DC were stimulated by LPS for 24 h. DC phenotype was then assessed by flow cytometry (maturation markers: CD80, CD83, CD86, HLA-DR, and CCR7). Data were normalized to control DC (= 100%). Data are from six different experiments and mean values are shown as percentages of positive cells ± standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001). (B) IL-10 and IL-12 production by both control DC and DC cultured with RANKL. Data are from 11 different experiments and mean values are shown as cytokine concentration (pg/mL) ± standard deviation (***P < 0.001).

Figure 4.

OPG inhibits RANKL effect on DC. (A) OPG restores the phenotype of DC cultured in the presence of RANKL. DC were cultured either alone or in the presence of OPG alone, human recombinant RANKL (0.5 μg/mL) or RANKL and OPG. At day 6, DC were stimulated by LPS for 24 h. The expression of DC maturation markers (CD80, CD83, CD86, HLA-DR, and CCR7) was assessed by flow cytometry. Data were normalized to control DC (= 100%). Data are from five different experiments and mean values are shown as percentages of positive cells ± standard deviation (*P < 0.05). (B) IL-10 and IL-12 production by control DC and DC cultured with RANKL (0.5 μg/mL) or with RANKL and OPG. Data are from eight different experiments and mean values are shown as cytokine concentration (pg/mL) ± standard deviation (*P < 0.05). ns: not significant; OPG: osteoprotegerin.

Figure 5.

RANKL inhibition is sufficient to reverse the tolerogenic profile of DC. (A) Analysis of DC phenotype after coculture in the presence of SCC cell lines treated or not with human OPG. After 6 d of coculture and 24 h of incubation with LPS, the expression of CD80, CD83, CD86, HLA-DR and CCR7 was assessed by flow cytometry. Data are from seven independent experiments and were normalized to control DC (= 100%). Mean values are shown as percentages of positive cells ± standard deviation (*P < 0.05, **P < 0.01). (B) Secretion levels of IL-10 and IL-12p70 in supernatant of DC cocultured with cancer cell lines in the presence or not of OPG. The secretion levels were measured by ELISA after 6 d of coculture and 24 h of incubation with LPS. DC cultured alone or with OPG were used as controls. Data are presented as means ± standard deviation of six independent experiments (*P < 0.05; **P < 0.01). (C) FACS analysis of ILT3 expression on DC cocultured with SCC cell lines in the presence or not of OPG. Data were normalized to control DC (= 100%). Data are from five different experiments and mean values are shown as percentages of positive cells ± standard deviation (*P <0.05; **P < 0.01). (D) DC cocultured in the presence of OPG induced a lower Treg cell differentiation in vitro. Real-time RT-PCR analysis of FoxP3 expression in allogeneic CD4⁺ T cells cultured with DC isolated from previously described coculture experiments. DC cultured alone were used as control. Data represent mean ± standard deviation of four independent experiments (*P < 0.05; **P < 0.01).

Figure 6.

OPG expression is increased in (pre)neoplastic cervical lesions compared to normal tissues but, remains stable during cervical cancer progression. Representative images of OPG expression in exocervical epithelium (A), metaplasia (B), LSIL (C), HSIL (D) and SCC (E). The OPG immunoreactivity is observed in the epithelial compartment. (F) Semi-quantitative analysis shows a significant increased OPG expression in metaplasia (n = 9), LSIL (n = 8), HSIL (n = 11) and SCC (n = 10) compared to the exocervix (n = 5). Asterisks indicate statistically significant differences (**P < 0.01; ***P < 0.001). Original magnification: X100.