Although human papillomavirus (HPV) DNA is detected in the majority of squamous intraepithelial lesions (SILs) and squamous cell carcinomas (SCCs) of the uterine cervix, the persistence and progression of cervical lesions suggest that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most SILs show quantitative and functional alterations of Langerhans cells (LCs). The aim of this study was to determine whether modulation of E-cadherin-mediated homophilic and heterotypic interactions between keratinocytes and LCs is involved in these abnormalities of LCs in (pre)neoplastic cervical epithelium. Cell membrane expression of E-cadherin and the density of CD1a+ LCs were low in the epithelium of SILs and SCC biopsy specimens, compared with normal exocervical epithelium. Dendritic cells (DCs) and LCs generated in vitro were randomly distributed throughout the full thickness of organotypic cultures of E-cadherin- HPV-transformed cells. In contrast, these cells rapidly adhered to the keratinocyte cell layers when HPV-transformed cells transfected with E-cadherin were used. These data suggest that the E-cadherin-mediated contact between keratinocytes and LCs is potentially important for initiating or maintaining the immune response during chronic HPV infection.

The purpose of this study was to investigate the ability of CD1a+ Langerhans/dendritic cells (LCs/DCs) to infiltrate human papillomavirus (HPV)-associated (pre)neoplastic lesions of the uterine cervix. Migration of LCs/DCs in the presence of keratinocytes derived from normal cervix and HPV-transformed cell lines was evaluated in Boyden chambers and in organotypic cultures and correlated with granulocyte/macrophage colony-stimulating factor (GM-CSF) production by the cells, as determined by ELISA. Conditioned media of HPV-transformed keratinocytes contained lower amounts of GM-CSF and induced a decreased motile response of LCs/DCs in the Boyden chamber assay compared with those of normal cervical keratinocytes. The migration of LCs/DCs in the presence of conditioned media from normal keratinocytes could be blocked by an anti-GM-CSF antibody, and the migration of LCs/DCs in the presence of conditioned media from HPV-transformed keratinocytes could be increased by supplementing the media with recombinant GM-CSF. GM-CSF was also a potent factor in enhancing the colonization of LCs/DCs into organotypic cultures of HPV-transformed keratinocytes, as the infiltration of LCs/DCs in the in vitro-formed (pre)neoplastic epithelium was minimal under basal conditions and dramatically increased after the addition of GM-CSF to the cultures. These results suggest that GM-CSF could play an important role in the recruitment of LCs/DCs into the HPV-transformed (pre)neoplastic cervical epithelium and be useful as a new immunotherapeutic approach for cervical (pre)cancers.

Although human papillomavirus (HPV) antigens are expressed in a majority of (pre)neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix, progression to invasive cancer may occur, which suggests that the presentation of viral antigens to the immune system is deficient in some SILs. To determine whether professional antigen-presenting cells die in SILs, we assayed for the apoptosis of immature dendritic cells (DC) in organotypic cultures of HPV-transformed keratinocytes, which reproduce many features of in vivo observed SILs. Unexpectedly, the infiltration of organotypic cultures by DC specifically induced the apoptosis of HPV+ tumor cells, whereas DC were not affected. In the same conditions and in coculture experiments, apoptosis was not observed in normal keratinocytes. The induction of apoptosis required membrane contacts between DC and HPV-transformed keratinocytes. Although the HPV+cell lines were sensitive to the effects of TRAIL, soluble TRAILR2-Fc did not block the DC-induced apoptosis. Furthermore, although FasL and Fas were detected on DC and HPV+ cell lines, respectively, functional analysis revealed that this pathway is not responsible for the apoptosis induced by the DC. All together these results suggest that DC may be at the interface between innate and adaptive immunity by inducing the apoptosis of (pre)neoplastic cells.

Langerhans' cells (LCs) are a subset of immature dendritic cells (DCs) and play a key role in the initiation and regulation of immune responses. Functional studies of these cells have been hampered by difficulties in generating a large number of LCs in vitro. We describe a new method to efficiently generate immature DCs exhibiting morphological, immunohistochemical, and ultrastructural features of LCs (CD1a and CCR6þ ) from a limited number of CD34 þ cord blood progenitors. This method is based on a two-step procedure consisting of an amplification phase followed by a terminal differentiation induction. The amplification step is initiated with a combination of hematopoietic growth factors (thrombopoietin/stem cell factor/fetal liver tyrosine kinase-3 ligand), cytokines (granulocyte-macrophage colonystimulating factor, tumor necrosis factor-a, and interleukin-4), and 5 ng/ ml of transforming growth factor (TGF)-b1. The differentiation is induced by increasing the concentration of TGF-b1 to 12.5 ng/ml. These culture conditions were efficient for generating a large number of immature LCs (8.74 Â 10 6 + 3.2) from 15 Â 10 4 CD34 þ progenitor cells. In addition, these LCs were shown to be able to infiltrate an in vitro reconstructed epithelium. Because LCs play an important role in the mucosal immunity, this technique could be useful to study their interactions with epithelial pathogenic agents and to perform pharmacological, toxicological, and clinical research.

Dendritic cells (DC) are the most e cient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and accessibility in normal tissue. Substantial numbers of DC can be generated from peripheral blood cultured in the presence of interleukin-4 (IL-4) and granulocyte/ macrophage-colony-stimulating factor (GM-CSF). We showed in this study that substantial numbers of DC can be obtained from the peripheral blood of patients with (pre)neoplastic lesions of the uterine cervix. The procedure required relatively small blood samples (10 ml) and the presence of 100 U/ml IL-4 and 800 U/ml GM-CSF in the culture medium. There was no signi®cant di erence in the morphology, yield, phenotype and function of generated DC between patients with cervical (pre)-neoplastic lesions and healthy individuals. When the hematopoietic factor Flt3 ligand (Flt3L, 40 ng/ml) was added, there was an average increase in the DC population of 26% compared to cultures with GM-CSF and IL-4 alone. Approximately 1.2´10 6 cells with the characteristics of dendritic cells could be obtained when Flt3L was included in the medium. The addition of Flt3L did not modify the phenotypic pro®le of DC (HLA-DR + , CD1a + , CD4 + , CD54 + , CD80 + , CD86 + , CD40 + , CD3 A and CD14 A ). In addition, Flt3L generated functional DC capable of stimulating the proliferation of alloreactive T cells. These results suggest that Flt3L, in association with GM-CSF and IL-4, provides an advantageous tool for the large-scale generation of DC and that an immunotherapy based on the use of DC generated in vitro is possible in patients with (pre)neoplastic lesions of the uterine cervix.