High risk human papillomavirus (HPV) infected cervical epithelium is relatively devoid of professional antigen-presenting cells (APCs) and this has been attributed to the expression of HPV encoded gene products. Langerhans cells (LCs) are professional APCs that are critical in the induction of immune response to viral infection of the skin. However, there is little information on the function of these cells in HPV-related cervical cancer. HPV16 E7 transgenic (also known as K14E7) mice are used in our laboratory to investigate immunotherapy targeted at skin expressing antigen in keratinocytes or epithelial tumors. The aim of this study is to investigate whether E7 protein of HPV16 affects the numbers, activation markers and functional properties of LCs in the skin and skin draining lymph nodes (LN). Besides that, this study also examines the possible mechanisms of the E7-expressing keratinocytes altering the LC functions. To answer these questions, epidermal cells and LN cells from E7 transgenic mice were analysed by flow cytometry and were further cultured for in vitro functional properties.
It is shown in this study that there was an increase in absolute numbers of LCs in E7 transgenic epidermal skin compared to the syngeneic strain. Phenotypic analysis showed that LCs in E7 epidermis have higher expression of all APC-activation markers (co-stimulatory molecules and MHC molecules) compared to non-transgenic mice and OVA transgenic mice. Double transgenic (K14E7xK5mOVA) mice also demonstrated increased expression of the activation markers, which is evidence of the E7-induced alteration. In epidermal cell cultures, these ‘activated’ E7 LCs are able to take up the OVA antigen more efficiently compared to LC from non-transgenic mice, suggesting that the LCs from E7 skin are functioning well for capturing the antigen. Furthermore, the LCs from E7 transgenic mice were able to stimulate OVA-specific CD8+ T cells (OT-I) and OVA-specific CD4+ T (OT-II) as shown by in vitro proliferation assay. The ability of LCs to stimulate cytokine production was measured from the same assay by detecting the cytokines in the supernatant but there were no significant finding.
Consistent with the findings in the epidermis, LCs that migrated to the skin-draining LN are higher in E7 transgenic mice compared to wild-type controls. In the LN, the LCs displayed activated phenotypes relative to the LCs in the skin for both E7 transgenic and syngeneic strain. In addition, the level of maturation markers on these LCs was comparable to wild-type control, except the upregulation of MHC class I suggesting that the presence of E7 protein might have some influence on the MHC class I machinery. From the in vivo migration assay, LCs that migrated from the skin to the LN in response to the contact sensitizer is altered in the E7 transgenic mice demonstrated by lower percentage of FITC+ bearing LCs in comparison to non-trangenic mice. Furthermore, the LCs from K14E7 also exhibit unaltered stimulatory capacity showed in the proliferation assay and cytokine production of OT-I and OT-II responder cells in vitro. By using K14E7xK5mOVA double transgenic mice (both antigens presented on the same keratinocytes) in OVA-specific T cells transfer experiment, skin APCs of these mice are able to drive proliferation of OVA specific CD8+ T cells in vivo more efficiently in comparison to OVA transgenic mice. However, the proliferation of OVA-specific CD4+ T cells is less efficient in both double transgenic and OVA transgenic mice. These findings might indicate that E7 might induce enhancement of MHC class I pathway.
Subsequently, the examination on lymphoid deficient mice (Rag1-/-) with or without E7 proteins showed that LC numbers in the epidermal skin and LN are not much affected in comparison to wild-type strain (K14E7 and C57BL/6, respectively). However, the expressions of MHC class II on LCs in the Rag1-/-xK14E7 mice were altered in the epidermis and LN. In draining LN of E7 immunodeficient mice, higher expression of MHC class I and CD80/86 molecules on LCs might indicate higher maturation status; however, the changes of MHC class II expression revealed the lymphoid-dependent and E7-mediated LC alteration. Finally, it is also shown that the presence of keratinocytes is important for observation of co-stimulatory molecule expression on the LCs. In summary, these results provide some insight into how HPV16 E7 may influence the specific immune response through the alteration of LC population.