HPV 16 E6/E7 expressing microparticles alter functional phenotype of Langerhans cell-like cells
High-risk human papillomavirus (HPV) infections can lead to the development of cervical cancer, the fourth most common cancer in women worldwide. High-risk HPV type 16 (HPV16) is responsible for over 50% of cervical cancers and persistent over-expression of the oncoproteins E6 and E7 is necessary for carcinogenesis. One hallmark of HPV infection is the evasion of innate immune recognition and failure to activate adaptive immune responses. Previous work done in the Hibma laboratory has attributed potential escape from immune surveillance of HPV to interactions with microparticles (MPs; Zhang et al., 2018). These small membrane fragments (0.1 – 1 um) released from HPV16 E7-expressing cells were able to suppress T cell responses caused by the downregulation of important signalling molecules (e.g. CD40 and IL-12) on Langerhans cells, the antigen presenting cells implicated in HPV pathogenesis. Our aim is to investigate whether these effects can be found in the presence of both E6 and E7 oncoproteins, and to improve our understanding of whether MPs can affect immune cells beyond the site of infection. Species-restricted effects of MPs will also be tested. Through differential centrifugation steps, we have produced MP from mouse and human cell lines expressing the HPV16 E6/E7 oncoproteins (TC-1, CaSki and SiHa) and control cells (PDV and C33a). Antigen presenting cells (APC), such as Langerhans cell-like cells (LCLC) and dendritic cells (DC), were differentiated from murine bone marrow precursors following cytokine supplementation. The effect of MP on Langerhans and dendritic cells’ functional phenotype (MHC-I/II, CD40 and IL-12) was investigated following 48 hours of co-culture and flow cytometric analysis. After sizing bead and annexin V analysis for MP detection, we have found that all cell lines produce similar number of MP, and the E6 and E7 have a role in the regulation of MP release. MP from E6/E7 expressing cell lines downregulated CD40 expression in lipopolysaccharide (LPS) stimulated LCLC. This result confirmed our hypothesis that HPV16 E6 and E7 expressing MP have an immune suppressive effect on the phenotype of LCLC. Furthermore, MP derived from either human or mouse cell lines downregulate IL-12 production independent of HPV16 E6 and E7 expression in DC. Findings here suggest MPs have an immune modulatory role and can suppress adaptive immune responses against HPV-infected cells, and this warrants further investigation of the lipid, gene and post-transcriptional profiles of MPs.
Advisor: Hibma, Merilyn
Degree Name: Bachelor of Biomedical Sciences with Honours
Degree Discipline: Pathology
Publisher: University of Otago
Keywords: Human papillomavirus; E6/E7 oncoproteins; Microparticles; Langerhans cells; Antigen-presenting cells
Research Type: Thesis