Extracellular Vesicles produced by Human Papillomavirus E6-E7-expressing cells have effects on T and B cells.

BRAVO-MIANA ROCIO DEL CARMEN; MOTRICH, RUBÉN DARÍO; RIVERO, VIRGINIA ELENA; MOSSMAN JESSICA; CUFFINI, CECILIA; OLIVERA CAROLINA; PAIRA DANIELA ANDREA; DONADIO ANA CAROLINA

Buenos Aires

Workshop; Extracellular Vesicles in Immunology; 2020

International Society for Extracellular Vesicles

HPVs are a large family of small DNA viruses characterized by their tropism to skin or mucosa. High risk HPVs are the etiological agents of cervical cancer and exert their dangerous functions mainly through E6 and E7 proteins that interact with numerous cellular molecules finally fostering viral immune-evasion and tumor development. In contrast to the increasing knowledge of the intracellular activities of E6 and E7 proteins, the possible effects on the intercellular communication is not yet well known. It has been described that Extracellular Vesicles (EVs) take part in cell-to-cell communication between infected cells and immune cells. With that in mind, we wish to determine if EVs were shed from C33-A carcinoma cervical cells expressing or not E6/E7 proteins from High risk HPV 16. To stablish if EVs were produced from E6/E7 expressing C33-A and control C33-A cells, equal numbers of cells were cultured until 70-80% confluence and maintained in serum free medium for the last 48 hours. Later, E6/E7 EVs and Control EVs were purified from supernatants using ultracentrifugation standard protocols. We observed by Transmission Electron Microscopy that E6/E7 expression increases the relative number of small-EVs (0-150nm) produced by C33-A cells when compared with EVs-size found in control EVs. We then questioned whether exposure of murine T and B lymphocytes to both EVs might have effects on its activation. To test this, murine spleen mononuclear cells were co-cultured with E6/E7 EVs or control EVs and then activated with the TLR4 ligand lipopolysaccharide (LPS) or plate bound anti-CD3/CD28 antibodies. After 24 hours of activation expression different markers were measured using flow cytometry. The presence of E6E7 EVs and control EVs produced changes in the expression of Ki67 and CD69 on T lymphocytes. Moreover, E6E7EVs and control EVs induced the expression of CD69 and CD86 on B lymphocytes. These preliminary results showed that C33-A cells expressing E6 E7 proteins from High risk HPV16 produce EVs with different characteristics to those shed by C33-A control cells that may alter the responsiveness of T and B lymphocytes and would alter the virus persistence. .