ANALYSIS OF TAP INDEPENDENT PRESENTAITON IN A HERPESVIRUS INFECTION

We are interested in studying a type of herpesvirus in a mouse model to understand the immune response to infection. We focus on studying CD8 ⁺ T cells as this herpesvirus is a potential candidate as a vaccine vector for CD8 ⁺ T cell-controlled pathogens. In the classical antigen presentation pathway the cell utilizes the transporter associated with antigen processing (TAP) to deliver peptides to MHC class I molecules, and presents them on the surface to activate CD8 ⁺ T cells during viral infection. It has been shown that there are peptides that are presented independently of TAP, which can be important for potential vaccine development. Therefore, we hypothesize that there is TAP-independent presentation of peptides in this herpesvirus infection. To test this, we infected wild type (WT) C57BL/6 mice with this herpesvirus and stimulated them with infected WT and TAP ^-/- bone marrow dendritic cells (BMDCs). At 1, 3, and 7 weeks post infection we took the spleen from the infected mice and obtained purified CD8 ⁺ T cells from the splenocytes. We co-cultured the infected BMDCs with the purified CD8 ⁺ T cells in presence of brefeldin A to inhibit the secretion of interferon. After incubation, we stained the cells with anti-CD8- APC and IFNγ-PE and were able to measure the CD8 ⁺ T cell response using flow cytometry. Results showing that TAP ^-/- infected BMDCs are able to stimulate CD8 ⁺ T cells from WT infected mice, would indicate that there is TAP-independent presentation in a herpesvirus infection. The next step in our study was to analyze the CD8 ⁺ T cell response against different peptides to determine which ones are presented in absence of TAP. We infected wild type and TAP ^-/- mice with this herpesvirus and at 1 and 7 weeks post infection we isolated their splenocytes and stimulated the cells with 5 peptides representing described virus epitopes presented by MHC class I molecules. Our results using flow cytometry did not detect any CD8 ⁺ T cell response in TAP ^-/- mice, therefore the peptides we tested are presented in a TAP-dependent manner. We plan to test a variety of peptides to potentially identify peptides from this herpesvirus that are presented independently of TAP using predictions based on biochemical properties of the peptides.