814 Immune Evasion by Kaposi's Sarcoma-associated Herpesvirus During Latency C. Tomescu*, W. K. Law, D. H. Kedes Univ of Virginia, Charlottesville
Background: Under selective pressure from host cytotoxic T-lymphocytes (CTLs), many viruses have evolved to downregulate MHC class I and/or T-cell co-stimulatory molecules from the surface of infected cells. Recently Kaposi's Sarcoma-associated Herpesvirus (KSHV), a newly identified human gamma herpes virus and the etiological agent responsible for several AIDS-associated tumors, has been shown to encode two proteins, MIR-1 and MIR-2, that serve this function during lytic replication. However, KSHV exists in a predominantly latent state with less than 5% of infected cells expressing discernable lytic gene products. Thus, lytic-dependent mechanisms of immune protection are not likely to be active in most KSHV infected cells. As a result, we have searched for evidence of such defensive strategies in 2 endothelial cell culture systems that strongly favor latent KSHV infection.
Methods: Utilizing a panel of antibodies against several endothelial cell surface proteins, we investigated the potential down-regulation of immune-modulatory proteins by KSHV in 2 complimentary endothelial cell culture systems: primary dermal microvascular endothelial cells (pDMVEC) infected with cell associated virus and telomerase immortalized endothelial (TIME) cells infected directly with cell free KSHV. Infected cells within the population were identified with a novel intra-nuclear flow cytometry technique utilizing a fluorescently labeled monoclonal antibody against the latent-associated nuclear antigen (LANA) of KSHV. Immunofluorescence assays (IFA) were then used to confirm the low level of lytic protein expression following de novo infection.
Results: We observed a dramatic down-regulation of MHC class I, CD31 (PE-CAM), and CD54 (ICAM-I), but not CD58 (LFA-1) or CD95 (Fas), shortly following de novo infection with KSHV. Intranuclear flow cytometry confirmed that down-regulation occurred predominantly on KSHV infected (LANA-positive) cells, but not on uninfected neighbors. Importantly, while the vast majority of KSHV infected cells showed surface down-regulation of immune-modulatory proteins, less than 3% expressed lytic proteins such as MIR-1 and MIR-2 by IFA.
Conclusions: Together, these results suggest that latent infection with KSHV results in the modulation of immune regulatory protein expression on the surface of infected endothelial cells. This may facilitate viral escape from CTLs, and suggests that KSHV possesses two complimentary mechanisms, one lytic and one latent, in outwitting host immune defenses.
