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The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Immunology II Abstract 46 CYTOKINE RESPONSES BY CD8 T LYMPHOCYTES TO KSHV/HHV8 LYTIC AND LATENCY PROTEINS Lauren Lepone, Giovanna Rappocciolo, Emilee Knowlton, Paolo Piazza, Mariel Jais, Frank J. Jenkins and Charles R. Rinaldo Dept of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA Abstract Objectives: CD8 T cells producing more than one cytokine or cytotoxicity factor, i.e. polyfunctional, have been shown to relate to control of HIV-1 infection. Based on our previous findings that dendritic cells (DC) are optimally required as antigen presenting cells to reveal MHC class I epitopes of KSHV/HHV-8 glycoprotein B (gB) (Blood 99:3360, 2002), we determined CD8 T cell cytokine responses to multiple lytic and latency viral proteins. Methods: T cells were stimulated with DC from HHV-8 seropositive, HLA A*0201 donors that had been loaded with overlapping peptides derived from gB and K8.1 lytic proteins, and K12 and LANA latency proteins, and screened for IFNγ production by ELISPOT. Positive peptides were assessed for IFNγ, IL2, TNFα, MIP1β, and CD107a production by flow cytometry. Results: ELISPOT and flow cytometry revealed new, potential HLA A*0201 9mer epitopes for gB (5), K8.1 (4), K12 (2) and LANA (5). In HHV-8 seropositive healthy donors controlling infection, the predominant CD8 T cell response to protein epitopes was monofunctional, with a portion of CD8 T cells being polyfunctional. Conclusions: This demonstrates that HHV-8 proteins predominantly induce single cytokine or cytotoxicity factors in CD8 T cells from healthy, HHV-8 seropositive, HIV-1 negative adults, with a portion of T cells producing 2-3 of these factors. These immunogenic regions of the viral proteins could be important in immunopathogenesis of KSHV/HHV-8 infection and progression to Kaposi’s sarcoma. Presenting author Email: LML33@pitt.edu 72
The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Immunology II Abstract 47 INFECTION OF LYMPHOID CELLS BY KSHV IN CULTURED PRIMARY HUMAN TONSILLAR LYMPHOID CELLS EX VIVO Jinjong Myoung and Don Ganem HHMI and Departments of Microbiology and Medicine, UCSF Abstract KSHV is known to infect B cells in vivo and is a causative agent of rare human B cell lymphomas (PELs). Paradoxically, however, established B cell lines are resistant to infection in vitro, which has greatly retarded progress in understanding the biology of KSHV in its natural lymphoid compartment. We prepared primary human lymphoid aggregate cultures (HLACs) from tonsils, and examined their infectability by KSHV. Recombinant virus (rKSHV.219) expressing GFP under the EF1α promoter was prepared from induced Vero cells latently infected with this virus. Tonsil cells were infected with rKSHV, then subjected to flow cytometric analysis. When tonsil cells were infected with an MOI of 0.75, 15% of CD19+ cells became GFP-positive. Surprisingly, in the same culture many more T cells (54% of CD3+ cells) were shown to be GFP+; CD8+ T cells were consistently more susceptible than CD4+ T cells. This difference in infectivity in T cell subsets became more prominent when PHA was used to activate the T cells prior to infection. When exposed to chemical inducers of lytic growth, infected HLAC cultures displayed enhanced production of infectious KSHV.214, demonstrating that HLACs are competent to support the complete lytic cycle of KSHV. No immortalization or blastic transformation has yet been observed in infected HLACs. However, GFP+ B cells were significantly enriched when assessed at d13 post-infection, while the proportion of GFP+ T cells decreased significantly by d13 PI. These data suggest that KSHV infection provides survival advantage in B cells, but not in T cells. Presenting author Email: jinjong.myoung@ucsf.edu 73
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Keiji Ueda Dept.of Infectious Disea
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