Training Plan: Cytotoxic T lymphocytes (CTL) are an important host immune response which retards disease progression in human immunodeficiency virus HIV-1 infection. Studies to date on HIV-1 specific CTL have utilized recombinant methods of antigen infection, but no detailed studies on whole virus infected cells have been performed. Investigation of HIV-1 inhibition have been clouded by the use of bulk uncloned cells, with poorly defined cell populations. The availability of cloned CTL of known specificity and HIV- 1 permissive cell lines of known MHC type provide the opportunity to clarify the role of CTL activity in HIV-1 infection. Relevant issues to be explored in the proposed research include: (a) examining the ability of specific CTL clones to recognize infected cells, (b) analyzing the ability of different CTL clones to inhibit the replication of HIV-1, exploring the effect of epitope specificity on these two abilities, and (d) determining the effects of target epitope sequence variation on CTL activity against infected cells. Clonal target and effector cells will allow control of factors such as viral strain, input inoculum, MHC matching, and effects of CTL epitope specificity. Recognition of infected cells will be explored by acute synchronous infection of target cells followed by daily use of these cells as target cells for different MHC matched CTL clones. Flow cytometry, supernatant ELISA p24 measurement and virus yield assays, and northern blots will allow measurement of issues such as the kinetics of infection and virus production, expression of surface adhesion molecules important for CTL recognition, and kinetics of expression of different HIV-1 proteins. Inhibition of HIV-1 replication in infected cells will be studied using the same targets and effectors, with low MOI infections followed by co-culture with CTL clones. Supernatant will be harvested at intervals for ELISA p24 measurement. Magnetic bead separation will allow determination of the reversibility of observed inhibition, 0.4 micron transwell membranes will allow evaluation for the role of soluble factor(s) in inhibition, and cell free CTL supernatant will be analyzed for such factor(s). The effect of epitope variation will be explored by constructing point mutants of HIV-1 containing known naturally occurring epitope variations. Use of these mutants in the same recognition and inhibition assays described above, including dual infections with mutant and wild type, will allow analysis of the mechanisms of escape from CTL. Better understanding of the role of CTL, in controlling HIV-1 infection will have potential application in future therapeutic strategies for immunotherapy and vaccine design.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Clinical Investigator Award (CIA) (K08)
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Special Emphasis Panel (ZAI1-PRJ-A (80))
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Massachusetts General Hospital
United States
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