MACROPHAGE ANTIGEN PROCESSING OF HIV SUBTYPES

CD8+, MHC-1 restricted cytotoxic T
lymphocytes (CTL) are responsible for controlling viremia associated with
human immunodeficiency virus (HIV) infection. CTL activity is directed
against epitopes from lymphocytotropic (T-tropic) and monocytotropic (M-tropic)
strains. T-tropic HIV strains do not cause a productive infection in
human monocytes and macrophages. However, we have shown that macrophages
exposed to T-tropic or M-tropic HIV are equally able to induce a primary CTL
response, indicating that processing of viral antigens is occurring after
exposure to both strains. Thus we hypothesize that macrophages can support
both entry and processing of T-tropic HIV and subsequently elicit MHC class I-restricted
cytotoxic T lymphocyte responses to specific viral epitopes in the
absence of a productive infection. We will test this hypothesis by examining
each of the steps involved in antigen processing using an in vitro fixed cell
model of HIV infection. The following specific aims will be tested: 1) To
determine the significance of various entry mechanisms by T-tropic and M-tropic
HIV into alveolar macrophages and monocyte derived macrophages on
subsequent CTL responses, 2) To determine whether MHC class I-restricted HIV
epitopes are generated in the cytoplasmic compartment by proteasomes or in
endosomes, 3) To determine if HIV peptide-MHC class I coupling requires
synthesis of new MHC class I molecules or can occur with preexisting molecules
via a regurgitant type pathway, 4) To determine specific epitopes recognized
by CTL primed in vitro, and 5) To determine if specific HIV epitopes require
transporter associated with antigen processing (TAP)-dependent MHC class I
processing. Understanding these pathways may provide insight to novel
therapies aimed at enhancing HIV antigen presentation and the subsequent
cellular immune response in HIV.
The candidate is currently a pulmonary fellow in the Department of Medicine at
Indiana University. At the proposed start-up time the candidate will be a
Lecturer on the faculty in the Pulmonary and Critical Care Division with 75
percent protected time allocated for research. To date the candidate has
trained in the laboratory of Dr. Homer Twigg, acquiring basic immunologic
knowledge and laboratory skills. This proposal is a logical mechanistic
extension of this work designed to allow the candidate to develop a basic
understanding of antigen processing pathways. Importantly, in this proposal
the candidate will develop new research skills by working in the laboratories
of Dr. Homer Twigg (CTL cloning, CTL generation and assays), Dr. Janice Blum
(intracellular antigen processing pathways, transfection techniques using TAP-deficient
cells), Dr. Randy Brukiewicz (working with vaccinia virus and
constructs to study specific CTL epitopes), and Dr. Douglas Perry (liposome
biology, phagocytic pathways). Each of these investigators have the expertise
and resources (money and laboratory space) necessary to ensure successful
completion of the training program. By acquiring the knowledge and skills
outlined in this proposal, the candidate hopes to fulfill his career goal of
becoming a full-time, funded researcher in an academic pulmonary division.