The dynamics of T lymphocytes have moved to center-stage in HIV immunopathogenesis and therapy. Recent work has led to a high turnover model of CD4 depletion. This model is based on inference and extrapolation rather than direct measurement, however, because methods fro direct measurement of lymphocyte proliferation and destruction (turnover) directly, in a variety of clinical settings, to test basic hypotheses concerning the immunopathogenesis of HIV disease. We have developed an endogenous labeling technique, using stable (non- radioactive) isotopes with mass spectrometric (MS) analysis, for measuring proliferation and turnover of cells, including T cells in humans. T cell DNA is labeled in vivo by the de novo nucleotide synthesis pathway, by administering [2H] or [13C]-glucose to label the deoxyribose (dR) moiety, which is then analyzed by MS. Labeling of dR in this manner has important advantages over previous techniques used in animals from nucleosides (e.g., 3H-thymidine).
Our specific aims relate to 3 general areas.
Aim 1 is to test the high CD4-turnover model. CD4 proliferation rates will be compared in AIDS patients (CD4 less than200/mm3) to seronegative controls. Next, the mechanism by which anti-retroviral therapy increases CD4 counts in AIDS will be determined (reduced destruction, disinhibited differentiation and/or proliferation, altered distribution). The kinetics of naive (CD45RA+CD62L+) vs memory (CD45RA-CD62L-) CD4+ and CD8+ T cell subsets will also be measured, to determine functional consequences for peripheral immune repertoire and the relationship between plasma viral load and the production of CD4+ and CD8+ T cells. Second, earlier changes in lymphocyte dynamics in progression of HIV disease will be measured (CD4 counts greater than 500 and 200-500/mm3) to establish the burden on CD4 production early. The kinetics o naive and memory CD8+ and CD4+ cells will also be measured over the range of CD4 counts (cross-sectionally and longitudinally), to relate dynamics to changes in subset proportions. Inter-individual variability for T cell dynamics in subjects matched for virologic parameters will be analyzed (host-virus interactions). The isotopic method also allows kinetics of newly produced T cells to be measured, to assess selective destruction of dividing cells. Finally, we will test predictions (e.g. dynamics in long-term survivors s more rapid progressors; the mechanism by which interleukin-2 increases CD4 counts in AIDS). Measurement of the dynamics of HIV-virions with stable isotopes will also be pursed, to develop more completer computer models. In summary, the goal of this project is to measure lymphocyte dynamics directly in humans, using a new technique recently developed in our laboratory, in order to test several fundamental hypotheses concerning the immunopathogenesis and treatment of HIV disease.
