The central mission of my laboratory in the field of HIV/AIDs is to develop techniques that allow direct testing in vivo of hypotheses relating to the immunopathogenesis and therapy of HIV infection. At the time that this award started, we had developed a stable isotope-mass spectrometric technique for measuring T-cell dynamics in humans, but there were several limitations and questions that could not be addressed. Over the past 3-4 years, my laboratory has worked systematically to advance the technology so that more complex, nuanced models of HIV pathogenesis can be tested experimentally. Technical progress has been extremely satisfying. By combining our heavy water (2H2O) labeling approach, greatly reduced requirements for cells and multiparameter cell sorting, a number of questions have been or can now be addressed. A protocol for identifying surface phenotypic markers of long-lived ("central memory") and short-lived ("effector memory") T-cells has been established. The markers tested so far (e.g., CCR7, CD28) have revealed only modest differences in turnover. The utility of T-cell turnover as a prognostic metric of viral pathogenicity has been explored in early HIV-infection and the kinetic consequences of chronic immune activation have been established in a mouse model. Kinetic measurements were also used to dissect out the contributions from local clonal expansion vs. cell recruitment into lymph nodes (LN), in the lymphoproliferative response to antigen (Ag) stimulation and to identify agents that specifically inhibit T-cell recruitment. Finally, ancillary aspects of the immunopathogenesis of HIV-infection, such as LN fibrogenesis, are now measurable concurrently, by heavy water labeling. In the next phase of this project, we propose to build on these methodologic advances to address a number of pathogenic questions. First, two remaining technical advances will be pursued (reducing cell numbers required to 10-100 cells;and reducing the time required for heavy water administration in humans to 1-3 days). We will continue to test different surface markers (e.g., integrin-a4b7, CD27, IL-7 receptor, IL-15R9, and IL-18R) as markers of central vs effector memory T-cells. Once identified, these sort-purified subpopulations will be further characterized molecularly and functionally, with the McCune lab. Also, the kinetics of Ag-specific T-cells and other rare subpopulations can now be characterized, due to the technical advances noted, in several settings: i) the response to yellow fever vaccination (with R. Ahmed);ii) HIV-gag specific T-cell kinetics in untreated seropositive patients, compared to anti-CMV-specific T-cells in HIVnegative subjects (with R.Sekaly);iii) response to hepatitis B and influenza vaccine and kinetics of T-regulatory cells (M.McCune);and iv) Kinetics of clones of T-cells transferred into mice in very low numbers (M. Jenkins). Two important clinical questions will also be addressed, using these subtle metrics of T-cell dynamics: are there residual immune abnormalities in HIV-infected person after 10-15 years of effective ARV therapy (compared to age matched seronegatives, with S.Deeks and P. Hunt)? And can the turnover of phenotypicallyspecific T-cell subpopulations serve as a marker of immune activation or viral pathogenicity? For the latter, HIV-infected subjects who are untreated, effectively ARV treated, or virologically resistant will be studied, and T-cell subpopulation kinetic markers will be compared to other metrics of immune activation. Finally, the kinetic techniques will be applied to lymphoid tissue: establish egress vs death of CD62L-CD44 bright T-cells in mice (W. Paul);and kinetics of T-cells and collagen (fibrogenesis) in gut lymphoid tissue in SIV-infection (S. Dandekar). In summary, the quanta! advances in measurement technology achieved in the first phase of this award now allow interesting and fundamental questions about HIV-immunopathogenesis to be addressed directly.