CCR5 and CXCR4 tropism and CD4 kinetics in HIV-1 infection

HIV infection causes disease by progressively depleting the number of a particular type of immune cells, ?CD4 cells?. We do not fully understand how infection results in loss of CD4 cells over time. Although HIV infects and destroys CD4 cells directly, the body normally just makes more cells to replace the lost ones.

We believe that what determines how HIV affects the body?s CD4 cells is the exact type of cell it infects. There are several sub-types of CD4 cells. Different strains of virus prefer to infect different sub-types of CD4 cell according to the receptors (like locking devices) on the cell surface. Two receptors, ?CCR5? and ?CXCR4?, appear particularly important. We hypothesise that the interaction between the viral CCR5/CXCR4 preference (?tropism?), the type of cell infected and the speed at which those cells are dividing determines the long-term effect of HIV on CD4 cell numbers.

To investigate this concept, we will measure how fast different sub-types of CD4 cells are dividing and disappearing within the body. Although much has been learnt from investigating cells and viruses in test tubes, answering such questions about CD4 loss can only be done by studying virus and CD4 cells within the body of people infected with HIV. We have recently developed a way of doing this using glucose containing an excess of deuterium. Deuterium is a naturally-occurring non-radioactive form (or isotope) of hydrogen which behaves exactly like hydrogen but can be measured using a mass spectrometer. We will give deuterium-labelled glucose (which is harmless) as a drink (half-hourly for 10 hours) to people with HIV infection, then take blood samples over the following 3 weeks. The glucose is used by dividing, but not non-dividing, cells to make new DNA. By separating the cells into their subtypes and measuring the deuterium in their DNA using a mass spectrometer, we can measure how fast CD4 subtypes divide and how long they survive in the body. We will also test the virus in the bloodstream for its CCR5/CXCR4 tropism. We will compare the results to those from a group of people without HIV infection.

We will use these measurements to create a mathematical picture or ?model? of how HIV interacts with different immune cells to explain why CD4 cells are destroyed slowly over time and why changes in viral tropism trigger accelerated loss of CD4 cells.

Progressive loss of CD4+ T cells is the cardinal feature of HIV infection. However, the pivotal mechanisms which drive CD4 cell loss remain poorly understood. Although immune activation and accelerated CD4 cell turnover are thought to be important contributors to such loss, the chemokine tropism of the dominant viral strain appears to be critical. HIV-1 entry into CD4+ T cells depends on interaction between envelope, CD4 and a co-receptor, CCR5 or CXCR4 for R5 and X4 viruses respectively. R5 viruses dominate early in disease but a switch to X4 triggers more rapid CD4 decline. We hypothesise that the dominant viral tropism and the rate of CD4 decline is determined by the kinetics of the T cell subpopulations for which the virus is tropic.

We propose to measure in vivo lymphocyte proliferation and death rates using deuterium-labelled glucose as a tracer for cell division in human volunteers. Specifically we aim to investigate: (1)The relative turnover rates of CCR5 and CXCR4 na?ve and memory lymphocytes in HIV-uninfected individuals, and (2) The effects of X4 and R5 viral strains on the turnover rates of CCR5 and CXCR4 na?ve and memory T cells in HIV-infected individuals before and after antiretroviral therapy.
From the above we propose to define a quantitative kinetic model to explain why the switch from R5 to X4 tropic viruses is associated with accelerated CD4 T cell decline.

Subjects will receive 6,6-D2-glucose as an oral solution half-hourly for 10 hours, a methodology we have previously validated. Cells will be sorted according to expression of CD4, CD45, CCR5 and CXCR4 by flow cytometry, DNA will be extracted and analysed by gas chromatography mass spectrometry for deuterium content. Data will be modelled to yield proliferation and disappearance rates of individual cell populations. Data will be related to the dominant viral tropism.

We believe that the findings will have important implications for understanding the pathophysiology of HIV infection and explaining how CD4 depletion occurs. Such data would form the basis for developing novel immunological strategies for HIV infection and understanding the potential cellular impact of novel agents currently being developed to interfere with virus-chemokine interactions.