The Turnover Rate of Human CD4+CD25+ T cells in vivo

Our bodies mount immune responses when attacked by infectious organisms. The central participants in this response are our white cells, the soldiers that combat the infective agents. However, these white cells are under strict orders not to attack helathy tissue by friendly fire and these commands come in part from a particular kind of white cell known as regulatory cells. Little is known about how these regulatory cells are trained in the body and where they are recruited from. The main objective of this project is to determine if these cells are recruited from the ranks of the responsive immune army and if so, what additional training does this involve ? We will also determine if the ageing and potentially dysfunctional immune army in elderly humans who are over 80 years old can be trained to become regulatory cells in the same way as those in young people. The timing of this study is highly opotimal as we will be using two different novel human experimental systems that we have already developed, that enable us to investigate immune cells and immune responses in humans in vivo.

A unique population of helper CD4+ T cells that express the CD25 surface marker has been shown to inhibit immune responses and prevent the development of inflammatory and autoimmune diseases. Where do these cells come from? In mice these cells have been shown to arise in the thymus. However the thymus involutes and thus cannot continuously generate these cells especially in humans who have a much longer lifespan. An alternative possibility is that CD4+CD25+ regulatory cells differentiate from responsive cells and some evidence for this exists in mice. However, until now, limitation in experimental procedures has precluded the investigation of these cells in humans in vivo. To test this hypothesis, we will first investigate the proliferation of these cells in vivo using novel technology that involves the safe labelling of proliferating cells with deuterated glucose in vivo. This is followed by analysis of extent of 2H labelling in sorted CD4+CD25+ and CD4+CD25- subsets in the same individuals at 4 different time points by mass spectroscopy. This will indicate the rate of turnover of these cells in vivo and enable us to conclude if these cells are generated sporadically or continuously throughout life. We will measure the impact of this turnover by measuring telomere length in the cells by fluorescence in situ hybridization coupled to flow cytometry, a technique already developed in house, and telomerase inducibility of these cells, using the telomere repeat amplification protocol (TRAP) assay. Thirdly, we will determine the clonal relationship between responsive and regulatory T cells which will enable us to determine if they are derived form the same original clones of cells. This will be performed using the heteroduplex method which we have used successfully in previous studies. Finally, we will explore gene changes using cluster analysis techniques that are associated with the induction of anergy and regulatory activity in antigen-specific responsive T cells.