Aging Aging

54 Pawelecdiluted cells on an irradiated feeder cell layer in the presence of chemicallydefined media supplemented with growth factors (e.g., IL-2). We wished toestablish whether culture aging of TCCs did occur, how it could be characterized,and whether it depended on the source and nature of the T-cells studied.The first question approached was to what extent culture conditions affectedcloning efficiencies and longevity of the TCC (8); most of our early data werethen obtained using a standard culture system employing medium supplementedwith human serum (HS) and natural IL-2 (9) and using feeder cellsconsisting of a pool of peripheral blood mononuclear cells (PBMCs) from >20random healthy donors. The T-cells to be cloned were derived from young adultdonors and were mostly prestimulated with alloantigens. Under these conditions,the type of T-cell predominantly derived was CD4 + and carried the α/βT-cell receptor (TCR2). This chapter therefore focuses on this type of TCC,and not on CD8 or TCR1 cells, which seem to behave somewhat differently inculture, but which we have not studied so extensively.For meaningful studies of T-cell aging in vitro, it is essential to know the invivo age of the starting T-cell population. This is impossible in a mixture ofT-cells from an adult individual, as separation of subsets according to naivecell and memory cell markers is a crude and inaccurate method. Assessing ageby measuring telomere lengths, even of individual cells, is also not satisfactory,as it cannot take into account whether telomerase has been activated atsome point in these cells (10). The only way to be sure that all T-cells beingstudied are of the same age at the beginning of the experiment is to isolateprecursors and cause them to differentiate into T-cells in vitro (11,12). Longevitycomparisons between these pre-T-cell-derived precursors and TCCs frommature T-cells of the same donors suggested that the latter have a shorter lifeexpectancy corresponding to the time required for the precursors to developinto T-cells in vitro (13). We therefore hypothesized that the T-cell “clock” wasfirst set at the time when fully mature T-cells were generated and not at sometime beforehand at the precursor or stem cell level (14). Going further back inthe T-cell differentiation pathway, CD34 + stem cells have the potential todevelop into T-cells in in vitro culture systems (15), and this property could beexploited to study T-cell aging. Thus far, cumbersome thymic organ culture orthymic stromal cell culture systems have been required for this; here we presenta variant TCC culture protocol that allows the generation of mature T-cells fromisolated CD34 + stem cells in liquid culture in the absence of thymic components.2. Materials1. T-cell growth factors (TCGFs): The quality and purity of the T-cell growth factorsemployed is critical. The main and most commonly used TCGF is IL-2 in theform of purified recombinant protein. Nowadays, many companies offer high-