Aging Aging

282 BeckmanWhatever the mechanism employed, it is important that the final T-cell fractionis free of B cells (and other T-cell subpopulations if desired), monocytes,and natural killer (NK) cells. For example, “young” T-cells in particular canrespond well to a single mitogenic signal such as phytohemagglutinin (PHA)with < 1% contaminating monocytes. NK cells, on the other hand, are oftenoverrepresented in the circulation of aged individuals and because NK cellscan be purified along with T-cells, they not only may skew the final “T” cellcount but also are a potent source of interferon-γ when activated.Some of the advantages of working with purified populations of T-cells arethat functional changes can be related to a specific cell type(s), and most importantly,when comparing young and aged subjects, the actual number of T-cells(and indeed, accessory cells) seeded into a given well can be controlled in allexperiments.The disadvantages of multiple purification procedures are low cell yields(despite relatively large volumes of blood) and increased risk of contamination.It is also important that the purification process per se does not preactivate thecells. For example, avoid using strongly mitogenic MAbs or sheep red bloodcells (SRBCs) during positive selection. Although T-cell binding to SRBCs(E-rosettes) is a well-known means of enriching for T-cells, very low numbersof SRBCs provide sufficient stimulus to activate purified T-cells in the presenceof PHA.Human peripheral T-cells are normally quiescent. They can be activated tosecrete interleukin-2 (IL-2) and proliferate by a variety of stimuli. In general,complete T-cell activation requires two signals. The first is delivered throughthe T-cell receptor (TCR)–CD3 complex by small antigenic peptides in associationwith HLA molecules. This is best achieved in the laboratory usingreagents such as superantigens or MAbs directed against TCR or CD3. Thefirst signal can also be triggered by ligands that bind the surface receptor CD2.Second signals follow engagement of ligands on the surface of antigen presentingcells (APCs) with T-cell surface receptors (TCRs) such as CD28. However,a variety of molecules and in vitro systems can bypass the need for APCs orother accessory cells (ACs), making it possible to dissect specific T-cell–ACinteractions and identify actual sites of dysfunction.Two potent pan T-cell mitogens, namely MAb OKT3 (anti-CD3) and thesuperantigen, staphylococcal enterotoxin B (SEB), have an obligatory requirementfor ACs. However, OKT3-induced activation can be achieved in theabsence of ACs either using wells precoated with anti-mouse IgG, which promotescrosslinking of the TCR–CD3 complex, or using plate-bound OKT3 andfibronectin. The latter combination is a particularly strong T-cell stimulus.Alternatively, plate-bound 64.1 (an IgM anti-CD3 MAb) alone can induceproliferation in highly purified T-cells.