Aging Aging

T-Cell Function in the Aged 283With respect to CD2-induced stimulation, mitogens such as PHA and severalanti-CD2 MAbs that trigger the CD2 receptor are powerful activators.Interestingly, CD2-induced activation is markedly diminished in the elderlyand because PHA is relatively nonspecific, CD2 activation is probably bestobserved using the anti-CD2 MAb pair T112 and T113. Together they arestrongly mitogenic for T-cells in the presence of ACs. More importantly, whenused with purified T-cells at relatively low concentrations, this pair of MAbsprovides a potent signal 1 without causing IL-2 secretion. Full activation isachieved, however, when used in combination with any one of a number ofdifferent costimulatory factors or cells. To this end, various cofactors can easilybe tested for their ability to provide an effective signal 2.Proliferation or activation can be determined in a number of ways. Clearly, thechoice of the primary readout system is best governed by the type of informationrequired. Other factors, including the availability of technical expertise, the sensitivityand specificity of the assay, cost, and time are also important considerations.The simplest approach is to measure tritiated thymidine incorporation. Probablythe best marker of T-cell activation, however, is the production of IL-2 andother T-cell-derived cytokines. Resting unstimulated human T-cells remaintranscriptionally silent even after several days of in vitro culture. Cytokinescan be detected at the mRNA level by reverse transcriptase-polymerase chainreaction (RT-PCR), in situ hybridization using labeled probes, or by in situPCR. The RT-PCR method (described in detail below) is relatively simple toperform; it is also fast, sensitive and semiquantitative (if required). Moreover,it allows the expression of a large range of activation-associated genes to beexamined simultaneously using very small cell numbers, for example,cytokines and their receptors, other immune function genes (e.g., HLA DR,LFA–1, transferrin receptor, adhesion receptors, CD80, CD86, CD40), cellcycle-associatedgenes, proto-oncogenes etc.At the protein level, cytokines are readily detected by enzyme-linkedimmunosorbent assay (ELISA) (using cell supernatants) and in situ immunohistochemistry.The latter technique is very impressive (particularly when coupledto double or triple labeling and flow cytometry allowing the accurate enumerationof specific cell types, e.g., IL-4 producing CD4 + CD45RO + T-cells) but thetrick to getting it to work consistently is obtaining the right MAbs (e.g., see refs.3–5). It is definitely a procedure worthwhile pursuing in the aging area.Immunophenotyping and cell-cycle analysis using BrdU or propidium iodidecould also be used to complement the above readouts. Wherever possible, utilizea number of different detection systems when comparing T-cell proliferationin aged and young subjects. Finally on the subject of read-out, if “old”T-cells do not appear to respond to a particular stimulus, check if they haveactually apoptosed.