Aging Aging

86 Kirk and Millerexperiments on unseparated T-cell pools are likely to confound age effects onactivation pathways with the effects of subset transitions. Aging leads to anincrease in memory cells in both the CD4 and CD8 pools as measured by theincrease in CD44 hi cells (4). The naïve and memory pool can be further subdividedbased on differences in expression of the membrane glycoprotein, P-glycoprotein,which also shows increased expression with age (5). It thus seemsreasonable to study activation pathways first in separated CD4 and CD8 subsets,and to include studies of separated subsets wherever practical.Purification of these subsets from spleens begins with the depletion of erythrocytesand B cells by differential centrifugation and panning on anti-IgGcoatedplates, respectively (6). These procedures typically produce 25–35 ×10 6 T-cells from a single mouse spleen, among which 85–95% express the CD3ε-chain characteristic of T-cells. For CD4 + T-cell purification, as is describedin this chapter, CD8 cells are depleted by incubation with anti-CD8 antibodiesfollowed by removal with anti-IgG coated magnetic beads. Multiple subsetscan be depleted simultaneously; for example, addition of anti-CD44 and anti-CD8 can be used to purify CD4 naïve cells (i.e., CD4 + CD45RB hi ). Yields of15–20 × 10 6 CD4 + T-cells are usually obtained from a single spleen. The numbersof memory and naïve cells that can be obtained from a single spleen varydepending on the age of the mouse (see previous paragraph); experimentsinvolving cell types present only at low frequencies, such as memory cells fromyoung mice, may require pooling spleens of mice of the same age in individualexperiments.1.2. Stimulation of T-Cellswith Monoclonal Antibodies to Cell DeterminantsBecause the number of cells obtained from a single spleen is on the order of5–20 × 10 6 depending on the subset isolated, it is necessary to develop experimentalconditions that permit the stimulation and assay of samples as small as2–5 × 10 6 cells. Therefore, choosing appropriate stimuli as well as optimizingstimulation conditions will influence the level of activation of the target enzymeand are important factors in developing reliable assays for aging studies.Activation of T-cells can be achieved through the use of lectins, such asconcanvalin A or phytohemagglutinin, that bind to unknown cell surface determinants.T-cells can also be stimulated using monoclonal antibodies specific forcomponents of the TCR and for other cell surface markers (e.g., CD4 or CD28),or stimulated using pharmacological agents such as phorbol esters and calciumionophores. We describe here the use of monoclonal antibodies to the ε-chain ofthe TCR/CD3 complex and CD4 to stimulate CD4 + T-cells isolated from youngand old mice. We feel that this provides a physiologically relevant stimulationfor a polyclonal population of T-cells. Studies using T-cells from transgenic