Effect of age on the repertoire of cytotoxic memory (CD8+CD45RO+) T cells in peripheral blood: the use of rearranged T cell receptor gamma genes as clonal markers.

We have established a method to estimate the number of clones in peripheral blood, using rearranged T cell receptor gamma genes as clonal markers, selecting cells at random, and establishing the sizes of the clones to which they belong. Clone sizes were quantified by a clone-specific PCR test based on the VNJ junctional sequence, which typically detects 1-2 copies of its target gene. All clones chosen for study were subsequently quantified in blood, and sizes ranged from 3 x 10(-6) (1 cell in 330,000 CD8+CD45RO+ cells) to 3.5 x 10(-2) permitting numbers of clones to be estimated from the harmonic mean of clone size. Two independent estimates from a healthy young adult (20-30 years old) gave repertoires of 94,000 and 110,000 clones. Two other healthy young adults gave repertoires of 40,000 and 55,000 clones. Repertoires in four healthy active older (>75 years old) adults were more variable but generally lower, being 3600, 5500, 14,000 and 97,000 clones, despite enlarged clones making up >1% of the compartment in the last individual. Overall, young adults had smaller clones (p=0.026, non-directional Mann-Whitney U-test). If the human body contains 5 l of blood, clones have 2 x 10(3)-1.0 x 10(7) cells in blood. These results confirm a diverse repertoire of rearranged T cell receptor gamma genes. The number of clones thus defined are broadly consistent with other estimates of repertoire, despite differences in marker genes used and subsets of cells studied.