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

• Published in: Journal of immunological methods 2006-01, Vol.308 (1), p.1-12
• Main Authors: Dare, Raellene, Sykes, Pamela J., Morley, Alexander A., Brisco, Michael J.
• Format: Article
• Language: English
• Subjects: Adult; Aged; Aged, 80 and over; Aging - genetics; Aging - immunology; Biological and medical sciences; Clone Cells; Cloning, Molecular; Female; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; Genetic Markers; Human; Humans; Immunologic Memory; Leukocyte Common Antigens - metabolism; Male; Memory; Molecular immunology; Molecular Sequence Data; Repertoire development; T cell receptors; T lymphocytes; T-Lymphocyte Subsets - cytology; T-Lymphocyte Subsets - immunology; T-Lymphocytes, Cytotoxic - cytology; T-Lymphocytes, Cytotoxic - immunology; Techniques
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2005.08.016

We have established a method to estimate the number of clones in peripheral blood, using rearranged T cell receptor γ 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 × 10 − 6 (1 cell in 330,000 CD8+CD45RO+ cells) to 3.5 × 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 × 10 3–1.0 × 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.