COMPARISON OF 2 TECHNIQUES FOR THE MOLECULAR TRACKING OF SPECIFIC T-CELL RESPONSES - CD4(-CELL CLONES PERSIST IN A STABLE HIERARCHY BUT AT A LOWER FREQUENCY THAN CLONES IN THE CD8(+) POPULATION() HUMAN T)

Oligoclonal or clonal T-cell expansions, presumed to be antigen driven , are frequently sought and followed for diagnostic and prognostic pur poses, as well as to understand more about their natural history. Tech niques based on conservation of T-cell receptor CDR3 length are increa singly widely used, often without assessment of sensitivity or specifi city. We present a comparative evaluation of a novel modified heterodu plex technique and a CDR3-length-based assay. Dilution of a known clon e in a mixed T-cell population shows that in our hands the heteroduple x technique is at least 10-fold more sensitive than the CDR3-length-ba sed assay. However, even with this level of sensitivity, we do not det ect clonal expansions in unstimulated CD4(+) T cells. This contrasts w ith the frequent detection of CD8(+) clones in fresh samples and sugge sts different mechanisms of clonal homeostasis in the two subsets. We show that both techniques detect functional expansions after in vitro stimulation with a recall antigen. The distinct molecular footprint se en with the heteroduplex technique allows reproducible follow up of sp ecific clonal expansions. We have exploited this to demonstrate that t he repertoire of clones expanded by in vitro tetanus toroid stimulatio n shows stability within an individual, implying long-term maintenance of multiple CD4(+) clones.