HEALTHY-HUMAN T-CELL RECEPTOR BETA-CHAIN REPERTOIRE - QUANTITATIVE-ANALYSIS AND EVIDENCE FOR J-BETA-RELATED EFFECTS ON CDR3 STRUCTURE AND DIVERSITY

Analysis of TCR repertoire usage and clonality is of potential value i n understanding the pathogenesis of a number of human immune-mediated diseases. In diseases that are likely to be dependent on antigen-drive n T cells, it has been suggested that particular TCR junctional region or CDR3 sequences may be critical. Rigorous methods for TCR analysis which are both quantitative and qualitative are therefore required. Of those commonly available, only anchor and inverse PCR are capable of giving high-quality information on V, D, N, and J region usage, but it has not been established whether both methods are quantitatively or a nalytically equivalent. We show here that both methods detected consid erable variability in the usage of V beta and J beta segments in the p eripheral blood repertoire of a normal individual. No preferential V-J pairing could be demonstrated. An excess usage of J beta 2 family mem bers was indicated by both methods, although the relative usage of dif ferent J beta 2 families differed between the two techniques. The pred ominantly used V beta usage showed that for some families, estimates o f their frequency in the repertoire differed significantly between the anchor and inverse libraries. When sampling relatively few clones the variation between V beta families estimated using the two methods can be considerable. This is likely to be a result of sampling error from a large gene family. Large-scale screening of several thousand clones is recommended to confirm the absolute values obtained from sequencin g. Variation in CDR3 length appeared to be normally distributed, sugge sting that a statistically optimal junctional region length may have b een selected for contact with antigen. CDR3 length distribution differ s significantly between receptors, which have rearranged to J beta 1 v ersus J beta 2 families, with the J beta 2-associated CDR3 on average between 0.5 and 1.2 of an amino acid longer. Thus the TCR beta junctio nal region repertoire of humans is subject to structural constraints a ssociated with J beta usage. It will be important to establish whether variation in CDR3 length and J beta usage exists between subsets of h uman T cells in order to interpret TCR repertoire data from disease an d control tissues.