Molecular Analysis of the Complementarity Determining Region 3 of the Human T Cell Receptor Beta Chain. Establishment of a Reference Panel of CDR3 Lengths from Phytohaemagglutinin Activated Lymphocytes

The T cell receptor (TCR) repertoire of a lymphocyte population may be characterized by the distribution of lengths of the hypervariable fragment known as the complementarity determining region 3 (CDR3). Immunological activity leading to clonal predominance will result in an over-representation of given CDR3 lengths and a distortion of the CDR3 length distribution. CDR3 length distribution may be studied by the in vitro amplification of TCRB cDNA followed by gel electrophoresis of the resulting product. We have established a simple, robust method for the evaluation of CDR3 length distribution in human lymphocyte samples. The CDR3 length distribution in phytohaemagglutinin (PHA) activated lymphocytes from a large number of healthy donors was established as a reference panel for each of 22 human TCR beta variable (BV) families. We propose that an abnormal CDR3 length distribution be defined as one in which one or more CDR3 lengths exceed the upper confidence limit (5% significance, one-sided test) given by this PHA reference population. Using this criterion in titration experiments, we were able to identify a clone when it constituted 2% of the cells analyzed. Over-dilution of cellular material or cDNA may produce falsely abnormal CDR3 length distributions. A nested technique using two separate amplification steps was found to yield results comparable in quality to the single amplification technique. When few cells are available, the nested method gives more material for CDR3 length analyses. However, it does not reduce the likelihood of a falsely abnormal distribution being recorded when the cellular material is too scarce.