T‐Cell Receptor Diversity Expressed by CD4+ T Cells Activated by Primary Allogeneic HLA‐DR Stimulation: Estimation of the Degree of CDR3 Diversity

In order to analyse the diversity of T-cell receptors (TCRs) expressed by the T-cell population activated by allogeneic HLA-DR stimulation, TCRβ cDNA was synthesized from mRNA of human CD4+ T cells that had been stimulated in a primary mixed lymphocyte reaction (MLR). The TCRβ cDNA was amplified by the polymerase chain reaction (PCR), subjected to bacterial cloning, and sequenced from Vβ through Jβ. Twenty-six different Vβ genes and 10 different Jβ segments were detected among 56 randomly selected cDNA clones. Occurrences of Vβ17.1 and Jβ1.5 were higher than those found in the CD4+ T-cell population activated with a CD3-specific antibody. A total of 53 different CDR3 sequences, two of them occurring more than once, were detected among the 56 cDNA clones. In order to estimate the degree of CDR3 diversity, amino acid similarity in the CDR3 region of the cDNA was calculated and compared with those of the anti-CD3-activated T-cell sequences as well as those of various published T-cell clone sequences, each directed to either alloantigens or single antigenic peptides. It was found that the similarity score among CDR3 sequences obtained from the MLR (56.4 ± 10.3) was comparable to those of anti-CD3-activated T cells (55.7 ± 10.7) and those of T-cell clones directed toward alloantigens (range, 48.4 ± 12.4−59.4 ± 13.1), but significantly smaller than those of T-cell clones directed toward single antigenic peptides such as those derived from myelin basic protein (75.6 ± 17.9) and cytochrome c (76.9 ± 20.5). These results provide quantitative proof that TCRs of T cells activated by primary allogeneic HLA-DR stimulation have a larger diversity than those recognizing single antigenic peptides.