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

Abstract

In order to analyse the diversity of T-cell receptors (TCRs) expressed by the T-cell population activated by allogeneic HLA-DR stimulation, TCR beta cDNA was synthesized from mRNA of human CD4+ T cells that had been stimulated in a primary mixed lymphocyte reaction (MLR). The TCR beta cDNA was amplified by the polymerase chain reaction (PCR), subjected to bacterial cloning, and sequenced from V beta through J beta. Twenty-six different V beta and 10 different J beta segments were detected among 56 randomly selected cDNA clones. Occurrences of V beta 17.1 and J beta 1.5 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.

T‐Cell Clonal Change after Allo‐Kidney Transplantation in Humans

“…basic in-vitro studies, Onda et al demonstrated that T cells stimulated in vitro with primary allogeneic HLA-DR antigens show as great a CDR3 diversity as those stimulated nonspecifically with anti-CD3 specific antibody [10]. In contrast, T cells stimulated with a single peptide as an antigen showed more restricted CDR3 diversity, which implies that TCRs involved in direct allo-recognition can exhibit much greater diversity compared with those involved in indirect allo-recognition [10,11]. Even when the single mismatched HLA locus (HLA-DR1) was targeted, a highly variable CDR3 or junctional sequence was shown for allo-reactive T-cell clones [12,13].…”

Analysis of the T‐Cell Receptors Utilized for Allogeneic HLA‐DR Recognition: Comparison of Different Responder‐Cell Donors Possessing an Identical HLA‐DR Allele

“…Random cDNA clone sequence analysis. Double-stranded TCRA and TCRB cDNAs were synthesized and amplified by the anchor-ligation PCR method, as described previously [10,18]. Briefly, a synthetic double-stranded anchor was added to the upstream region of the cDNA by blunt-ended ligation and the anchored cDNA was amplified by 30 PCR cycles using a primer complementary to the anchor in combination with either the TCRAC primer FPR16 or TCRBC primer FPR27.…”

“…Next, we carried out random cDNA cloning of the TCRs utilized for allogeneic HLA-DRB1*0406 recognition by the responder cells. The TCR cDNAs prepared from the MLR-activated CD4 þ T cells of 1CF, HID and KT14 were subjected to the anchorligation PCR, in which a synthetic anchor ligated in the upstream region of the cDNA enabled PCR-amplification of TCR cDNAs with different TCRV segments to be achieved equally efficiently [10,18]. Of the 41-44 randomly selected clones, those derived from 1CF, HID and KT14 yielded 39, 40 and 39 productive TCRA cDNA clones and 40, 41 and 43 productive TCRB cDNA clones, respectively.…”

“…We and other investigators have carried out sequence analysis and demonstrated that TCRs expressed by alloreactive T cells are highly diverse with respect to both their variable (TCRV) segment usage and third complementarity-determining region (CDR3) sequences. These findings indicate that each alloreactive T-cell population consists of a large number of different T cells, each type of which may be directed against a distinct alloantigen structure [3][4][5][6][7][8][9][10].…”

“…In addition to the basal diversity of alloreactive TCRs, our previous study revealed that several TCR-CDR3 sequences occurred more than once in CD4 þ T cells activated with allogeneic HLA-DR molecules by the primary mixed lymphocyte reaction (MLR) [10]. This result suggested that certain T-cell clones had been utilized preferentially for allorecognition.…”