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In order to evaluate the T cell receptor (TCR) usage by clones of human allograft infiltrating lymphocytes, this study utilized polymerase chain reaction (PCR) amplification of TCR transcripts from five clones which were previously shown to react with a human leucocyte antigen (HLA)-DR3 mismatch between a living related kidney donor and recipient. The five CD4+ (CD8-) clones, which were selected for TCR analysis, proliferated in response to HLA-DR3 and three of the clones were also cytotoxic against the same target cells. After identification of the TCRAV and TCRBV usage of the clones, the sequence of the TCR alpha and beta were determined by direct sequencing of the PCR product. The results indicate that several different TCRAV and TCRBV gene segments are used among the different clones, but the two clones that were both cytotoxic and proliferative in response to HLA-DR3 shared identical TCRAV27-J42-C and TCRBV13-D1-J1S2-C1 transcripts. The additional three clones showed various TCRAV and TCRBV transcripts, but evaluation of the CDR3 region of the TCR beta chain, corresponding to the peptide antigen binding sites, demonstrated shared amino acid motifs which resulted both from germline sequences and combinations of n-region and germline-derived codons. These results suggest that the repertoire for anti-HLA-DR3-reactive clones can include a diverse expression of TCR, but there may be selection for some clones, as well as conserved motifs in the CDR3 region of anti-DR3 specific clones.

Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.

T lymphocyte regulation of immunoglobulin production may be abnormal in some patients with common variable immunodeficiency (CVI). Phenotypic analysis of peripheral blood T lymphocytes from nine patients with CVI was conducted to examine whether an abnormal distribution could be detected in a functionally distinct T lymphocyte subpopulation. The percentage of CD8+ lymphocytes proved to be increased in some patients and decreased in others. In comparison with normal controls, many patients with CVI had reduced percentages of lymphocytes expressing both CD4 and CD45RA, a phenotype associate with naive CD4+ cells. There was no significant difference in CD4+ populations bearing CD29 or leucocyte adhesion molecule-1 (LAM-1) antigens. The pattern of gene rearrangement of the T cell antigen receptor (TCR) was studied using peripheral blood lymphocytes from these patients with CVI. Genomic DNA from freshly isolated lymphocytes as well as from selectively propagated CD4+ or CD8+ populations were examined using Southern blot analysis and a probe for the beta chain of the TCR. A polyclonal pattern of TCR gene rearrangement, without the appearance of dominant non-germline bands, was demonstrated in all patient samples. These data suggest that the T lymphocytes in patients with CVI have a polyclonal pattern of TCR rearrangement despite an abnormal distribution of T cell subpopulations in some patients.

Graft-infiltrating lymphocytes from both human renal and cardiac allografts were propagated in interleukin 2 in order to evaluate rearrangements in the T-cell receptor (TcR) beta-chain genes. Individual biopsies from renal allografts during episodes of cellular rejection were examined as well as multiple biopsies of heart transplant patients from whom endomyocardial samples were taken prior to, during, and after episodes of rejection. TcR beta-chain rearrangements were evaluated in Southern blots using DNA extracted from interleukin 2-propagated cells and digested with restriction endonucleases permitting assessment of rearrangements to both C beta 1 and C beta 2. Rearrangements shared among greater than 5% of the "bulk" culture appear as nongermline bands when hybridized with a C beta probe. Single-cell progeny were generated from limiting dilution, and the rearrangements among the cloned progeny compared to the "bulk" of the cultured progeny of graft-infiltrating lymphocytes. The results indicate that "dominant" rearrangements are a common feature of renal allograft-infiltrating lymphocytes (14 of 15 cases examined). Since the number of cells which can be recovered from a given cardiac biopsy may be limiting, evaluation of clonal dominance from these cultures is more difficult to evaluate. However, sharing of "dominant" rearrangements among multiple biopsies from the same cardiac allograft patient indicates an in vivo selection for T cells with the same receptor rearrangement. Analysis of individual clones showed 3/33 clones from a renal allograft sharing the "dominant" rearrangement noted in the bulk culture, but none of these "dominant" clones showed antidonor specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies to the clonally unique variable-region determinants (idiotype) of the antigen-specific alpha beta heterodimeric receptor of a clone of cytotoxic T cells (CTLs) were shown previously to render diverse cells, regardless of their own surface antigens, susceptible to lysis by that clone of CTLs. To extend these findings, we have sought to develop a general means for targeting cells for destruction by any CTL, without regard to its alpha beta idiotype and specificity for antigen. We explored the use of heteroantibody duplexes formed by joining covalently an antibody to the T3 complex (anti-T3), which is associated with the alpha beta receptors on all human mature T cells, and a second antibody, specific for an antigen on the intended target cell. The second antibody selected in this study was specific for the idiotype (Id) of the surface immunoglobulin of a human B-lymphoma (anti-Ig Id). In the presence of the anti-T3/anti-Ig Id heteroantibody duplex the B-lymphoma cells were lysed by a clone of human T8+ CTLs (of unrelated specificity) but not by a noncytotoxic clone of human T4+ helper T cells, and lysis by the CTLs was specifically blocked by the uncoupled anti-T3 or the uncoupled anti-Ig Id antibodies. The extent of the heteroantibody-dependent cytolysis depended both on the heteroantibody concentration and on whether the intended target cells or the CTL effectors were initially preincubated with the heteroantibody. Under optimal conditions, heteroantibody-dependent lysis of the surrogate target (B-lymphoma) cells by the CTLs compared favorably with lysis of their natural target cells by the same CTLs. Overall, our findings suggest that heteroantibody duplexes containing anti-T3 antibody may be capable of targeting selected cells, such as tumor cells, for destruction in vivo by the body's CTLs.