Human T-cell Receptor Beta-chain Research Articles

Compositional characteristics of human peripheral TRBV pseudogene rearrangements

The diversity of the T cell receptor (TCR) complementarity determining region 3 (CDR3) repertoire is the result of random combinations, insertions and deletions during recombination of the germline V, D and J gene fragments. During evolution, some human TCR beta chain variable (TRBV) pseudogenes have been retained. Many previous studies have focused on functional TRBV genes, while little attention has been given to TRBV pseudogenes. To describe the compositional characteristics of TRBV pseudogene rearrangements, we compared and analysed TRBV pseudogenes, TRBV open reading frames (ORFs) and functional TRBV genes via high-throughput sequencing of DNA obtained from the peripheral blood of 4 healthy volunteers and 4 patients. Our results revealed several differences in J and D gene usage. The V deletion distribution profile of the pseudogenes was significantly different from that of the ORFs and functional genes. In addition, arginine, lysine and cysteine were more frequently used in putative CDR3 pseudogene rearrangements, while functional rearrangements used more leucine. This study presents a comprehensive description of the compositional characteristics of peripheral TRBV pseudogene rearrangements, which will provide a reference for further research on TRBV pseudogenes.

Structural basis of T-cell specificity and activation by the bacterial superantigen TSST-1

Superantigens (SAGs) bind simultaneously to major histocompatibility complex (MHC) and T-cell receptor (TCR) molecules, resulting in the massive release of inflammatory cytokines that can lead to toxic shock syndrome (TSS) and death. A major causative agent of TSS is toxic shock syndrome toxin-1 (TSST-1), which is unique relative to other bacterial SAGs owing to its structural divergence and its stringent TCR specificity. Here, we report the crystal structure of TSST-1 in complex with an affinity-matured variant of its wild-type TCR ligand, human T-cell receptor beta chain variable domain 2.1. From this structure and a model of the wild-type complex, we show that TSST-1 engages TCR ligands in a markedly different way than do other SAGs. We provide a structural basis for the high TCR specificity of TSST-1 and present a model of the TSST-1-dependent MHC-SAG-TCR T-cell signaling complex that is structurally and energetically unique relative to those formed by other SAGs. Our data also suggest that protein plasticity plays an exceptionally significant role in this affinity maturation process that results in more than a 3000-fold increase in affinity.

A conserved tissue-specific structure at a human T-cell receptor beta-chain core promoter.

The T-cell receptor (TCR) beta-chain promoters have been characterized as nonstructured basal promoters that carry a single conserved ubiquitous cyclic AMP-responsive element. Our investigation of the human TCR beta gene uncovers a surprisingly complex and tissue-specific structure at the TCR Vbeta 8.1 promoter. The core of the promoter (positions -42 to +11) is recognized by the lymphoid cell-specific transcription factors Ets-1, LEF1, and AML1 as well as by CREB/ATF-1, as is demonstrated in gel shift and footprinting experiments. With the exception of LEF1, these factors activate transcription in T cells. Binding sites at the core region show little conservation with consensus sites. Nonetheless, CREB, Ets-1, and AML1 bind and activate cooperatively and very efficiently through the nonconsensus binding sites at the core promoter region. Moderate ubiquitous activation is further induced by CREB/ATF and Sp1 factors through proximal upstream elements. The tissue-specific core promoter structure is apparently conserved in other T-cell-specifically expressed genes such as the CD4 gene. Our observations suggest that both the enhancer and the promoter have a complex tissue-specific structure whose functional interplay potentiates T-cell-specific transcription.

Transactivation of the Moloney murine leukemia virus and T-cell receptor beta-chain enhancers by cbf and ets requires intact binding sites for both proteins.

The Moloney murine leukemia virus (Mo-MLV) enhancer contains binding sites (LVb and LVc) for the ets gene family of proteins and a core site that binds the polyomavirus enhancer-binding protein 2/core-binding factor (cbf) family of proteins. The LVb and core sites in the Mo-MLV enhancer contribute to its constitutive activity in T cells. All three binding sites (LVb, LVc, and core) are required for phorbol ester inducibility of the Mo-MLV enhancer. Adjacent binding sites for the ets and cbf proteins likewise constitute a phorbol ester response element within the human T-cell receptor beta-chain (TCR beta) enhancer and contribute to constitutive transcriptional activity of the TCR beta enhancer in T cells. Here we show that the CBF alpha subunit encoded by the mouse Cbfa2 gene (the murine homolog of human AML1) and three ets proteins, Ets-1, Ets-2, and GA-binding protein (GABP), transactivate both the Mo-MLV and mouse TCR beta enhancer in transient-expression assays. Moreover, we show that transactivation by Cbf alpha 2 requires both intact ets and cbf binding sites. Transactivation by Ets-1, Ets-2, and GABP likewise requires intact binding sites for ets proteins and CBF. Supportive biochemical analyses demonstrate that both proteins can bind simultaneously to a composite enhancer element. These findings suggest that ets and cbf proteins cooperate in vivo to regulate transcription from the Mo-MLV and TCR beta enhancers.

PCR-based genotyping and haplotype analysis of human TCRBV gene segment polymorphisms.

There are at least 63 tandemly arranged human T-cell receptor (Tcr) beta-chain variable region (BV) gene segments, which have presumably arisen by repeated gene duplication events. The 5'-most half of the TCRBV gene loci is particularly complex in organization due to the presence of multiple interspersed members of the largest BV subfamilies, BV5, BV6, and BV13. Polymorphism and linkage relationships among these genes has been poorly characterized in part due to the high similarity of these duplicands. Germline DNA polymorphisms were specifically examined in the exons and introns of these and other BV gene segments distributed across 240 kilobases (kb) in this 5'-most region. Polymerase chain reaction restriction enzyme-based assays were used to genotype ten point mutations in seven of the BV gene segments. Eight of these polymorphisms altered an amino acid of the BV gene segment. In addition, length polymorphisms due to simple sequence repeats were noted in the introns of six BV6 subfamily members. Approximately 250 unrelated haplotypes were constructed by segregation analyses of fifteen of these TCRBV polymorphisms. Linkage disequilibrium analyses indicated that haplotypic relationships are not detectable over a distance of more than 55 kb in this genomic region. These TCRBV polymorphisms, and the haplotypic analysis, provide important resources and guidance for future attempts to associate Tcr germline DNA differences in the human population with immune response differences, such as might occur in some autoimmune diseases.

Nucleotide sequence analysis of 77.7 kb of the human V beta T-cell receptor gene locus: direct primer-walking using cosmid template DNAs.

The nucleotide sequence of 77.7 kb from the human T-cell receptor beta-chain locus was determined directly from three overlapping cosmid clones using the primer-walking approach. Computer-aided analyses of this sequence reveal the presence of at least 11 genic regions that are closely related to the human T-cell receptor beta variable region (TCRBV) gene family. These include five germline sequences that have previously been determined, V beta 21.2, V beta 8.1, V beta 8.2, V beta 8.3, and V beta 16, and four whose sequences have partially been determined at the mRNA level, V beta 6, V beta 23, V beta 12.2, V beta 24. The two remaining V beta Tcr-related sequences have eluded discovery by cDNA and RT-PCR cloning and genomic blot hybridization methods. These two V beta Tcr-related genes lack > 75% nucleotide sequence identity with any other V beta Tcr gene member and therefore, by convention, are referred to as new subfamily members V beta 25 and V beta 26. This lack of shared identity with other subfamily members explains why they were not detected by hybridization. The promoter regions of these V beta Tcr genes contain the conserved Tcr decamer element located between 80 and 110 bp 5' of the translation start site, generally near a putative TATAA promoter element. Our sequence analysis also reveals that a 3.3-kb duplication unit was involved in the recombination event that produced the closely related V beta 8.1 and 8.2 gene subfamily members. This sequenced region of the V beta locus contains an average number of repetitive DNA elements (21 Alu, three L1, three MER, and three retrovirus-related elements.

A method for producing monoclonal antibodies to human T-cell-receptor beta-chain variable regions.

Study of the T-cell repertoire in humans has been hampered by the lack of monoclonal antibodies (mAbs) to the T-cell receptor (TCR) variable region (V) gene products. We describe a method for producing mAbs to the human TCR beta-chain V (V beta) gene products in which mice were immunized with a rat basophil cell line (RBL-2H3) transfected with the extracellular domain of the TCR heterodimer fused to the lambda chain of CD3. These cells acted as excellent immunogens for raising anti-TCR mAb and also formed the basis of a rapid screening assay. We generated mAbs against V beta protein of the TCR, showed that these mAbs stained approximately 1% of peripheral blood T cells, and further showed that the mAbs could stimulate proliferation of these T cells. We then characterized the mAbs by amplifying TCR cDNA derived from mAb-stimulated cells and sequencing the beta chain. All clones sequenced used the V beta 7.1 chain, proving conclusively that the mAbs generated were specific for V beta 7.1 subfamily. This method generates mAbs to human TCR V beta proteins efficiently and might allow production of a complete panel of mAbs directed against human TCR V beta proteins.

A model of in vivo cell proliferation of adult T-cell leukemia

We have made a model of in vivo cell proliferation of leukemic cells from adult T-cell leukemia (ATL) patients using severe combined immunodeficiency (SCID) mice. Peripheral blood mononuclear cells (PBMC) or lymph node cells (LNC) depleted of B cells and monocytes were intraperitoneally injected into SCID mice treated with antimurine interleukin-2 receptor (IL-2+) beta chain monoclonal antibody (MoAb)(TM- beta 1), followed by daily injection of human recombinant IL-2 until 60 days after cell injection. SCID mice injected with ATL cells from 6 of 8 ATL patients were found to have the tumor or leukemia 5 to 7 weeks after the inoculation of cells. Serum levels of soluble form of human IL-2R alpha chain (Tac) were markedly elevated in such mice. The cells recovered from the mice injected with leukemic cells from four different ATL patients had the same cell surface phenotype as that of original leukemic cells which were CD4+Tac+. Furthermore, we detected the same integration site of human T-cell leukemia virus type I (HTLV- I) provirus and the same rearrangement pattern of human T-cell receptor (TCR) beta chain gene as those of ATL cells by Southern blot hybridization, indicating that the cells proliferating in SCID mice were derived from the original ATL cell clone. Histologic examination showed that the pattern of the infiltration of ATL cells into various organs in SCID mice was similar to that of an ATL patient. Such a model of in vivo cell proliferation of ATL cells will be useful for the study of the mechanism of neoplastic cell proliferation and for the development of a new and effective treatment of ATL.

A Model of In Vivo Cell Proliferation of Adult T-Cell Leukemia

We have made a model of in vivo cell proliferation of leukemic cells from adult T-cell leukemia (ATL) patients using severe combined immunodeficiency (SCID) mice. Peripheral blood mononuclear cells (PBMC) or lymph node cells (LNC) depleted of B cells and monocytes were intraperitoneally injected into SCID mice treated with antimurine interleukin-2 receptor (IL-2+) beta chain monoclonal antibody (MoAb)(TM-beta 1), followed by daily injection of human recombinant IL-2 until 60 days after cell injection. SCID mice injected with ATL cells from 6 of 8 ATL patients were found to have the tumor or leukemia 5 to 7 weeks after the inoculation of cells. Serum levels of soluble form of human IL-2R alpha chain (Tac) were markedly elevated in such mice. The cells recovered from the mice injected with leukemic cells from four different ATL patients had the same cell surface phenotype as that of original leukemic cells which were CD4+Tac+. Furthermore, we detected the same integration site of human T-cell leukemia virus type I (HTLV-I) provirus and the same rearrangement pattern of human T-cell receptor (TCR) beta chain gene as those of ATL cells by Southern blot hybridization, indicating that the cells proliferating in SCID mice were derived from the original ATL cell clone. Histologic examination showed that the pattern of the infiltration of ATL cells into various organs in SCID mice was similar to that of an ATL patient. Such a model of in vivo cell proliferation of ATL cells will be useful for the study of the mechanism of neoplastic cell proliferation and for the development of a new and effective treatment of ATL.

Unusual organization of the human T-cell receptor beta-chain gene complex is linked to recombination hotspots.

Two rare Sfi I polymorphisms of 360 kb and 280 kb present within the human T-cell antigen receptor beta-chain gene complex were revealed by pulsed-field gel electrophoresis. They represent allelic variants of the polymorphic 330- and 300-kb Sfi I fragments previously described. The 360-kb polymorphism results from duplication of the 30-kb DNA fragment responsible for the 330/300-kb insertion/deletion-related polymorphism. The 280-kb polymorphism results from a 20-kb deletion from the 300-kb SfiI allele. The rare polymorphisms also map on either side of a Sal I site located near a recombination hotspot, suggesting that germline duplications and deletions arose from nonhomologous crossover events.

The human T-cell receptor beta-chain repertoire: longitudinal fluctuations and assessment in MHC matched populations.

The influence of the environment and of the major histocompatibility complex (MHC) in shaping the human T-cell receptor beta-chain variable region (TCRBV) repertoire has not been systematically studied. Here, expression of TCRBV gene families was estimated by a sensitive polymerase chain reaction (PCR)-based method. Serial studies of peripheral blood, performed at 2-week intervals over a 3-month period, revealed that fluctuation in the expression of many TCRBV genes occurred in healthy individuals and in the absence of clinically evident infections. Fluctuation of TCRBV4, TCRBV5.2, TCRBV9, and TCRBV13.1 genes were present in all subjects. Additional TCRBV genes fluctuated in some but not in other individuals. Comparison of the TCRBV repertoire between these unrelated individuals indicated differences in the mean expression of TCRBV5.1, TCRBV9, TCRBV11, TCRBV15, TCRBV17, and TCRBV20 genes. For any TCRBV gene, intersubject differences were generally of a magnitude of twofold or less. Larger differences characterized the TCRBV repertoire of CD4 compared to CD8 cells. Some differences, for example over-representation of TCRBV2 and TCRBV5.1 on CD4, and TCRBV10, TCRBV14, and TCRBV16 on CD8 cells, were present in most subjects. Individuals homozygous for DR2- or DR3-bearing extended MHC haplotypes displayed similar individual variability of TCRBV expression. These data indicate that the circulating TCRBV repertoire in humans is both dynamic and diverse. Both environment and MHC effects contribute to the diversity of TCRBV expression.

Organization of human T-cell receptor beta-chain genes: clusters of V beta genes are present on chromosomes 7 and 9.

To ascertain the extent and organization of the germ-line human T-cell receptor (TCR) beta-chain gene repertoire, beta-chain variable region (V beta) genes were mapped by pulsed-field gel electrophoresis, cosmid cloning, and in situ hybridization. Probes derived from the 24 known V beta families were mapped to a total of six Sfi I fragments in DNA samples from multiple individuals representing all possible haplotypes of TCR V- and C (constant)-region insertion/deletion-related polymorphisms. Four of the Sfi I fragments were linked to one another to develop an extended map of the TCR beta-chain gene complex previously localized to chromosome 7q35. The remaining two Sfi I fragments, containing 6 V beta genes, could not be linked to the TCR beta-chain gene complex. Using human-hamster somatic cell hybrids and in situ hybridization, these orphon genes were localized to chromosome 9p. Nucleotide sequences of the orphon V beta genes, derived from cosmid clones, were 93-97% identical to V beta genes in the TCR beta-chain gene complex. Open reading frames in three of the orphon V beta genes were intact as were the recombination signal sequences. As expected, based on their orphon status, none of the V beta genes of chromosome 9 was detected in transcripts containing C beta. These results indicate that the functional germ-line V beta repertoire in humans is substantially (10%) smaller than previously estimated.

A phorbol ester response element within the human T-cell receptor beta-chain enhancer.

The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 and beta E3 Ets site mutations also profoundly affected activity and inducibility of the enhancer. In contrast, enhancer activity but not its inducibility was affected by mutation of the beta E2 core-binding factor site. Cotransfection studies showed that Ets-1 specifically repressed activity of the multimerized beta E2 element and the complete T-cell receptor beta-chain enhancer. These data show that the T-cell receptor beta-chain enhancer responds to protein kinase C-mediated activation signals via a functional domain, composed of two elements, which contains binding sites for Ets transcription factors and which is negatively regulated by Ets-1.

Identification of novel human T-cell receptor V beta gene segments by the anchored-polymerase chain reaction.

We have studied the diversity of the expressed human T-cell receptor (TCR) beta-chain repertoire by analysis of mRNA from unstimulated peripheral blood T-cells. The anchored-polymerase chain reaction (PCR) was used to isolate TCRB transcripts. Of 20 full or near full-length functional transcripts sequenced, two were novel TCRVB gene segments. They have strong sequence similarities to the known TCRBV5, and 8 subfamilies. Southern blot analysis and sequence-specific oligonucleotide hybridization confirmed: a) that these sequences are present in genomic DNA; b) their relationship to the known TCRBV families. A TCRBV sequence similar to a recently identified novel TCRBV24 subfamily was also found. We show by southern blotting that this sequence forms a single member subfamily, and by deletion analysis of T-cell lines, we have mapped this sequence to lie between the genes which encode the TCRBV8.1 and TCRBV5.3 gene segments. The results show that the anchored PCR is a powerful tool in the analysis of the TCR repertoire, which may contain more V gene segments than previously defined.

A method for production of antibodies to human T-cell receptor beta-chain variable regions.

Mouse T-cell hybridomas bearing human V beta elements were produced by transfection of human/mouse hybrid T-cell receptor beta-chain genes into a mouse T-cell hybridoma lacking an endogenous beta-chain gene. These hybridomas were entirely mouse in origin except for the human V beta region. These cells were used to immunize mice against human V beta elements. Mouse monoclonal antibodies have thus been generated against human V beta 13.1 and -13.2. We expect that the method outlined in this paper will be useful in the production of monoclonal antibodies specific for other human V beta or V alpha elements.

Sequence and expression of a novel human T-cell receptor ?-chain variable gene segment subfamily

We describe the characterization of a novel subfamily of human T-cell receptor beta-chain variable TCRBV gene segments, detected through homology to a mouse Tcrb-V13 gene segment probe. The new human TCRBV gene segment subfamily TCRBV21 contains three members, all of which have been characterized with respect to nucleotide sequence, chromosomal location, and expression in human thymocytes and peripheral T lymphocytes. Our results indicate that one member of the TCRBV21 subfamily is not expressed in either tissue type although its coding region, promoter, recognition, and splice sequences all appear to be functional at the nucleotide level, possibly indicating regulation at the level of transcription.

Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay.

DNA diagnostics, the detection of specific DNA sequences, will play an increasingly important role in medicine as the molecular basis of human disease is defined. Here, we demonstrate an automated, nonisotopic strategy for DNA diagnostics using amplification of target DNA segments by the polymerase chain reaction (PCR) and the discrimination of allelic sequence variants by a colorimetric oligonucleotide ligation assay (OLA). We have applied the automated PCR/OLA procedure to diagnosis of common genetic diseases, such as sickle cell anemia and cystic fibrosis (delta F508 mutation), and to genetic linkage mapping of gene segments in the human T-cell receptor beta-chain locus. The automated PCR/OLA strategy provides a rapid system for diagnosis of genetic, malignant, and infectious diseases as well as a powerful approach to genetic linkage mapping of chromosomes and forensic DNA typing.

Identification and functional characterization of the human T-cell receptor beta gene transcriptional enhancer: common nuclear proteins interact with the transcriptional regulatory elements of the T-cell receptor alpha and beta genes.

A transcriptional enhancer has been mapped to a region 5.5 kilobases 3' of the C beta 2 gene in the human T-cell receptor (TCR) beta-chain locus. Transient transfections allowed localization of enhancer activity to a 480-base-pair HincII-XbaI restriction enzyme fragment. The TCR beta enhancer was active on both the minimal simian virus 40 promoter and a TCR beta variable gene promoter in both TCR alpha/beta + and TCR gamma/delta + T cells. It displayed significantly less activity in Epstein-Barr virus-transformed B cells and K562 chronic myelogenous leukemia cells and no activity in HeLa fibroblasts. DNA sequence analysis revealed that the enhancer contains a consensus immunoglobulin kappa E2 motif, as well as an AP-1-binding site and a cyclic AMP response element. DNase I footprint analyses using Jurkat T-cell nuclear extracts allowed the identification of five nuclear protein-binding sites, T beta 1 to T beta 5, within the enhancer element. Deletion and in vitro mutagenesis studies demonstrated that the T beta 2- and T beta 3- and T beta 4-binding sites are each required for full transcriptional enhancer activity. In contrast, deletion of the T beta 1- and T beta 5-binding sites had essentially no effect on enhancer function. Electrophoretic mobility shift assays demonstrated that TCR alpha/beta + and TCR gamma/delta + T cells expressed T beta 2-, T beta 3-, and T beta 4-binding activities. In contrast, non-T-cell lines, in which the enhancer was inactive, each lacked expression of at least one of these binding activities. TCR alpha and beta gene expression may be regulated by a common set of T-cell nuclear proteins in that the T beta 2 element binding a set of cyclic AMP response element-binding proteins that are also bound by the T alpha 1 element of the human TCR alpha enhancer and the decamer element present in a large number of human and murine TCR beta promoters. Similarly, the T beta 5 TCR beta-enhancer element and the T alpha 2 TCR alpha-enhancer element bind at least one common T-cell nuclear protein. Taken together, these results suggest that TCR beta gene expression is regulated by the interaction of multiple T cell nuclear proteins with a transcriptional enhancer element located 3' of the C beta 2 gene and that some of these proteins may be involved in the coordinate regulation of TCR alpha and beta gene expression.

Identification and Functional Characterization of the Human T-Cell Receptor β Gene Transcriptional Enhancer: Common Nuclear Proteins Interact with the Transcriptional Regulatory Elements of the T-Cell Receptor α and β Genes

A transcriptional enhancer has been mapped to a region 5.5 kilobases 3' of the C beta 2 gene in the human T-cell receptor (TCR) beta-chain locus. Transient transfections allowed localization of enhancer activity to a 480-base-pair HincII-XbaI restriction enzyme fragment. The TCR beta enhancer was active on both the minimal simian virus 40 promoter and a TCR beta variable gene promoter in both TCR alpha/beta + and TCR gamma/delta + T cells. It displayed significantly less activity in Epstein-Barr virus-transformed B cells and K562 chronic myelogenous leukemia cells and no activity in HeLa fibroblasts. DNA sequence analysis revealed that the enhancer contains a consensus immunoglobulin kappa E2 motif, as well as an AP-1-binding site and a cyclic AMP response element. DNase I footprint analyses using Jurkat T-cell nuclear extracts allowed the identification of five nuclear protein-binding sites, T beta 1 to T beta 5, within the enhancer element. Deletion and in vitro mutagenesis studies demonstrated that the T beta 2- and T beta 3- and T beta 4-binding sites are each required for full transcriptional enhancer activity. In contrast, deletion of the T beta 1- and T beta 5-binding sites had essentially no effect on enhancer function. Electrophoretic mobility shift assays demonstrated that TCR alpha/beta + and TCR gamma/delta + T cells expressed T beta 2-, T beta 3-, and T beta 4-binding activities. In contrast, non-T-cell lines, in which the enhancer was inactive, each lacked expression of at least one of these binding activities. TCR alpha and beta gene expression may be regulated by a common set of T-cell nuclear proteins in that the T beta 2 element binding a set of cyclic AMP response element-binding proteins that are also bound by the T alpha 1 element of the human TCR alpha enhancer and the decamer element present in a large number of human and murine TCR beta promoters. Similarly, the T beta 5 TCR beta-enhancer element and the T alpha 2 TCR alpha-enhancer element bind at least one common T-cell nuclear protein. Taken together, these results suggest that TCR beta gene expression is regulated by the interaction of multiple T cell nuclear proteins with a transcriptional enhancer element located 3' of the C beta 2 gene and that some of these proteins may be involved in the coordinate regulation of TCR alpha and beta gene expression.

Haplotyping the human T-cell receptor beta-chain gene complex by use of restriction fragment length polymorphisms.

We have studied the genetic segregation of human T-cell receptor beta-chain (TCR beta) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). We constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCR beta gene complex. Analysis of allele distributions between TCR beta genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequillibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCR beta gene complex. Our results should provide new insight into recent reports of disease associations with the TCR beta gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome.