T-cell receptors in immune responses.
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Biomedical subjects
M A Robinson
Publications and source records attributed to M A Robinson.
TCRBV20S1 allele frequencies vary among human populations.
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TCRBV12 genes are polymorphic but the protein products encoded by each gene are conserved.
The human TCRBV gene complex contains three BV12 genes and all are polymorphic. There are two alleles of BV12S4; one allele contains a stop codon in the coding region which precludes its expression. There are four alleles of BV12S2 and two alleles of BV12S3 genes; however, the substitutions present in both genes are conservative. Although allelic variants exist for each of the three BV12 genes, only one protein product is encoded by each gene.
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The influence of non-HLA genes on the human T-cell receptor repertoire.
We previously demonstrated a central role for HLA genes in determining the T-cell receptor (TCR) repertoire. However, these studies also suggested that other genetic factors might also play a role in the development of this repertoire. In order to assess the role of non-HLA genes in the development of the TCR repertoire, we have analysed and compared the TCR repertoires of individuals in three families consisting of both monozygotic twins as well as an HLA-identical sib. TCR repertoire analysis was performed with both V-segment-specific MoAb and the polymerase chain reaction using TCRBV-segment-specific oligonucleotide primers. We observed that in every case the TCR repertoires of identical twins were more similar to each other than to their HLA-identical sib. Furthermore, in one family we were able to show by genotype analysis that most of the differences in repertoire between the identical twins and their HLA-identical sib were caused by polymorphisms in the TCR genes that influence expression levels. These studies document an important role for non-HLA genes in determining the TCR repertoire in man and raise the possibility that such TCR polymorphisms may play a significant role in determining disease susceptibility.
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Characterization of an infectious molecular clone of human T-cell leukemia virus type I.
An infectious molecular clone of human T-cell leukemia virus type I (HTLV-I) was derived from an HTLV-I-transformed rabbit T-cell line, RH/K30, obtained by coculture of rabbit peripheral blood mononuclear cells (PBMC) with the human HTLV-I-transformed cell line MT-2. The RH/K30 cell line contained two integrated proviruses, an intact HTLV-I genome and an apparently defective provirus with an in-frame stop codon in the env gene. A genomic DNA fragment containing the intact HTLV-I provirus was cloned into bacteriophage lambda (K30 phi) and subcloned into a plasmid vector (K30p). HTLV-I p24gag protein was detected in culture supernatants of human and rabbit T-cell and fibroblast lines transfected with these clones, at levels comparable to those of the parental cell line RH/K30. Persistent expression of virus was observed in one of these lines, RL-5/K30p, for more than 24 months. Biologic characterization of this cell line revealed the presence of integrated HTLV-I provirus, spliced and unspliced mRNA transcripts, and typical extracellular type C retrovirus particles. As expected, these virus particles contained HTLV-I RNA and reverse transcriptase activity. The transfected cells also expressed surface major histocompatibility complex class II, whereas no expression of this molecule was detected in the parental RL-5 cell line. Virus was passaged by cocultivation of irradiated RL-5/K30p cells with either rabbit PBMC or human cord blood mononuclear cells, demonstrating in vitro infectivity. The virus produced in these cells was also infectious in vivo, since rabbits injected with RL-5/K30p cells became productively infected, as evidenced by seroconversion, amplification of HTLV-I-specific sequences by PCR from PBMC DNA, and virus isolation from PBMC. Availability of infectious molecular clones will facilitate functional studies of HTLV-I genes and gene products.
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A genetically determined insertion/deletion related polymorphism in human T cell receptor beta chain (TCRB) includes functional variable gene segments.
Polymorphism in the human T cell receptor beta chain (TCRB) gene complex includes haplotypes with different numbers of TCRBV genes. An insertion/deletion related polymorphism (IDRP) in the human TCRBV region was found to involve TCRBV gene segments. Inserted TCRB haplotypes contain an additional 21.5 kb in which three TCRBV genes are encoded, members of the TCRBV7, TCRBV9, and TCRBV13 families. Two TCRBV gene segments were present only in inserted haplotypes; one of these, TCRBV7S3, is a functional gene and the other, TCRBV9S2(P), is a pseudogene because of an inframe termination colon. In addition, inserted haplotypes contain two identical copies of the TCRBV13S2 gene, whereas deleted haplotypes have only one copy. Deleted haplotypes could be subdivided into two types, deleted*1 and deleted*2, on the basis of sequence variations in TCRBV6S7 and TCRBV13S2 genes. Both deleted*1 and deleted*2 haplotypes contained the same number of TCRBV genes; both contain 60 genes of which 50 are functional, whereas, inserted haplotypes contained 63 genes of which 52 are functional. Comparisons of inserted region sequences with the homologous region in a deleted haplotype, and with sequences surrounding related TCRBV genes, revealed patterns of similarity that suggest insertion as well as deletion events have occurred in the evolution of the TCRBV gene complex. These data indicate that the genomic TCR repertoire is expanded in individuals who have inserted TCRBV haplotypes. The presence of additional TCRBV genes or, alternatively, the absence of certain TCRBV genes may have an impact upon immune responses and susceptibility to autoimmune diseases.
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Level of human TCRBV3S1 (V beta 3) expression correlates with allelic polymorphism in the spacer region of the recombination signal sequence.
One of the causes of variations in the expressed human T cell receptor (TCR) BV (V beta) repertoire is genetic variation in the germline DNA. Herein evidence is provided that allelic polymorphism may affect recombination frequency for a specific V gene. Two alleles of the TCR BV3 differ only at a single nucleotide position (C/T) within the 23-bp spacer region of the recombination signal sequence. These alleles are associated with variable percentages of BV3 cells in the peripheral blood, as shown in families and in unrelated normal donors. Individuals homozygous for allele 2 have a mean of 8.1% BV3 cells, heterozygous individuals have a mean of 4.7% BV3 cells, and homozygotes for allele 1 have a mean of 1.2% BV3 cells in CD3+ CD4+ peripheral blood T cells. Since the correlation is tight in unrelated individuals and other genetic differences were not found in the vicinity of BV3, we suggest that the spacer region sequence itself modifies recombination efficiency. This allelic system provides an example of a novel mechanism by which cis-acting genetic elements may affect recombination in a natural in vivo system.
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Molecular weight dependent tissue accumulation of dextrans: in vivo studies in rats.
The effects of molecular weight (M(r)) on the serum and urine pharmacokinetics and tissue distribution of dextrans, potential macromolecular carriers for drug delivery, were studied in rats. A single 5 mg/kg dose of fluorescein-labeled dextrans (FDs) with a M(r) of 4000 (FD-4), 20,000 (FD-20), 70,000 (FD-70), or 150,000 (FD-150) was administered into the tail vein of separate groups of rats. At different times after the administration of each FD, animals were sacrificed, and blood, urine, and various tissues were obtained. The concentrations of FDs in the samples were subsequently determined by using a sensitive and specific high performance size exclusions chromatographic method. Among the tissues studied, high accumulation of dextrans was found only in the liver (liver:serum AUC ratios < or = 29) and spleen (spleen:serum AUC ratios < or = 10), with high concentrations in these tissues persisting even at the last sampling time (96 h). In contrast, the serum concentrations of the studied FDs were not measurable beyond 12 h. The serum and urine kinetics and the liver, spleen, and kidney accumulation of FDs demonstrated a significant degree of M(r) dependency. The total and renal clearance of FDs consistently decreased with an increase in M(r). However, the effects of M(r) on the tissue accumulation of dextrans was tissue dependent. For the liver, the tissue:serum AUC ratios increased from 0.346 for FD-4 to 15.2 for FD-20 and 28.8 for FD-70, while a further increase in M(r) to 150 kDa (FD-150) resulted in lowering the ratio to 8.59 in this tissue. For the spleen, the ratios increased from 0.095 for FD-4 to 9.56 for FD-150.(ABSTRACT TRUNCATED AT 250 WORDS)
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The extent of the human germline T-cell receptor V beta gene segment repertoire.
An assessment of the size of the human TCRBV gene segment repertoire based on the identification of TCRBV gene segments in genomic DNA was undertaken. PCR amplification from cloned and uncloned genomic DNA sources, nucleotide sequencing, Southern blot hybridization, and cosmid cloning were used to identify TCRBV gene segments in multiple unrelated individuals. The key advantages to this approach were: 1) TCRBV gene segments which are expressed only at very low levels in cDNA libraries were still detectable, and 2) it was possible to discriminate between alleles at the same locus vs products of different loci. A total of 63 unique TCRBV gene segments were identified and sequenced. Six of these TCRBV gene segments had not been previously described. Thirty-four cosmid clones containing 51 of the 63 identified TCRBV gene segments were isolated and screened for the presence of additional novel TCRBV subfamily members. These results, obtained by a variety of complementary approaches, indicate that the human TCRBV germline repertoire encodes at least 63 TCRBV gene segments of which 52 are functional. The availability of the majority of these TCRBV gene segments on cosmid clones should facilitate further investigation of germline TCRBV gene segment polymorphism and putative disease associations.
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Frequent recombination in the human T-cell receptor beta gene complex.
Although individual TCRVBV gene segments exhibit limited polymorphism, human T-cell receptor beta (TCRB) haplotypes are characterized by multiple different combinations of allelic markers. This observation suggests that genetic recombination may have played a role in the generation of these haplotypes. Meiotic recombination in a region spanning approximately 250 kilobases (kb) at the 3' end of the TCRB gene complex was investigated by extended family studies and by analysis of single sperm. Segregation patterns of polymorphic TCRB markers in families allowed the assignment of TCRB alleles to parental haplotypes and detection of recombinants among the offspring. Among the 178 informative paternal meioses, four (approximately 2%) were recombinant, whereas no recombinants were found in the 199 maternal meioses. In addition, segregation of two allelic markers was examined in a total of 1101 individual sperm from two heterozygous donors to detect exchange events in this region. The results revealed a similar rate of recombination, approximately 1.3%, which, along with the family data, suggests that at, least in males, meiotic recombination in this 250 kb region may be six times higher than the "average" rate of 1% per 10(6) bases that has been estimated for the human genome.
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The human T-cell receptor variable gene segment TCRBV6S1 has two null alleles.
The extent of polymorphism in TCRBV6S1 was examined by screening 203 individuals of diverse ethnic backgrounds by using SSCP. Three alleles were detected, including two that were described previously (TCRBV6S1*1 and *2P). The third allele (TCRBV6S1*3P), identified in these studies, is a pseudogene because, similar to allele *2P, it contains a substitution of a highly conserved cysteine residue near CDR3. Among a panel of 126 Caucasian donors, alleles *1, *2P, and *3P were observed to have frequencies of 0.72, 0.12, and 0.16, respectively. The extent of this survey suggests that it is unlikely for there to be additional common variants of TCRBV6S1. The approach used here enables rapid typing for polymorphism in a TCRBV gene that results in an allelically determined hole in the TCR repertoire.
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Enantioselective kinetics of verapamil and norverapamil in isolated perfused rat livers.
The kinetics of the individual enantiomers of verapamil (VER) and its metabolite, norverapamil (NOR), were studied in isolated perfused rat livers (IPRLs) after administration of racemic drug or the preformed metabolite. After constant infusion of 20 micrograms/min of racemic VER to single-pass IPRLs, the hepatic availabilities (F) of the enantiomers were low (S-VER, 0.069 +/- 0.030; R-VER: 0.046 +/- 0.025) and stereoselective (S:R ratio, 1.6 +/- 0.2). After administration of similar doses, the F values of the preformed NOR enantiomers (S-NOR: 0.24 +/- 0.04; R-NOR, 0.10 +/- 0.02) were higher than those of the VER enantiomers. However, the stereoselectivity in F of NOR (S:R ratio, 2.2 +/- 0.1), was in the same direction of that of VER. Further, the fractions of R enantiomers unbound to bovine serum albumin in the perfusate were higher than those of their antipodes for both VER (R:S ratio, 1.9 +/- 0.1) and NOR (R:S ratio, 2.6 +/- 0.2). Therefore, for unbound moieties, modest stereoselectivity in the metabolism of VER in favor of the S-isomer and no stereoselectivity in the metabolism of NOR were observed. Overall, our data suggest that the stereoselective protein binding is a primary determinant of stereoselectivity in the hepatic availability of VER and NOR in IPRLs.
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Patient satisfaction with an antepartum home-care program.
Evaluation of patient satisfaction with alternative health-care delivery models is an important component of program evaluation. Patient satisfaction occurs when there is congruence between expected care and the care received. This descriptive study investigated patient satisfaction with the Antepartum Home Care Program in Winnipeg, Canada. A convenience sample of 66 pregnant women completed the Antepartum Home Care Program Evaluation Questionnaire. Most respondents were very satisfied with the physical and emotional care they received at home, including the information they received. Ninety-eight percent of the women indicated that the care they received from the program met their expectations. Being able to receive care at home, rather than in the hospital, was the most frequently identified benefit of the Antepartum Home Care Program. These results will assist program planners with decisions concerning program improvements and growth.
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Community-based home-care program for the management of pre-eclampsia: an alternative.
OBJECTIVE: To evaluate the safety, acceptability and cost of a community-based home-care program for the management of mild pre-eclampsia. DESIGN: A descriptive study of outcomes between Apr. 1, 1985, and Dec. 31, 1989. SETTING: St. Boniface General Hospital, Winnipeg. PATIENTS: Urban Winnipeg residents between 27 and 40 weeks' gestation with mild pre-eclampsia who demonstrated acceptance and compliance with home-care management; 321 patients of 1330 were enrolled in the program. INTERVENTIONS: Bed rest at home with daily biochemical and biophysical follow-up protocol and weekly clinic visits; patient education; hospital admission for labour, induction, worsening pre-eclampsia or noncompliance with rest at home. OUTCOME MEASURES: Patterns of referral to the program; clinical, biochemical and biophysical profiles; incidence of severe complications; reduction in total hospital stay and cost analysis. RESULTS: As many women were referred from physicians' offices as were referred from the hospital's antepartum unit, the average gestational age at referral being 36 weeks. Most (205 [64%]) of the women were nulliparous. The average length of stay in the program was 11.5 days. The program's availability resulted in a reduction of 2 days (from 5.7 days to 3.7 days) on average in the length of hospital stay when analysed for all 1330 women with pre-eclampsia. Of the 321 patients in the program 137 (43%) were admitted to hospital for worsening pre-eclampsia; severe pre-eclampsia developed 4 days after admission in 9. No patient suffered eclampsia, disseminated intravascular coagulopathy, abruption or fetal loss related to pre-eclampsia while in the program. The estimated cost saving in the management of pre-eclampsia was over $700,000 over the study period. CONCLUSION: The community-based home-care program is a safe, feasible and less costly alternative to hospital admission in the management of mild pre-eclampsia.
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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.
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Variation in HTLV-I sequences from rabbit cell lines with diverse in vivo effects.
Comparison of nucleotide sequences determined for HTLV-I integrated provirus from two rabbit cell lines, RH/K30 and RH/K34, revealed greater than 99% identity to one another. Substitutions encoding amino acid interchanges were observed in the gag, pol, and rex regions whereas the env and tax proteins were identical in the two lines. Comparison with the human prototypic HTLV-I sequence revealed considerably more variation, especially in the viral envelope region where the rabbit sequences are identical. The HTLV-I lines differed in their potential to cause disease in rabbits: injection of the RH/K34 cell line caused human adult T-cell leukemia/lymphoma-like (ATLL) disease which was fatal within 10 days, whereas all rabbits injected with the same or higher doses of RH/K30 survived with a low-grade leukemia that showed evidence of acute rejection. Correlation of lethality with viral sequence was tested by injection of rabbits with two other rabbit cell lines with HTLV-I provirus identical to RH/K34 in LTR, gag, and env regions. The fact that only one of these lines produced fatal disease suggests that pathogenic determinants lie outside of these regions or, alternatively, that the structure of the integrated virus is not the sole factor in the cell lines' ability to cause ATLL-like disease.
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[Allelic polymorphism in the coding region of human T cell receptor V beta 12 gene].
Sequence substitutions in T cell antigen receptor (TCR) beta chains have been shown to have a profound impact upon the fine specificity of T lymphocytes. It is, therefore, of interest to characterize allelic variations in the TCR gene segments that assemble to encode TCR molecules. In the present report, the sensitive technique of single stranded conformational polymorphism (SSCP) was used to screen for allelic sequence variations in the coding region of TCR V beta 12 gene. TCR V beta 12 specific primers were used to PCR amplify and 32P label a 273bp segment of the variable (V) gene segment. Sequence differences between different allelic forms were detected by denaturation of the DNA and separation on a non-denaturing, high resolution acrylamide gel. Using this rapid approach, four alleles of the TCR V beta 12 gene were detected in a population of 90 ethnically diverse individuals. Correct Mendelian segregation of the four alleles was demonstrated in family studies (n = 23) and allele frequencies were 0.4722, 0.3166, 0.2056, and 0.0056 respectively. Sequence analysis revealed sequence differences of 1-3 nucleotides involving three separate codons. However, predicted amino acid sequences of all four alleles are identical. These data strongly suggest that the TCR V beta 12 protein sequence is highly conserved.
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Usage of human T-cell receptor V beta, J beta, C beta, and V alpha gene segments is not proportional to gene number.
Certain T-cell receptor (TCR) beta-chain variable (V), joining (J), and constant (C) gene segments, as well as TCR alpha-chain V gene segments, are disproportionally represented in TCR alpha and beta cDNA libraries derived from PHA-stimulated peripheral blood lymphocytes. Sequences of 138 TCR alpha clones and 96 TCR beta clones were determined and of these 128 TCR alpha clones and 88 TCR beta clones were found to contain unique combinations of V, J, and C gene segments or to display diversity in N region nucleotides. The frequency of the V, J, and C genes used in the assembly of unique transcripts was ascertained. Of the 24 reported V beta gene families, 21 were observed among the 88 TCR beta clones including four V beta families (V beta 1, V beta 2, V beta 3, and V beta 4) that were represented in the sample 2 1/2-5 times more frequently than would be expected on the basis of copy number within the gene complex. Seventy-eight percent of the clones were positive for C beta 2 and more than half of the clones (53%) used one of two J beta 2 genes: J beta 2.1 was present in 27 clones and J beta 2.7 in 20 clones. TCR V alpha families were also disproportionately represented in this sample. Twenty-five of 30 V alpha families were observed in the sample of 128 clones including six recently reported V alpha families. Three V alpha families, V alpha 2, V alpha 8, and V alpha 23, accounted for approximately 40% of the TCR alpha clones and were represented at 18%, 9.4%, and 13.3%, respectively. Both V alpha 2 and V alpha 8 gene families contain more than one gene; thus the number of clones observed in these families may, in part, be related to gene number. However, V alpha 23, which appears to be a single-copy gene family, is significantly overrepresented in this sample. Although disproportional usage of V beta genes may be accounted for by superantigen exposure, reasons for disproportional usage of J beta, C beta, and V alpha genes are presently unknown.
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