Identification of dog T-cell receptor β chain genes

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers D16409, D16410 and D16412     

Sequence analysis of sheep T-cell receptor β chains

The nucleotide sequences reported in this paper have been submitted to the GenBank database and have been assigned the accession numbers M94181-M94183.     

Analysis of sheep T-cell receptor β-chain heterogeneity

 We analyzed nucleotide and deduced amino acid sequence heterogeneity of sheep T-cell receptor β-chain cDNAs isolated from an anchored-polymerase chain reaction library. Evaluation of 34 individual rearrangements has defined 18 new β-chain variable region sequences which have been clustered into 13 families. Presumptive allelic polymorphisms of four of these variable regions have been defined, as well as ten distinct β-chain joining region sequences. The present analysis indicates that sheep T-cell receptor β-chains are composed of characteristic leader, variable, joining, and constant region sequences, and that imprecise joining and N-region addition contribute significantly to diversity in the third hypervariable region. Thus, it appears that sheep, like all other mammals studied to date, employ somatic rearrangement of multiple germline genes to create β-chain heterogeneity. These findings have allowed us to estimate the diversity of the sheep T-cell receptor β-chain variable region repertoire, and they provide information that will permit the evaluation of the role that specific T-cell populations play in naturally occurring and experimental diseases of sheep. Received: 20 October 1997 / Revised: 20 April 1998     

Aberrations of chromosome 3. A marker of T-cell lymphomas?

Aberrations of chromosome occur in different malignancies, but they are more frequent in lymphoproliferative ones than in the others. In this study here four out of five T-zone lymphomas had abnormalities of chromosome 3. This lead to the question of whether aberrations of chromosome 3 might be a marker of T-cell lymphomas. The conclusion which can be drawn from the cases described in the literature, the own unpublished cases, and these four T-zone lymphomas is that abnormalities of chromosome 3 cannot be regarded as a discriminative marker of T-cell derived malignancies. It is suggested that the relationship between chromosome aberrations and type of disease is not necessarily a direct one. One possible model of the genesis of chromosome aberrations in malignant diseases is presented.     

Isolation of monotreme T-cell receptor α and β chains

Monotremes are an ancient mammalian lineage that last shared a common ancestor with the marsupial and eutherian (placental) mammals about 170 million years ago. Characterization of their immune genes is allowing us to gain insights into the evolutionary processes that lead to the mammalian immune response. Here we describe the characterization of the first cDNA clones encoding T-cell receptors from a monotreme. Two TCR -chain cDNAs (TCRA) from the short-beaked echidna, Tachyglossus aculeatus, containing complete variable, joining and constant regions were isolated. The echidna TCRA constant region shares approximately 37% amino acid identity with other mammalian TCRA constant region sequences. The two variable regions belong to the TCRAV group C, which also contains V genes from humans, mice, cattle and chickens. One echidna TCR -chain cDNA (TCRB) containing the entire constant region was isolated and sequenced. It shares about 63% identity with other mammalian TCRB constant region sequences. The echidna TCRBV belongs to TCRBV group A, which also contains V genes from various eutherian species. Southern blot analysis indicates that, like in other mammalian species, there is only one TCRA constant region copy in the echidna genome, but at least two TCRB constant regions.     

Regulation of naïve and memory T-cell homeostasis

Recent work has confirmed the existence of homeostatic mechanisms that regulate the overall size and composition of the mature T-cell pool. Homeostatic mechanisms not only control total T-cell numbers but appear to act differently on na?ve vs. memory cells. The roles of self-MHC/peptide ligands and certain cytokines in T-cell homeostasis are discussed.     

Fine specificity and T-cell receptor β-chain gene rearrangements of five H-2Db-specific cytotoxic T-cell clones

A panel of cytotoxic T lymphocyte clones that recognize H-2^b target cells has been established. Six different clones were distinguished according to the following criteria. First, the fine specificity of the clones was determined by testing proliferation and cytotoxicity on target cells of recombinant mice. Clone 221 recognized H-2K^b, and five other clones recognized H-2D^b. Clone 433 distinguished itself from the other five D^b-specific clones by cross-reacting with an antigen on H-2^k cells. Second, the presence of an idiotypic determinant as defined by the 3179 clone-specific monoclonal antibodies was investigated in cytotoxicity inhibition experiments. One of the D^b-specific clones, 653, was inhibited by these antibodies and was therefore clearly different from the other D^b-specific clones. The third criterion involved the rearrangement pattern of the DNA coding for the chain of the T-cell receptor. Southern blot analysis showed that each clone had a unique pattern. Interestingly, clone 653 , which expresses the same idiotypic determinant as clone 3F9, had deleted the C 1 gene cluster, whereas this gene is functionally expressed in clone 3179.Abbreviations used in this paper C constant gene segment - Con A concanavalin A - CTLs cytotoxic T lymphocytes - D diversity gene segment - ³H-dThd tritiated thymidine - J joining gene segment - kb kilobase pairs - LPS lipopolysaccharide - MHC major histocompatibility complex - MLC mixed lymphocyte culture - SDS sodium dodecyl sulfate - V ariable gene segment     

Alleles of the rat T-cell receptor β chain gene complex

Inbred rat strains provide a rich source of genetic diversity in immunologically relevant gense. We have characterized the alleles of one of these genes, encoding the rat T-cell receptor C1 chain, by Southern blots and nucleic acid sequencing. The Cb1 gene segments from DA and LEW rats display complex allotypic variation: both coding and noncoding regions contain multiple nucleotide substitutions. In addition, there is a polymorphic insertion of a rat repetitive LINE element 3 to the coding region. The Cb1 alleles are one part of larger Tcrb haplotypes, containing V, D, and J elements; complete Cb1 genomic nucleotide sequences, and a partial list of the strain distribution of the two alleles, are described in this report.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M65136.     

Subgroups of Tcr α chains and correlation with T-cell function

T-cell receptor (Tcr) chains are classified into four subgroups (I, II, III, and miscellaneous) based on the amino acid residues at positions 61 and 62. Subgroup I has Gly Phe at these positions, subgroup II has Arg Phe, subgroup III has Arg Leu, and subgroup miscellaneous has several other combinations. Variability plots for subgroups I, II, and III sequences show higher values around positions 93–103, 105, 108, 111, 113, and 115, suggesting that these positions may interact with the processed antigen molecules. Smaller peaks are present at various other regions which may bind the major histocompatibility complex class I or II molecules. The patterns of variability within one subgroup are similar for all species, for human alone, and for mouse alone. These subgroup patterns appear much less complicated than patterns for sequences in all subgroups taken together, implying that subgroups may be related to Tcr functions. Among 83 mouse chains, 15 are from cytotoxic cells and 40 from helper cells. Of the 15 from cytotoxic cells, 11, 2, 0, and 2 are in subgroups I, II, III, and miscellaneous; and of the 40 from helper cells, 9, 16, 12, ans 3 are in subgroups I, II, III, and miscellaneous, respectively. Thus, a correlation between sequence and function of Tcr chains seems possible. Address correspondence and offprint requests to: M. Schiffer.     

Sequence and diversity of bovine T-cell receptor β-chain genes

The nucleotide sequences of 38 T-cell receptor (Tcr) -chain cDNA clones which were isolated from a cDNA library (2 × 10⁶ plaques) constructed from bovine peripheral blood lymphocytes were determined. Of 38 cDNA clones, 22 were rearranged and contained the functional variable (V) gene segments. These clones were tentatively divided into nine Tcrb-V gene families which correspond to the human Tcrb-V family. Among them, a Tcrb-V12 gene segment was isolated from 9 out of 22 clones, suggesting that this Tcrb-V family was expressed in the bovine peripheral blood lymphocytes. Two different constant (C) geen segments were found, and both C regions were composed of 178 amino residues. The amino acid sequences of bovine Tcrb-C regions are approximately 80%–82%, 78%, and 78% similar to those from human, mouse, and rabbit, respectively. To estimate Tcrb-V-associated restriction fragment length polymorphisms (RFLPs), Southern blot analysis was performed using liver DNAs from four bovine breeds, Holstein, Angus, Hereford, and Japanese Black. However, no significant difference was observed among genomic DNAs of Tcrb-V loci from these four breeds.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers D90121-40. Address correspondence and offprint requests to: N. Ishiguro.     

Will telomere erosion lead to a loss of T-cell memory?

Evidence is accumulating that elderly individuals are more susceptible to infection with organisms to which they were previously immune. This indicates that there might be a limit to the persistence of immune memory. This fact is particularly disturbing because the average life expectancy of humans has almost doubled in the past 200 years and is still increasing. We discuss mechanisms that might constrain the persistence of memory T cells and consider whether humans will suffer from memory T-cell exhaustion as life expectancy increases.     

Sequence and diversity of rhesus monkey T-cell receptor β chain genes

We have sequenced 23 rearranged T-cell receptor chain (Tcrb) cDNA clones derived from peripheral blood lymphocytes (PBL) of a rhesus monkey. All of the clones have a variable-diversity-joining-constant (V-D-J-C) rearrangement similar to that of humans. Two rhesus constant (C) region genes were found, each closely reasembling human Cb 1 and 2. All of the rhesus J region sequences align well with ten of the 13 reported human J regions. 17 of the 23 rhesus V region sequences could be assigned to families homologous with eight different human families (Vb 1, 2, 6, 7, 8, 9, 13, and 14). The remaining six V region sequences are more distantly related to human Vb 1 and 13. Thus, the organization and sequences of studied rhesus Tcrb chains resemble human homologs. An evolutionary tree analysis revealed paralogous relationships between specific members of the rhesus and human V region families. Analysis of synonymous and nonsynonymous nucleotide sequence differences indicated that the evolution of the presumed major histocompatibility complex (MHC)-contact regions of the Tcrb chains is less constrained than that of the framework regions.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M60529-M60554.     

T-cell receptor V Tβ genes in natural populations of mice

The composition of 15 V _T gene subfamilies has been examined by Southern hybridization among a broad spectrum of colony bred rat and mouse species extending phylogenetically from Rattus to Mus musculus domesticus. Most mouse species contain a similar content of V _T genes as determined by the number of hybridizing restriction fragment (RF) bands. Furthermore, the extent of restriction fragment length polymorphism (RFLP) appears to be limited. Some V _T gene families, however, are missing from Rattus (VT7, V _T12) and M. shortridgei (V _T9, V _T16). Extension of the V _T survey to a panel of 38 wild-caught mice reveals that nearly a third lack specific hybridization to the V _T5 probe. Previous reports have established that the mouse inbred strains SJL, C57BR, C57L, and SWR lack 50% of their V _T repertoire, including V _T5 (Behlke et al. 1985). This study demonstrates that natural populations of mice also carry a significantly reduced V _T gene repertoire.     

Haplotypic origin of β-chain genes expressed by human T-cell clones

A contribution of allelic variation of T-cell receptor (Tcr) genes to the immune response has not been studied. Here we report that the presence of insertion-deletion-related polymorphisms (IDRP) of the Tcr chain (Tcrb) can be utilized to distinguish the parental origin of the gene complex that undergoes rearrangement in individual T-cell clones. Phytohemagglutinin stimulated clones from an individual heterozygous for an IDRP located between the variable(V) and diversity (D)-joining (J) region genes were studied for the presence of V to DJ rearrangements in each of the two parental chromosomes. Results indicate that single rearrangements were present in the majority of clones. in contrast to the double rearrangements of D to J genes that were generally present. In this individual, V to DJ rearrangements also occured with different frequencies on each of the two germline genes. IDRP clonotyping of the Tcrb complex should prove generally applicable to the study of the influence of allelic variation of Tcrb genes in selection of the expressed T-cell repetoire.     

Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-γ (IFN-γ) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected¹. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- γ secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-γ secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- γ ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)² in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 10⁵ cells per well typically used in ELISPOT assays^1,3, 1,000 T-cell clone cells in the presence of 1 x 10⁵ autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-γ. This assures successful performance of IFN-γ ELISPOT assay. Similarly, IFN- γ ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- γ ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes⁴.