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Results for “TCR specificity prediction”

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PubMed2005

Resolving T-cell receptor clonality in two and genotype in four multiplex polymerase chain reactions.

BACKGROUND AND OBJECTIVES: The diagnosis of T-cell neoplasia requires the use of immunohistochemistry on tumor sections or molecular genetic analysis of T-cell receptor (TCR) clonality. Multiplex polymerase chain reactions (PCR) offer a sensitive and expeditious approach to determining clonality early in the diagnostic work-up. We determined the sensitivity and specificity of four multiple PCR for genotyping lymphoid neoplasms at the TCR loci gamma (TCRG), delta (TCRD) and beta (TCRB, including complete [Vbeta-Jbeta] and incomplete [Dbeta-Jbeta] rearrangements). DESIGN AND METHODS: Template DNA was derived from frozen or formalin-fixed tissue and from imprints of aspirates or cut tissue surface on a FTA MicroCard. Each multiplex PCR was performed for 36 cycles in a single tube with multiple previously reported fluorescently labeled primers (TCRG and TCRD) or novel homologous primers (TCRB) and analyzed on electropherograms (Genescan), applying stringent criteria for interpreting clonal peaks. Two hundred and eleven clinically and immunohistochemically well-characterized benign and malignant non-T-cell lymphoid proliferations, including 138 B-cell lymphomas, were analyzed to determine specificity. The results were compared with those of 28 peripheral and immature T-cell neoplasms and two NK/T-cell lymphomas to determine sensitivity and compute predictive values. RESULTS: In all T-cell tumors, one or more TCR loci showed clonal rearrangement, which was not evident in two NK/T-cell lymphomas. TCRG was the single most informative locus (clonal rearrangement in 89%), followed by TCRB (79%) and TCRD (39%). Multiplex PCR targeting of TCRG and TCRD together resolved clonality in all T-cell neoplasms, whereas the TCRB locus was clonal in two of three cases with polyclonal TCRG. Unexpectedly, in B-cell lymphomas single clonal incomplete TCRB (Dbeta-Jbeta) peaks were 20 times more likely to occur than clonal TCRG. INTERPRETATION AND CONCLUSIONS: Clonality can be accurately determined in nodal T-cell lymphoma with two single-tube multiplex PCR targeting TCRG and TCRD. TCRB analysis should be considered in equivocal cases in which a polyclonal background may obscure clonal TCRD, but clonal incomplete TCRB rearrangement alone is insufficient for presuming T-cell lineage. In the absence of objective evidence of B-cell neoplasia, multiplex PCR of T-cell receptor genes may be used early in the diagnostic work-up, including for fine needle aspirates.

Adolescent↗

PubMed1995

Microvariation creates significant functional differences in the DR3 molecules.

Two DR3 molecules differ by four amino acids whose side chains point into the DR antigen-binding groove. To begin to assess the role of microvariation on DR3 function, DRB1*0302 residues were replaced with DRB1*0301 residues at beta-chain positions 26, 47, 86, and 47 plus 86. Murine fibroblast cell lines expressing DR(alpha, beta 1*0301), DR(alpha, beta 1*0302), and the four mutant 0302 molecules were examined for alloproliferative DR(alpha, beta 1*0302)-specific TLC stimulation and peptide binding. Changing position 26 had the most profound effect on T-cell recognition (seven of nine TLCs did not respond). Two TLCs did not respond to the mutant 0302V86 molecule and four TLCs that did respond to this mutant lost responsiveness when positions 47 and 86 were mutated together. These data suggest that each of these variant residues, including position 47, influence T-cell recognition. Surprisingly, none of the mutations had an effect on the absolute binding of HA 307-319 (DR[alpha, beta 1*0302] specific) and HSP 3-13 (DR[alpha, beta 1*0301] specific); however, the mutant 0302 molecules changed at position 86 (glycine to valine) consistently bound HA 307-319 at significantly higher levels than DR(alpha, beta 1*0302). These data for position 86 are in contrast to other DR molecules and indicate that peptide contact residues for a specific DR molecule cannot be predicted based on binding results obtained with other DR molecules. These data suggest that each of these variant groove residues, although not accessible to the TCR, contribute to the significant functional differences between the DR3 microvariants through subtle influences on the DR3-peptide complex.

Amino Acid Sequence↗

PubMed2002

Self epitopes shared between human skeletal myosin and Streptococcus pyogenes M5 protein are targets of immune responses in active juvenile dermatomyositis.

OBJECTIVE: To identify self T cell epitopes associated with proinflammatory immune responses and clinically active juvenile dermatomyositis (juvenile DM). The target of our search for relevant epitopes was represented by amino acid sequences shared between human skeletal myosin and Streptococcus pyogenes M5 protein. The long-term objective of the project is to identify suitable targets for immunotherapy of the disease. METHODS: We used computerized algorithms to identify putative agretopes on both the human myosin and Streptococcus M5 proteins. Direct binding assays for homolog peptides were used to confirm such predictions. Antigenicity and functional cross-reactivity were evaluated by cytotoxicity assays and by measurement of cytokine levels. Specific T cells were isolated by T cell capture, and T cell receptor (TCR) V(beta) gene usage was identified by reverse transcriptase-polymerase chain reaction. RESULTS: We identified peptides that are targets of disease-specific cytotoxic T cell responses. T cell reactivity against the self peptides correlates with clinical signs of early, active myositis. Such reactivity is accompanied by production of proinflammatory cytokines, which may contribute to the damage. T cell cross-recognition of bacterial and human homologs was shown functionally as well as by sorting peptide-specific T cells and identifying oligoclonal and largely overlapping TCR V(beta) gene usage. CONCLUSION: These findings represent the first identification of a self epitope in juvenile DM, providing a potential candidate for antigen-specific immune therapy.

Bacterial Proteins↗

PubMed1993

Direct evidence for the role of COOH terminus of mouse mammary tumor virus superantigen in determining T cell receptor V beta specificity.

It has recently been shown that open reading frames in the 3' long terminal repeats of mouse mammary tumor viruses encode superantigens. These viral superantigens (vSAGs) stimulate most T cells expressing appropriate V beta s almost regardless of the rest of the variable components of the T cell receptors (TCR) expressed by those cells. vSAGs produce a type II integral membrane protein with a nonessential short cytoplasmic domain and a large glycosylated extracellular COOH-terminal domain, which is predicted to interact with major histocompatibility complex class II molecules and the TCR. The transmembrane region of vSAG also has an internal positively charged lysine residue of unknown significance. A set of chimeric and mutant vSAG genes has been used in transfection experiments to show that only the extreme COOH-terminal portion of vSAGs determine their TCR V beta specificities, and to show that the lysine residue in the transmembrane domain is not essential for the function of vSAG.

Amino Acid Sequence↗

PubMed1994

Influence of human leukocyte antigen genes on TCR V gene segment frequencies.

Human leukocyte antigen (HLA)-dependent selection mechanisms exerted during thymic maturation are supposed to be main contributing factors to the genetic predetermination of the TCR repertoire and may have a detectable effect on adult peripheral blood lymphocyte V segment frequencies. Here, we analyzed whether polymorphic or non-polymorphic HLA determinants are associated with selected expression of some V gene segment specificities. We first examined the reactivity of 17 V segment specific mAb on purified CD4+ and CD8+ cell fractions in 10 unrelated people. We found a significant overexpression of only three V segment products (V beta 2, V beta 5.1 and V beta 6.7) in CD4+ and none in CD8+ cell fractions in most individuals. Skewing of certain V beta segments by non-polymorphic HLA determinants (i.e. class II for CD4+ and class I for CD8+ cells) is therefore more limited (3/17) than previously thought. Considering the effects of polymorphic HLA determinants, we compared TCR V segment frequencies in HLA-identical siblings to sibling pairs who differ at one or both HLA haplotypes, using 13 V beta specific mAb. In pairwise comparisons, we found that the HLA complex had no detectable effect on TCR repertoire in five large families with multiple siblings. Together, these observations suggest that HLA-predicted selection mechanisms exerted during thymic maturation might not have a predominant influence shaping the TCR repertoire of normal adults.

Antibodies, Monoclonal↗

PubMed1995

Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif.

In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4+8+ thymocytes upon TCR cross-linking. In contrast, expression of Tgtp is peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of approximately 50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation.

Amino Acid Sequence↗

PubMed1996

Specificity and binding properties of a single-chain T cell receptor.

The specificity of a T cell is dictated by an alpha beta T cell receptor (TCR) that recognizes a complex of peptide and a product of the major histocompatibility complex (MHC). Recent studies have begun to characterize the affinities and kinetics of these interactions, but details of the alpha beta TCR structure and function are not known. To examine some of these issues we focus in this report on a TCR derived from the T cell clone 2C. This TCR binds to a complex of the nonapeptide QL9 and the class I MHC product Ld with the highest affinity of any known TCR/ligand interaction (KD approximately 10 (-7) M). Circular dichroism showed that a single-chain TCR (scTCR) containing linked V alpha and V beta regions from T cell 2C and refolded from Escherichia coli inclusion bodies exhibited the characteristic beta-sheet structure of immunoglobulins. A sensitive assay that is capable of detecting the interaction of soluble scTCR with peptide /MHC ligand on the surface of target cells was used to demonstrate that the peptide specificity of this scTCR reflects that of the TCR found on the surface of 2C. Analysis of several scTCR V alpha region mutants confirmed that the V alpha domain is critical for the specificity of scTCR binding. Finally, we identified some notable differences in the complementarity determining regions (CDR) of the 2C TCR compared to the CDR of previously characterized, cytochrome- specific TCR. These differences are discussed in the light of what is known about antibody binding sites, the high affinity of the 2C TCR, and the nature of the residues on QL9 that are predicted to interact with the TCR.

Amino Acid Sequence↗

PubMed2006

A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition.

Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.

Animals↗

PubMed1996

Structural analysis of gamma delta TCR using a novel set of TCR gamma and delta chain-specific monoclonal antibodies generated against soluble gamma delta TCR. Evidence for a specific conformation adopted by the J delta 2 region and for a V delta 1 polymorphism.

We recently showed that secretion of non-chimeric disulfide-linked human gamma delta TCR ('soluble' TCR, sTCR) comprising V gamma 9 and V delta 2 regions could be achieved by simply introducing translational termination codons upstream from the sequences encoding TCR transmembrane region. Here we extended these findings by demonstrating efficient secretion and heterodimerization of gamma delta sTCR comprising V gamma 8, V delta 1 and V delta 3 regions, obtained via the same strategy. After immunization against immunoaffinity-purified soluble TCR, several hundreds of TCR-specific monoclonal antibodies (mAb) were generated, which fell in at least seven groups. One set of mAb was directed against a V gamma 8-specific epitope. Strikingly, despite the high degree of sequence homology between V gamma 8 and other V gamma I domains, none of these mAb were crossreactive with other members of the V gamma I family. Three other sets of mAbs were shown to recognize delta chains comprising V delta 1, V delta 2 and V delta 3 regions respectively, regardless of their junctional sequence or of the gamma chain to which they were paired. Among the V delta 1-specific mAb, some specifically recognized V delta 1D delta J delta C delta chains while others reacted with both V delta 1 D delta J delta C delta and V delta 1J alpha C alpha chains, which suggested V domain conformational alterations induced by the C region. Moreover, reactivity of one V delta 1-specific mAb (#R6.11) was affected by a polymorphic residue located on the predicted CDR4 loop of the V delta region. Two delta chain-specific mAb (#178 and #515) showed a highly unusual reactivity, which was negatively affected by particular V delta and J delta sequences: (i) mAb #515 and #178 recognized all TCR delta chains except those comprising V delta 1 or V delta 2 regions, respectively and (ii) within TCR delta chains carrying 'permissive' V delta regions, none of those comprising the J delta 2 region were recognized by #515 and/or #178 mAbs, which suggested a highly specific conformation adopted by this particular J delta sequence. Apart from its usefulness in TCR structural studies, this novel set of mAb represents an important tool for the characterization and isolation of gamma delta T cells expressing particular combinations of V gamma/V delta regions and for analysis of V alpha/V delta usage by alpha beta T cells. Moreover, since our present data strongly suggest that gamma delta TCR are easier to obtain in a soluble form than alpha beta TCR, an efficient strategy for the generation of V alpha region-specific mAb might be to immunize with chimeric gamma delta sTCR comprising particular V alpha regions.

Amino Acid Sequence↗

PubMed1990

Lysine 271 in the transmembrane domain of the T-cell antigen receptor beta chain is necessary for its assembly with the CD3 complex but not for alpha/beta dimerization.

The T-cell antigen receptor (TcR) complex present on most T-cells is formed by a clone-specific disulfide-linked alpha/beta heterodimer noncovalently associated to the CD3 complex, the latter composed of five invariant polypeptides: gamma, delta, epsilon, zeta/zeta, or zeta/eta. The presence of conserved, oppositely charged, amino acids in the predicted transmembrane domains of all the subunits of the TcR.CD3 complex suggests that these residues may have a critical function in the assembly and/or stabilization of the complex. In order to analyze the role of the transmembrane-charged amino acids in the association and cell surface expression of the TcR.CD3 complex, we have carried out site-directed mutagenesis of Lys271 in the transmembrane domain of the TcR beta chain and analyzed the capacity of the altered chain to assemble in a TcR beta-negative T-cell line. Here we show that substitution of this positively charged residue by alanine or glutamine does not prevent cytoplasmic association of alpha and beta chains to form disulfide-linked heterodimers, but does abolish formation of an alpha/beta.CD3 complex and, consequently, its expression on the cell surface.

Antigens, CD↗

PubMed2004

Cytokine production by virus-specific CD8(+) T cells varies with activation state and localization, but not with TCR avidity.

The ability of virus-specific CD8(+) T cells to produce cytokines was studied in mice infected with lymphocytic choriomeningitis virus and vesicular stomatitis virus. Intracellular staining was used to visualize cytokine-producing CD8(+) and CD4(+) T cells. Overall, virus-specific CD8(+) T cells produce a similar range of cytokines (IFN-gamma, TNF-alpha, IL-2, GM-CSF, RANTES, MIP-1alpha and MIP-1beta) as CD4(+) T cells, but the relative distribution of cytokine-producing subsets is different. Moreover, cytokine-producing CD8(+) T cells were found to dominate numerically at all time-points tested. Co-staining for more than one cytokine revealed that while all cytokine-producing CD8(+) T cells synthesized IFN-gamma, additional cytokines were produced by partly overlapping subsets of this population. The frequency of cells producing more than one cytokine was higher in a tertiary site (peritoneum) and generally increased with transition into the memory phase; however, GM-CSF producing cells were only present transiently. Concerning factors predicted to influence the distribution of cytokine-producing subsets, IFN-gamma and IL-12 did not play a role, nor was extensive virus replication essential. Notably, regarding the heterogeneity in cytokine production by individual cells with similar epitope specificity, variation in TCR avidity was not the cause, since in vivo-activated TCR transgene-expressing cells were as heterogeneous in cytokine expression as polyclonal cells specific for the same epitope.

Animals↗

PubMed1992

T cell receptor alpha-chain pairing determines the specificity of residue 262 within the Kb-restricted, ovalbumin257-264 determinant.

Ovalbumin-specific, Kb-restricted T cells recognize the minimal fully active synthetic peptide ovalbumin (OVA)257-264. This sequence coincides with the eight residue, allele-specific peptide binding motif previously predicted from direct sequencing of naturally occurring Kb-associated peptides (Falk, K., Rotzscke, O., Stevanovic, S., Jung, G., and Rammensee, H.-G., Nature 351:290, 1990). T cell recognition of a panel of analogs with single residue substitutions between the two putative Kb anchor residues at OVA261 and OVA264 suggested that at least one residue, Glu at position 262, is involved in TCR interaction. OVA-specific cytotoxic T lymphocytes (CTL) derived from TCR beta-chain transgenic mice, where the beta-chain originates from an OVA-specific, Kb-restricted CTL B3, showed that differences in TCR alpha-chain pairing determined the specificity for OVA residue 262. These data support the notion that residue 262 of the OVA T cell determinant, corresponding to position 6 within the Kb-binding motif, represents a contact site for TCR. This residue interacts directly with the TCR alpha-chain or with a site on the TCR beta-chain whose conformation is affected by TCR alpha-chain pairing.

Amino Acid Sequence↗

PubMed2002

Solid-phase epitope recovery: a high throughput method for antigen identification and epitope optimization.

Self tolerance to MHC class I-restricted nonmutated self Ags is a significant hurdle to effective cancer immunotherapy. Compelling evidence is emerging that altered peptide ligands can be far more immunogenic than their corresponding native epitopes; however, there is no way to reliably predict which modifications will lead to enhanced native epitope-specific immune responses. We reasoned that this limitation could be overcome by devising an empirical screen in which the nearly complete combinatorial spectrum of peptides of optimal length can be rapidly assayed for reactivity with a MHC class I-restricted cytotoxic T cell clone. This method, solid-phase epitope recovery, quantitatively ranks all reactive peptides in the library and allows selection of altered peptide ligands having desirable immunogenic properties of interest. In contrast to rationally designed MHC anchor-modified peptides, peptides identified by the present method are highly substituted in predicted TCR contact residues and can reliably activate and expand effector cell populations in vitro which lyse target cells presenting the wild-type epitope. We demonstrate that solid-phase epitope recovery peptides corresponding to a poorly immunogenic epitope of the melanoma Ag, gp100, can reliably induce wild-type peptide-specific CTL using normal donor T cells in vitro. Furthermore, these peptides can complement one another to induce these responses in an overwhelming majority of normal individuals in vitro. These data provide a rationale for the design of superior vaccines comprising a mixture of structurally diverse yet functionally convergent peptides.

Antigen Presentation↗

PubMed1991

Development of the CD4 and CD8 lineage of T cells: instruction versus selection.

T cells bearing the alpha beta T cell receptor (TCR) can be divided into CD4+8- and CD4-8+ subsets which develop in the thymus from CD4+8+ precursors. The commitment to the CD4 and CD8 lineage depends on the binding of the alpha beta TCR to thymic major histocompatibility complex (MHC) coded class II and class I molecules, respectively. In an instructive model of lineage commitment, the binding of the alpha beta TCR, for instance to class I MHC molecules, would generate a specific signal instructing the CD4+8+ precursors to switch off the expression of the CD4 gene. In a selective model, the initial commitment, i.e. switching off the expression of either the CD4 or the CD8 gene would be a stochastic event which is then followed by a selective step rescuing only CD4+ class II and CD8+ class I specific T cells while CD4+ class I and CD8+ class II specific cells would have a very short lifespan. The selective model predicts that a CD8 transgene which is expressed in all immature and mature T cells should rescue CD4+ class I MHC specific T cells from cell death. We have performed experiments in CD8 transgenic mice which fail to support a selective model and we present data which show that the binding of the alpha beta TCR to thymic class I MHC molecules results in up-regulation of the TCR in the CD4+8+ population. Therefore, these experiments are consistent with an instructive model of lineage commitment.

Animals↗

PubMed1995

Increased peptide promiscuity provides a rationale for the lack of N regions in the neonatal T cell repertoire.

Making use of mice deficient for terminal deoxynucleotidyl transferase (TdT) expression and a random peptide library, we have examined the diversity and peptide specificity of the neonatal T cell repertoire specific for a single H-2Db-restricted peptide. Consistent with the predicted decrease in repertoire diversity, polyclonal CTL lines and individual clones from different TdTo mice are more similar to each other than those from different wild-type mice in terms of their fingerprints of cross-reactivity to the library and their TCR sequences. We have also found that several TdTo CTL clones cross-react with many more library peptides than wild-type CTL clones. In a few instances, the degree of peptide promiscuity correlates with TCR sequence characteristics such as N region addition and homology-directed recombination, but not CDR3 loop length. Based on epitope titrations for each clone, TCR affinity for antigen is consistently high; thus, this reduced specificity for peptide may coincide with an accentuated affinity for the alpha helices of the MHC. Peptide promiscuity in the neonate may allow the relatively small numbers of T cells in the periphery to protect against a broader range of pathogens.

Amino Acid Sequence↗

PubMed2005

Diabetes is predicted by the beta cell level of autoantigen.

Two novel transgenic (Tg) strains were created expressing hen egg-white lysozyme (HEL) in a pancreas-specific fashion. RmHP.111 mice had levels of HEL per cell similar to that of the established ILK-3 strain, while RmHP.117 mice had 10-fold lower levels (50,000 molecules per cell). When bred to 3A9 TCR Tg mice, negative selection occurred equally in all three double-Tg combinations, yet only ILK-3 x 3A9 and RmHP.111 x 3A9 mice became diabetic. Additionally, activated 3A9 cells readily transferred diabetes into ILK-3 or RmHP.111 mice, but only marginally into the RmHP.117 strain. In the peripancreatic lymph node, division of naive 3A9 cells was similar between RmHP.111 and RmHP.117 strains, but pancreatic APCs from RmHP.111 x 3A9 mice stimulated HEL-reactive cells to a much greater degree than those from RmHP.117 x 3A9 mice. In this model, diabetes was dependent upon both initial priming in the peripancreatic lymph node and subsequent presentation in the pancreas, with disease incidence predicted by the beta cell level of autoantigen.

Animals↗

PubMed1996

Altered orientation of streptococcal superantigen (SSA) on HLA-DR1 allows unconventional regions to contribute to SSA Vbeta specificity.

Crystallographic studies reveal even distantly related bacterial superantigens (SAg) to adopt a common structural topology. Mutational analyses confirm that this shared folding pattern often confers a conserved function to analogous residues in different SAg, albeit with specificities for particular TCR or MHC class II molecules. It was thus surprising that the streptococcal SAg SSA differed from related SAg in the location of its Vbeta-determining residues. Because it seemed unlikely that SSA would deviate significantly from an SAg-like topology, we hypothesized that variations in SSA Vbeta-determining regions might result from differences in SSA-MHC class II interactions relative to other SAg during SSA presentation to the TCR. Comparison of the DR1-binding properties of SSA with its closest homologue SEB found different amino acid positions within SAg primary sequences to contribute to SSA-DR1 and SEB-DR1 interactions, and suggested that SSA bound DR1 with an altered orientation relative to SEB. The common involvement of DR1 alpha39K, however, predicted that the two SAg bound overlapping sites on DR1. Nevertheless, SSA and SEB did not effectively cross-compete for DR1 binding and had opposite patterns of DR1-binding affinity in the presence of distinct DR1-expressing cell lines. The data thus suggest that SSA and SEB bind not only with different orientations on DR1, but may bind preferentially to distinct DR1 subsets delineated by cell-specific factors. Differences in orientation of SSA on DR1 and/or interaction of SSA with particular DR1 subsets may explain why unconventional regions influence SSA TCR Vbeta specificity.

Animals↗

PubMed1994

Identification of T-cell receptor alpha-chain genes in the chicken.

T-cell receptor (TCR) alpha-chain (TCR alpha) and beta-chain (TCR beta) genes are well characterized in mammals, while only TCR beta genes have been identified in other vertebrates. To identify avian TCR alpha genes, we used monoclonal anti-CD3 antibodies to isolate chicken TCR alpha for peptide sequence analysis. Degenerate oligonucleotide probes were then used to isolate a candidate TCR alpha cDNA clone that hybridized with a 1.7-kb mRNA species present only in alpha beta T cells and in tissues populated by these cells. Southern blot analysis revealed gene rearrangement in thymocytes and alpha beta T-cell lines. The TCR alpha cDNA candidate encoded an open reading frame of 275 amino acids, the predicted variable (V)-, joining (J)-, and constant (C)-region amino acid sequences of which shared approximately 40%, 60%, and 25% homology with corresponding mammalian sequences. A single C alpha gene and approximately 25 V alpha genes were identified by using region-specific probes. The V alpha cDNA probe isolated from a V beta 1+ cell line reacted with transcripts from one of five V beta 2+ cell lines, suggesting shared use of V alpha genes by V beta 1+ and V beta 2+ T cells and the existence of other V alpha gene families. A genomic V alpha sequence was flanked by classical recombination signal sequences but, unlike previously defined V genes, the leader and V alpha region were encoded by a single exon. The data indicate evolutionary conservation of the basic TCR alpha gene structure in birds and mammals.

Amino Acid Sequence↗