Further characterization of T cell receptor chains of marsupials

cDNA clones encoding T cell receptor alpha (TCRalpha) and beta (TCRbeta) from the South American opossum, Monodelphis domestica were isolated and characterized. A single clone isolated encoding a TCRalpha chain was full length, containing the complete V (variable), J (joining) and C (constant) regions. Three partial cDNA clones were isolated for TCRbeta which contained complete C sequences. Phylogenetic analysis of the TCR Valpha revealed that the M. domestica sequence and a sequence from the Australian brushtail possum, Trichosurus vulpecula, belong to separate Valpha families and intersperse with sequences from eutherian mammals. Similar to results described for marsupial and eutherian light chains, diversity at the V region of the TCR is ancient and maintained. In contrast phylogenetic analysis of the TCR Calpha and Cbeta sequences from M. domestica, T. vulpecula, and other vertebrates revealed that the marsupial TCR C grouped together forming a sister group to eutherian mammals.     

Mutational analysis of conserved amino acids in the T cell receptor alpha-chain transmembrane region: a critical role of leucine 112 and phenylalanine 127 for assembly and surface expression

Correct assembly of all TCR complex polypeptides is essential for its cell surface expression and function. The transmembrane region of the TCRalpha chain is highly conserved and to gain insight into the structural and functional role of these residues, single amino acid substitutions were introduced and surface expression and signaling ability studied in T hybridoma cells. Introduction of acid residues within the TCRalpha chain transmembrane region were mostly tolerated, indicating that the net charge within this region of the TCR complex is not crucial to either assembly or signaling. However, mutations of leucine 112 or phenylalanine 127 to aspartic acids (L112D or F127D, respectively) resulted in dramatic loss of surface expression and, therefore, their signaling ability. Intracellular flow cytometry showed that the mutant TCRalpha polypeptides were present at levels comparable to wild-type, indicating that the reduced surface expression was not a consequence of impaired protein survival. The defect was characterized by immunoprecipitation and showed that residues L112 and F127 were involved in early interactions with the CD3 complex. A large proportion of the TCRalpha chain mutants L112D and F127D consisted of immature protein, indicative of a problem during early assembly of the TCR. Our findings provide evidence for the involvement of the conserved L112 and F127 residues of the TCRalpha chain transmembrane region in the assembly process of the TCR complex.     

Evolution of T cell receptor (TCR) alpha beta heterodimer assembly with the CD3 complex.

T cell antigen receptors (TCR) are composed of an antigen-recognizing unit, the TCRalpha beta heterodimer, and a signal transduction ensemble, the CD3 complex. Whereas mammals possess three CD3 dimers (delta epsilon, gamma epsilon, and zeta2), birds and amphibians have only two (delta/gamma-epsilon and zeta2). To understand evolutionary changes in TCR/CD3 assembly,a phylogenetic approach was employed to dissect the interaction of TCRalpha beta heterodimers with the CD3 components. While sheep and mouse TCRalpha and TCRbeta chains could replace the corresponding human chains in mutant human T cells to restore surface TCR/CD3 expression and function, chicken TCRalpha, TCRbeta and CD3delta/gamma chains were unable to replace the corresponding human chains in forming a chimeric TCR/CD3 complex. The inability of chicken TCR/CD3 components to replace the human molecules in T cells was found to result from the lack of interaction between chicken TCRalpha beta heterodimers and the human CD3 complex. In contrast, if no CD3 molecules are present (non-T cells), TCRalpha -TCRbeta chain pairing can take place in an apparently non-controlled way. Thus, the TCR-CD3 interactions have changed with the evolutionary divergence of two mammalian CD3gamma and CD3delta genes from a single prototypic chicken delta/gamma gene. Our data suggest that the structures in mammalian TCR.C regions, which distinguish between CD3delta and CD3gamma chains, have evolved with the appearance of two separate CD3delta and CD3gamma functions.     

T cell receptor DJ but not VDJ rearrangement within a recombination substrate introduced into a pre-B cell line

To elucidate mechanisms that regulate ordered and tissue-specific assembly of Ig and TCR variable region gene segments, we have introduced a recombination substrate comprised of germline TCR beta V, D, and J gene segments into an Abelson murine leukemia virus-transformed pre-B cell line that actively rearranges endogenous Ig H chain variable region gene segments but does not rearrange endogenous light chain or TCR variable region gene segments. We find that these cells efficiently join D beta segments to J beta segments within the mini-locus, but that they do not make any detectable site-specific rearrangements of the introduced V beta segment even though it is closely linked in the same construct to the D beta. These findings suggest that factors necessary for V beta to (D beta)J beta joining may be absent in these pre-B cells and also imply that the order in which TCR V beta, D beta, and J beta segments are rearranged can be influenced by factors other than the 12/23 recombination rule. Furthermore, in agreement with the an accessibility model of VDJ recombinase control, the D beta region of the construct was found to be relatively more sensitive to DNAase I digestion in isolated nuclei when compared to the unrearranged V beta region.     

Importance of the T cell receptor alpha-chain transmembrane distal region for assembly with cognate subunits

Antigen recognition by alphabeta T lymphocytes is mediated via the multisubunit TCR complex consisting of invariant CD3gamma,delta,epsilon and zeta chains associated with clonotypic TCRalpha and beta molecules. Charged amino acids located centrally within the TCRalpha transmembrane region are necessary and sufficient for assembly with the CD3deltaepsilon heterodimer. Previously, we have shown that deletion of 6-12 amino acids from the carboxy terminus of the TCRalpha-chain dramatically abrogates surface TCR expression, suggesting that the distal portion of the TCRalpha transmembrane region contains information that regulates the assembly and/or intracellular transport of TCR complexes. We have examined in more detail the molecular basis for reduced TCR expression in T cells bearing truncated TCRalpha chains. We found that in contrast to wild-type (wt), variant TCRalpha proteins missing the last nine C-terminal amino acids did not associate with core CD3gamma,delta,epsilon chains and were not assembled into disulphide-linked alphabeta heterodimers. The stability of newly synthesised wt and variant TCRalpha molecules was similar, showing that the abrogated surface TCR expression was not a consequence of impaired protein survival. Nevertheless, truncated TCRalpha chains still assembled with the chaperon protein calnexin in the endoplasmic reticulum, indicating that the distal portion of the TCRalpha transmembrane region is not essential for calnexin interaction. These data document a role for the distal portion of the TCRalpha transmembrane region in the assembly of TCR complexes and provide a molecular basis for reduced TCR expression in cells bearing truncated TCRalpha chains.     

Conserved nucleotide sequences at the 5' end of T cell receptor variable genes facilitate polymerase chain reaction amplification

Alignment of all available nucleotide sequences of mouse and rat alpha/beta T cell receptor (TcR) variable (V) regions revealed the presence of relatively conserved sequences at the 5' end of the V gene segments. Based on these conserved sequences, degenerate primers were developed for use in the polymerase chain reaction (PCR). The degenerate primers developed on the basis of the conserved sequences at the 5' end of rat and mouse V gene segments are expected to enable the amplification of all mouse and rat TcR alpha/beta chain V regions. To test their applicability, the primers were used for the amplification of the V region of the TcR alpha/beta expressed by rat T cell lines. After amplification, the TcR V regions expressed were cloned and sequenced. The Z1a T cell line was shown to use the same TcR V gene segments (V alpha 2 and V beta 8.2), as most other experimental allergic encephalomyelitis associated T cell lines, but had different D and J segments. In spite of these differences at the nucleotide level, a remarkable conservation of the amino acid sequence at the V beta D beta J beta junction was found. Alignment of a large number of human V alpha and V beta gene segments revealed the presence of similarly conserved sequences. Degenerate primers based on these conserved sequences enabled the amplification of TcR V regions of human T cell lines.     

T cell receptor CDR3 loop length repertoire is determined primarily by features of the V(D)J recombination reaction

The third complementarity-determining region (CDR) of the TCR alpha and beta chains forms loops that engage amino acid residues of peptides complexed with MHC. This interaction is central to the specific discrimination of antigenic-peptide-MHC complexes by the TCR. The TCRbeta chain CDR3 loop is encoded by the Dbeta gene segment and flanking portions of the Vbeta and Jbeta gene segments. The joining of these gene segments is imprecise, leading to significant variability in the TCRbeta chain CDR3 loop length and amino acid composition. In marked contrast to other pairing antigen-receptor chains, the TCR beta and alpha chain CDR3 loop size distributions are relatively narrow and closely matched. Thus, pairing of TCR alpha and beta chains with relatively similar CDR3 loop sizes may be important for generating a functional repertoire of alpha beta TCR. Here we show that the TCRbeta chain CDR3 loop size distribution is minimally impacted by TCRbeta chain or alpha beta TCR selection during thymocyte development. Rather, this distribution is determined primarily at the level of variable-region gene assembly, and is critically dependent on unique features of the V(D)J recombination reaction that ensure Dbeta gene segment utilization.     

TCR gamma chain diversity in the spleen of the duckbill platypus (Ornithorhynchus anatinus)

TCR gamma (TRG) chain diversity in splenic gammadelta T cells was determined for an egg-laying mammal (or monotreme), the duckbill platypus. Three distinct V subgroups were found in the expressed TRG chains and these three subgroups are members of a clade not found so far in eutherian mammals or birds. Each subgroup contains approximately five V gene segments, and their overall divergence is much less than is found in eutherians and birds, consistent with their recent evolution from an ancestral V gene segment. The platypus TRG locus also contains three C region genes and many of the residues involved in TCR function, such as interactions with CD3, were conserved in the monotreme C regions. All non-eutherian mammals (monotremes and marsupials) lacked the second cysteine residue necessary to form the intradomain disulfide bond in the C region, a loss apparently due to independent mutations in marsupials and monotremes. Monotreme TRGC regions also had among the most variation in the length of the connecting peptide region described for any species due to repeated motifs.     

Absence of clonal beta and gamma T-cell receptor gene rearrangements in a subset of peripheral T-cell lymphomas.

The authors describe a set of seven peripheral T-cell lymphomas that lack detectable rearrangements of T-cell receptor (TCR) genes. All cases showed antigenic profiles consistent with T-cell lymphoma, including expression of Leu-5 (CD2) antigen. However, few other T-lineage markers were found, and none of the cases tested (6 of 7) bound antibody recognizing the constant region of the beta TCR protein. Each case showed exclusively germline configurations of DNA for the beta TCR genes in Southern blot analyses with the use of several different combinations of restriction enzymes and DNA hybridization probes. One case contained clonal rearrangements of the gamma TCR gene and of the immunoglobulin heavy chain gene. Our results suggest that certain cases of peripheral T-cell lymphoma may lack rearrangements of TCR genes--particularly those cases expressing restricted numbers of T-lineage antigens. In view of these findings, failure to detect rearrangements of TCR genes by Southern blot analyses is not necessarily inconsistent with malignant lymphocytic proliferations in T-lineage neoplasia.     

Allelic polymorphism of TCRalpha chain constant domain genes in the bicolor damselfish

T cell receptor (TCR) chains are composed of two extracellular domains, the membrane-distal variable domain and the membrane-proximal constant domain. Data presented here show that the TCRA 'constant' (C) domain of damselfish exhibits considerable allelic polymorphisms that appear to be positively selected. Each of 32 damselfish TCRAC clones showed different patterns of atypical polymorphism in the constant region. Twenty-three of the 121 TCRalpha constant region amino acid residues show substitutions, clustered mainly in the loops between the beta strands. Coding regions of the TCRAC genes differ by up to 8% at the nucleotide level and 20% at the amino acid level. Southern hybridization, polymorphism segregation, and genomic cloning data suggest allelic polymorphism at two TCRAC genes, distinguished by a single amino acid. KA/KS ratios suggest that balancing selection is acting to maintain polymorphisms at the variable sites of one of these genes, but not the other, in a manner comparable to the peptide binding regions of MHC. Nonetheless, each TCRAC gene is spliced to variable and joining segments similar to those described in other species. These data suggest that our understanding of the function of the TCR constant domains of these vertebrates is incomplete.     

Detection of a T cell receptor delta chain with an anti-TCR alpha chain serum.

Two types of T cell antigen-specific receptors have been described. Most peripheral blood T lymphocytes express, at their surface, an antigen receptor consisting of alpha and beta subunits, while a small subset of thymocytes and a minority of mature T lymphocytes express a heterodimeric receptor termed gamma delta. Whereas the gene segments localization corresponding to the TCR gamma and beta chains are separate, genes encoding the joining and the constant regions of TCR delta chain are located between the TCR V alpha region and the J alpha-C alpha gene cluster. To determine whether V alpha gene segments are used by delta chains, immunoprecipitations from human TCR gamma delta expressing cell clones were performed with an anti-alpha serum. The results show that a rabbit antiserum raised against the purified REX TCR alpha subunit immunoprecipitates a TCR delta chain from the cell surface of only one human T cell clone termed SO1. However, since no SO1 RNA hybridization is observed with REX TCR V alpha probe and SO1 cloned cells do react with an anti-V delta 2 monoclonal antibody, we conclude that TCR delta and alpha chains expressed a limited structural homology and that REX TCR V alpha gene do not seem to be frequently used in a functional delta chain.     

A stable marker for specific T-cells: a TCR alpha/green fluorescent protein (GFP) fusionprotein reconstitutes a functionally active TCR complex

The detection of antigen specific clonal T-cell populations in vivo during T-cell selection and an immune responses is often hampered due to the lack of suitable clonotype specific monoclonal antibodies. In order to determine the potential usefulness of green fluorescent protein (GFP) to follow specific T-cells in vivo, we decided to express and analyze the function of a T-cell receptor (TCR) alpha chain-GFP fusionprotein. The TCRalpha and beta chain cDNAs of a Leishmania major-specific murine T helper 2 cell clone were cloned and inserted into the pHSE3' expression vector. Simultaneously, a TCRalpha expression vector was constructed containing a C-terminal in frame fusion with the open reading frame of the enhanced GFP (EGFP). TCRalpha/TCRbeta or TCRalpha-EGFP/TCRbeta constructs were expressed in T-cell hybridoma cells 58alpha(-)beta(-) which lack an endogenous TCR but still express CD3 components. The TCRalpha-EGFP fusionprotein was detected with the expected molecular weight by immunoprecipitation and Western Blot analysis. Surface staining of TCR components was detected in transfectants expressing the wild type TCR heterodimer and, with only a slight reduction in intensity, also in those expressing the TCR-EGFP complex. Hence, expression and transport to the outer cell membrane is possible despite the 27 kD C-terminal extension of the TCRalpha. Most importantly, the EGFP-tagged TCR was functional since the transfectants produced IL-2 in response to stimulation via their TCR. Thus, TCR-EGFP constructs represent attractive tools to study posttranslational regulation of TCR expression and ligand-induced TCR clustering as well as the fate of antigen specific T-cells during tolerance induction and immunity in transgenic mouse models.     

Complexity of the T cell receptor Cbeta isotypes in the Mexican axolotl: structure and diversity of the VDJCbeta3 and VDJCbeta4 chains

We have reported previously the presence of two T cell receptor beta-chain constant region (Cbeta) isotypes in the Mexican axolotl. Specific Dbeta and Jbeta segments were present at the Vbeta-Cbeta1 and Vbeta-Cbeta2 junctions and nine Vbeta families which associate with both isotypes were characterized. This report describes two new Cbeta isotypes, Cbeta3 and Cbeta4. About 70 % of the amino acids in Cbeta3 are identical to Cbeta1 and Cbeta2. A Dbeta3 and a single Jbeta3 were found at the Vbeta-Cbeta3 junctions. The Dbeta3 consensus core sequence (TACGTGGCTACGTGGG) differs to all the presently known Dbeta and the CDR3beta loops of the Vbeta-Cbeta3 junctions (mean: 11.1 amino acids) contain a majority of aromatic, small hydrophobic and basic residues. The CDR3beta loops of the other isotypes are shorter (mean: 8.5 amino acids), contain a majority of acidic residues and very few aromatic residues. The axolotl Cbeta4 sequence has about 46 % similarity to Cbeta1, Cbeta2 and Cbeta3. Dbeta4 is identical to Dbeta2 and six new Jbeta segments are used at the Vbeta-Cbeta4 junctions. Four new families of Vbeta segments (Vbeta10-Vbeta13) are preferentially associated to Cbeta4. A strong selective pressure must operate in most vertebrates to preserve the structural stability of the extracellular part of the Cbeta chain. The four axolotl Cbeta seem to have evolved more freely, perhaps to favor the early emergence of a large diversity of T cell receptors in an amphibian species which is not fully immunocompetent before the 5th month of development.     

Germline-encoded recognition of diverse glycolipids by natural killer T cells

Natural killer T cells expressing 'invariant' T cell receptor alpha-chains (TCRalpha chains) containing variable (V) and joining (J) region V(alpha)14-J(alpha)18 (V(alpha)14i) rearrangements recognize both endogenous and microbial glycolipids in the context of CD1d. How cells expressing an invariant TCRalpha chain and a restricted set of TCRbeta chains recognize structurally diverse antigens is not clear. Here we show that a V(alpha)14i TCR recognized many alpha-linked glycolipids by means of a 'hot-spot' of germline-encoded amino acids in complementarity-determining regions 3alpha, 1alpha and 2beta. This hot-spot did not shift during the recognition of structurally distinct antigens, suggesting that the V(alpha)14i TCR functions as a pattern-recognition receptor, conferring on natural killer T cells the ability to sense and respond in an innate way to pathogens displaying antigenic alpha-linked glycolipids.