The pre-B cell receptor and its function during B cell development

The process of B cell development in the bone marrow occurs by the stepwise rearrangements of the V, D, and J segments of the Ig H and L chain gene loci. During early B cell genesis, productive Ig H chain gene rearrangement leads to assembly of the pre-B cell receptor (pre-BCR), which acts as an important checkpoint at the pro-B/preB transitional stage. The pre-BCR, transiently expressed by developing precursor B cells, comprises the Ig muH chain, surrogate light (SL) chains VpreB and lambda5, as well as the signal-transducing heterodimer Ig alpha/Ig beta. Signaling through the pre-BCR regulates allelic exclusion at the Ig H locus, stimulates cell proliferation, and induces differentiation to small post-mitotic pre-B cells that further undergo the rearrangement of the Ig L chain genes. Recent advances in elucidating the key roles of pre-BCR in B cell development have provided a better understanding of normal B lymphopoiesis and its dysregulated state leading to B cell neoplasia.     

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.     

The variable,C(H)1, C(H)2 and C(H)3 domains of Ig heavy chain are dispensable for pre-BCR function in transgenic mice

The pre-BCR consists of Ig micro protein, the product of a heavy chain gene assembled by V(D)J recombination in pro-B cells, the surrogate light chains V(pre-B) and lambda 5, and the signaling chains Ig alpha and Ig beta. Signaling by the pre-BCR is a checkpoint required for further maturation of pro-B cells in the adult bone marrow. However, it is currently not known whether an extracellular ligand is required to initiate pre-BCR signaling. We reasoned that if the ectodomain of the pre-BCR is required to interact with a ligand, then a truncated heavy chain protein would not support B cell development. To test this notion, we produced transgenic mice expressing a heavy chain protein whose extracellular domains except for C(H)4 were replaced by an irrelevant Ig superfamily ectodomain from the human CD8 alpha protein. This transgene resulted in pre-BCR-like signaling since it rescued development of pre-B cells in recombinase-activating gene (RAG)1-deficient mice and resulted in allelic exclusion of the endogenous Ig heavy chain gene in RAG-proficient mice. These findings lead us to suggest that the majority of the extracellular region of the pre-BCR is not required for pre-BCR function and, thus, ligand binding is unlikely to be required for pre-BCR function.     

T-Cell Receptor Identification of an Oligodendrocyte-Specific Autoreactive Cytotoxic T-Cell Clone Without Self Restriction

In addition to myelin basic protein (MBP) and proteolipid protein (PLP), oligodendrocyte (Od) membrane autoantigens, such as the glycoprotein M2/MOG, could participate in the pathogenesis of autoimmune demyelinating diseases of the central nervous system (CNS), such as experimental allergic encephalomyelitis (EAE) or multiple sclerosis (MS). We have described an Od-specific autoreactive and cytotoxic T-cell clone, named C2, which recognized M2/MOG without conventional MHC restriction. In order to analyse the Od/C2 interaction, we determined the alpha/beta T-cell receptor (TCR) variable region usages and structures of C2. Monoclonal antibody stainings of C2 and nucleotide sequences show that the alpha chain is composed of a V alpha 5 and a J alpha identical to J alpha 18BBM142 gene segments, and that the TCR beta chain is composed of V beta 17a, D beta 2.1 and J beta 2.2 gene segments indicating that C2 used a conventional alpha/beta TCR for M2/MOG recognition.     

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.     

T cell receptor gamma chain variable gene rearrangements in acute lymphoblastic leukemias of T and B lineage

The status of immunoglobulin and T cell receptor genes in acute lymphoblastic leukemia (ALL) of T and B lineage has been studied. Our data indicate that illegitimate gene rearrangements at immunoglobulin heavy chain (in T cell ALL), and T cell receptor beta chain (in pre-B ALL) genes are only rarely found (2 out of 30 patients). In contrast, T cell receptor gamma chain gene rearrangements, characteristically found in T-ALL, are also present in 7 of 18 patients with pre-B ALL. Several features distinguish these illegitimate T cell receptor gamma chain gene rearrangements from those in normal and leukemic T cells. V gamma genes located far upstream of the J gamma/C gamma complexes (V gamma 2, V gamma 3, V gamma 4, V gamma 5) appear to be preferentially used in normal adult peripheral blood T cells. In contrast, V gamma genes located immediately 5' to the J gamma/C gamma complexes (V gamma 8, V gamma 9, V gamma 10, V gamma 11) predominate in V gamma -J gamma recombinations observed in T-ALL and pre-B ALL. Whereas the J gamma 2 region is primarily used in T cell receptor gamma gene rearrangements observed in T-ALL, those in pre-B ALL are confined mostly to the J gamma 1 region. These data suggest a limited accessibility of the T cell receptor gamma chain gene locus for recombination processes in early stages of B cell differentiation.     

T-cell antigen receptors in Atlantic cod (Gadus morhua l.): structure, organisation and expression of TCR alpha and beta genes

By using short degenerate primers complementing conserved T-cell antigen receptor (TCR) variable and constant region segments for PCR, we were able to isolate putative TCRalpha and beta chain full length cDNAs in Atlantic cod. The Valpha and Vbeta domains have the canonical features of known teleost and mammalian TCR V domains, including conserved residues in the beginning of FR2 and at the end of FR3. The Jalpha and Jbeta region possess the conserved Phe-Gly-X-Gly motif found in nearly all TCR and immunoglobulin light chain J regions. Similar to other vertebrates, the Atlantic cod Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic regions. The Atlantic cod Cbeta sequence lacks a cysteine in its connecting peptide region, but other motifs proposed to be important for dimerisation and cell surface expression are observed. Four different cod Cbeta sequences were identified, two of which share 3' untranslated regions different from one of the other two sequences, suggesting the existence of isotypic gene variants of Cbeta. Based on Southern blot analyses, the TCRalpha and beta gene loci appear to be arranged in translocon organisation (as opposed to multicluster) with multiple V gene segments, some (D) and J gene segments and a single or few C gene segments. Northern blot analyses show expression of the TCRalpha and beta chains in thymus, spleen and head kidney, expression of the TCRbeta chain was also detected in the ovary. Interestingly, no expression was detected in intestine even though the existence of T-cells in intestine has been proposed in other teleost species.     

Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen

BACKGROUND: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B- and T-cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti-TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex. RESULTS: The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two-hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co-immunoprecipitation, both in vitro and in vivo. TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation. CONCLUSION: TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.     

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.     

Diversity of novel recombining elements suggests developmentally programmed expression of the T cell receptor alpha/delta locus.

During fetal ontogeny, the first wave of gamma delta T lymphocytes appears in the thymus at day 14 of gestation assembling predominantly T cell receptors (TcR) with V gamma 3 and V delta 1. To identify V delta gene segments that are transcribed at day 16, subsequent to the first wave of V delta 1 expression, delta chain cDNA was amplified by the anchored polymerase chain reaction with single-sided specificity for C delta. Unexpectedly, most of the cDNA clones do not contain V gene segments. In some cDNA clones an alternative splice from the leader exon to the C delta exon has deleted the whole variable region exon. In other cDNA clones, multiple non-V-like elements are juxtaposed to the D delta 2 and J delta 1 gene segments. A large number of these diverse elements appear to be rearranged in fetal thymocytes, bringing V alpha gene segments located upstream of the recombining element into proximity to the J alpha locus. It is proposed that these rearrangements make irreversible the commitment to the TcR alpha beta lineage and determine a programmed read out of different clusters of V alpha gene segments.     

Onset of T-cell receptor Vbeta8.1 and Dbeta2.1 V(D)J recombination during thymus development of inbred mouse strains.

The assembling of T-cell receptor (TCR alpha/beta and gamma/delta) genes depends on the V(D)J recombination occurring in early thymocytes during thymus ontogeny. The V(D)J recombination reaction is directed by a recombinase complex from the RAG-1 and RAG-2 genes, and is modulated by several other gene products. Due to the essential role of the TCRbeta in thymocyte differentiation, it is important to define with precision the temporal emergence of the TCRbeta recombination in normal non-manipulated mouse strains. We analysed the onset of V(D)J recombination between TCRVbeta8.1 and Jbeta2.1 gene segments during fetal development of the thymus in three non-manipulated inbred strains of mice; BALB-c, C57BL/6 and CBA. We show that the emergence of the V(D)J recombination at the TCRbeta locus differs among strains, suggesting an in vivo role of the different genetic backgrounds in driving gene rearrangements.