Restriction of the T-cell receptor V delta gene repertoire is due to preferential rearrangement and is independent of antigen selection

To determine whether the limited V gene usage by the T-cell receptor delta (TCRD) chain is dictated by preferential rearrangement or by antigen selection, we characterized and compared the TCRDV gene repertoire of the productive with that of the unprotective allele in 80 human TCRG/TCRD clones. Six different V genes were found on the expressed allele; two of them, provisionally named DV7 and DV8, have not been described before on the surface of TCRG/TCRD T cells. Overall, six V genes and six non-V elements were isolated from the unproductive allele. Interestingly, the same set of genes was rearranged both in the productive and in the unproductive chromosome. These findings seem to suggest that antigen-independent mechanisms play a major role in the restriction of the TCRDV gene repertoire.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide database and have been assigned the accession numbers Z46643 (DV7-E2), Z46644 (DV8-E6), Z46645 (DV8-M1), Z46641 (AV12-E4), Z46642 (AV29-E5), Z46652 (DREC-E13), Z46637 (TCR-d), Z46638 (TCR-n), Z46639 (TCR-r), Z46653 (PSI-DVu), and Z46640 (TCR-w)     

V kappa gene usage, idiotype expression, and antigen binding among clones expressing the VHX24 gene family derived from naive and anti-idiotype immune BALB/c mice

The BALB/c myeloma protein ABPC48 binds beta(2-6)-linked fructosans and expresses genes derived from the VHX24 and V kappa 10 gene families. We have selected 30 hybridomas expressing the VHX24 gene family derived from mitogen-stimulated spleen cells of naive BALB/c mice and mice injected at birth with the syngeneic monoclonal anti-ABPC48Id, IDA10. The majority of mAb with kappa L chains uses V kappa 1. Antibodies reacting with IDA10 use both V kappa 10 and V kappa 1. Most of these VHX24+ mAb reacted with one or more members of a limited panel of predominantly polysaccharide Ag that have been previously observed to interact with antibodies expressing the VHX24 gene family. Nucleotide sequencing of selected VH and V kappa genes shows a very low frequency of somatic mutation. The effect of neonatal anti-Id injection on VHX24-V kappa pairing and Id expression is discussed.     

Immunoglobulin constant kappa gene alleles in twelve strains of mice

Genomic DNA for the immunoglobulin (Ig) constant kappa Igk-C gene region was amplified by the polymerase chain reaction (PCR) method and sequenced from twelve commonly used inbred mouse strains. PCR products were used directly as templates in dideoxy-DNA-sequencing, a method which avoids the sequencing errors caused by Taq polymerase, since no cloning step is required. In restriction fragment length polymorphism (RFLP) studies the SJL mouse strain has been shown to belong to a Igk-C allogroup different from other common inbred mouse strains. The BALB/c Igk-C region was sequenced earlier, but our Igk-C sequences clarify the situation and confirm the existence of three Igk-C alleles in inbred mice (Mus musculus domesticus). Mice belonging to the kappa (Igk) haplotype e (SJL) have allele c of the Igk-C gene. The strains belonging to the kappa haplotype [a albino strain, K subline (AKR), PL and d (C58)] have allele a, and all other eight strains belonging to three different Igk haplotypes (b, c, and f) use allele b of the gene. Allele b has at least one (possibly two) nucleotide differences from allele a in the Igk-C region, but five compared to allele c. The allelic sequences also predict two allotypic kappa polypeptide chains among twelve inbred strains. Alleles a and b encode identical polypetides, but allele c (SJL) has a conserved lysine to arginine substitution in residue 142.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X67002-X67012.     

Genetics and primary structure of V kappa gene segments encoding antibody to streptococcal group A carbohydrate. Comparison of V kappa gene structure with idiotope expression

Two gene segments, V kappa-25-39 and V kappa-25-47, that encode antibody to streptococcal group A carbohydrate in A/J mice were found to be more than 95% homologous in nucleotide sequence in both coding and noncoding regions. It was previously shown that V kappa-25-39 encodes immunoglobulins that express the IdX and IdI-1 idiotopes, whereas V kappa-25-47 encodes IdX+, and IdI-1- immunoglobulins. V kappa gene segments that were clearly allelic to V kappa-25-47 are used to encode IdX+, IdI-1- anti-group A carbohydrate antibodies by C.B20 mice and likely by C57BL/6 mice. Murine strains that are deficient in IdI-1 idiotope expression were investigated by Southern blotting with a 5' probe from V kappa-25-39. Two IdI-1-deficient strains, CE/J and C58/J, had a grossly altered V kappa gene segment structure compared with the A/J prototype. In contrast, the IdI-1-deficient strain, C57BL/6, was indistinguishable from A/J with the 5'V kappa-25-39 probe, indicating that more subtle genetic changes account for the loss of IdI-1 expression in C57BL/6 mice. The evolution of V kappa-25-39 and V kappa-25-47 gene segments was deduced by comparison with the homologous V kappa 24B gene segment of Mus pahari. V kappa-25-39 and V kappa-25-47 likely have recently duplicated once in A/J and related strains of laboratory mice and may have duplicated again in CE/J mice. Thus, individual members of the V kappa 24 gene family, to which V kappa-25-39 and V kappa-25-47 belong, are preserved while the number of gene copies expands or contracts. This fact is strong evidence that evolutionary forces have maintained the V kappa 24 gene family, all of which encode antibody specific for carbohydrate found in bacterial pathogens.     

Comparison of V kappa gene family expression in adult and fetal B cells

The functional B cell repertoires from adult and fetal mice were compared by examining V kappa gene family expression in individual cells. In addition, because little is known about the relative use of the various V kappa gene families in an immune response, adult B cells from several different strains of mice were analyzed. This was accomplished by stimulating B cells with the polyclonal activator, LPS. Activated cells were then analyzed for V kappa gene family expression at the single cell level by in situ hybridization using radiolabeled V kappa gene probes. It was found that all V kappa gene families tested were represented in the LPS-induced adult repertoire with V kappa 1, V kappa 4,5 and V kappa 19 being expressed to the largest degree in all strains tested. The LPS-induced adult V kappa gene family repertoire was then compared to the fetal repertoire and some differences were observed. In particular, a lower proportion of fetal B cells expressed V kappa 1 and a higher proportion of fetal B cells expressed V kappa 4,5 and V kappa 10. Importantly, compared with the adult response there was no evidence in the fetal response for an increased expression of V kappa 21, the family that maps closest to J kappa,C kappa. This is in contrast to what has been shown previously with H chain V region exons in which there was a clear preference for the VH gene families that mapped closest to DH.     

Human lupus anti-DNA autoantibodies undergo essentially primary V kappa gene rearrangements.

We have recently characterized the heavy chain variable region (VH) genes expressed by a panel of human anti-DNA antibodies derived from four patients with systemic lupus erythematosus and expressing an idiotypic marker representative of a subset of pathogenic autoantibodies. Here, we have cloned and sequenced the kappa chain variable region genes (V kappa) of the clones whose VH genes had been previously analysed. All the V kappa genes utilized map to the 280 kb portion of the 3' end of the locus, suggesting that they represent essentially the products of primary rearrangements. This proximal clustering of the V kappa genes used contrasts with the broad distribution of immunization-induced human antibody V kappa genes over 1400 kb of the locus. In addition, lupus autoantibodies show no tendency to express the downstream junctional (J kappa) exons--another indication of infrequent secondary variable gene assembly. Since successive rearrangements may extinguish high-affinity recognition of self antigens, we propose that this bias in V kappa and J kappa expression reflects a low rate of secondary light chain rearrangements among lupus autoantibodies. We also postulate that the corrective mechanism capable of editing potentially aggressive, self-reactive antibodies in these patients may be deficient--a deficit that could be genetically determined and/or somatically acquired.     

Utilization of V kappa families and V kappa exons. Implications for the available B cell repertoire

A molecular cloning approach was used to determine the relative utilization of 2 individual V kappa 21 genes, 13 V kappa gene families, and the 4 functional J kappa gene segments among splenic B cells of nonimmunized BALB/c mice. Based on the observed frequency of individual V kappa gene expression, we estimate that the mouse genome encodes 150 to 180 functional V kappa genes, and we suggest that most functional V kappa exons are expressed at comparable frequencies in the preimmune antibody repertoire. In contrast, clear differences in J kappa segment utilization were observed, J kappa 4 being consistently underrepresented with respect to the other J kappa segments.     

Structure of a germline rabbit immunoglobulin V kappa-region gene: implications for rabbit V kappa-J kappa recombination

Rabbit kappa-immunoglobulin chains exhibit diversity in the number of amino acids between the invariant residues Cys 88 and Phe 98; this length diversity is formally similar to that found in the human and mouse heavy chain systems, in which it results from interposition of the D element between V and J. To explore the molecular basis for this length diversity in rabbit kappa-chains we have determined the nucleotide sequence of a rabbit germline V kappa immunoglobulin gene. The spacing between the 7-mer and 9-mer signal elements of this gene suggest that it could recombine with J kappa without a D element. We discuss alternative explanations for the length diversity of rabbit kappa-chains.     

Ig V kappa family repertoire of plasma cells derived from autoimmune MRL mice.

V kappa gene family usage was determined in the resident in vivo-activated plasma cells of individual diseased MRL mice by using in situ hybridization. In this way, the entire autoimmune repertoire could be analyzed. Autoantibody levels and extent of glomerulonephritis were also measured, so that the severity of disease could be assessed. It was found that V kappa expression was highly variable from mouse to mouse. Some animals displayed a V kappa family repertoire similar to mitogen-stimulated cells and consistent with the size of the families. These animals tended to have lower disease indices. Other animals, which had higher disease indices, displayed considerable over- or underutilization of individual V kappa families. However, no particular V kappa families were repeatedly biased in their expression, as was found at the VH level with J558. Importantly, in the 10% of animals that expressed VH J558 exclusively, four or more V kappa families were expressed and multiple antiself specificities were produced. The data are most consistent with a number of J558 genes being expanded in a variety of self-specificities. However, because only VH J558 is expressed in these sicker animals, nonspecific polyclonal activation is highly unlikely. These results underscore the continuing evolution of the autoimmune repertoire, with considerable diversity at early stages followed by a highly selected repertoire in which a potential role for nonspecific polyclonal activation is virtually excluded.     

Defective T cell response to presented antigen in autoimmune mice

The effect of the single autosomal recessive gene lpr on antigen presentation was studied. MRL/Mp-lpr/lpr, C3H/HeJ-lpr/lpr, C57BL/6J-lpr/lpr, and their normal congenic partners were investigated. Mice bearing the lpr gene were unable to respond to TNP-KLH when presented by syngeneic antigen-presenting cells. The congenic normal partners gave a brisk response. Mixing experiments demonstrated that the defect resided with the lpr responding T cell and not with the lpr antigen-presenting cell. Antigen-presenting cells from lpr mice were capable of inducing T cell proliferation in normal congenic partners, whereas antigen-presenting cells from normal mice failed to stimulate lpr T cells. This defect was intrinsic to an Lyt-1+2- cell. Pharmacologic restoration was attempted by in vivo and in vitro administration of interleukin 2. However, cells from lpr mice remained unaffected. The relationship of these findings to autoimmunity is discussed.     

The human V kappa locus. Characterization of extended immunoglobulin gene regions by cosmid cloning

As part of the ongoing work in our laboratory on the structural organization of the human V kappa locus we screened cosmid libraries with V kappa gene probes and obtained numerous V kappa gene-containing cosmid clones. Several genomic regions of the V kappa locus were reconstructed from overlapping cosmid inserts and were extended by one step of chromosomal walking. The regions that are called Wa, Wb, Oa, Ob and Ob' comprise about 370 kb (10(3) bases) of DNA and contain 24 V kappa genes and pseudogenes. The V kappa genes belong to the three dominant subgroups (V kappa I, V kappa II, V kappa III) and are arranged to form mixed clusters with members of the different subgroups being intermingled with each other. The distances between the genes range from 1 to 15 kb. Three genes of the Wa and Wb regions that were sequenced turned out to be pseudogenes. Terminal parts of the regions Wa and Ob that do not contain V kappa genes of one of the known subgroups may represent extended spacer regions within the V kappa locus. Wa and Wb are duplicated regions located at different positions of the locus. Region Wb was found to comprise inversely repeated sections of at least 14 kb each that contain V kappa genes oriented in opposite polarity. This finding is consistent with inversion-deletion models of V-J joining; it also shows that the V kappa locus contains not only unique and duplicated but also triplicated parts. The data on the W and O regions are discussed together with those on the L regions and on other regions established in our laboratory. Although the picture of the human V kappa locus with, to date, about 70 different non-allelic V kappa genes is still incomplete, some general features with respect to the organization of the genes and the limited duplication of genomic regions have emerged.     

Modern evolution of a single-copy gene: the immunoglobulin C kappa locus in wild mice

The immunoglobulin kappa light-chain constant region gene (C kappa) has been cloned and sequenced from five wild mouse species. Analysis of these data has permitted an assessment of single-copy gene evolution during a limited time period as defined by the genus Mus. Sequence conservation was found to be as high (or higher) in the 5' and enhancer regions as in the coding region. The pattern of substitutions throughout these genes suggests that parallel evolution has occurred frequently and that substitutions at replacement sites have not decreased significantly, owing to saturation during this period of approximately 10 Myr. Phylogenetic relationships have been determined among these wild species as well as among members of the genus Rattus.      

The use of gene knockout mice in thermoregulation studies

As the use of gene knockout models in thermoregulation studies has gained popularity, the reported incidence of redundant or discrepant phenotypes between studies has also increased. Several gene knockout models mimic human processes and have provided valuable insight into the role of endogenous mediators in thermoregulatory processes. There are also many examples of mutant strains expressing virtually identical phenotypes as their wild-type controls, causing concern regarding the appropriateness of these models for the study of physiological processes. In some cases, discrepancies in results are being reported from different laboratories that are studying the same gene knockout model. While mutant strains provide a powerful tool for analysis of gene function in vivo, the breeding strategies used to generate the strain may have a profound impact on the expressed phenotype. This review examines the intricacies of working with a small rodent such as the mouse and discusses the advantages and disadvantages of using gene knockout models for thermoregulatory research. A number of experimental strategies that can be used to minimize the occurrence of redundant phenotypes are presented. The influence of background strain effects is also considered, since this may be one of the most important factors influencing a mutant phenotype. A future perspective is provided in which more advanced technologies using conditional gene inactivation and the production of rat knockout strains will improve current experimental design.     

Tramadol reduces the 5-HTP-induced head-twitch response in mice via the activation of mu and kappa opioid receptors

Tramadol, an atypical opioid analgesic, stimulates both opiatergic and serotonergic systems. Here we have investigated the effect of tramadol in mice on 5-hydroxyptrytophan (5-HTP)-induced head twitch response (HTR), which is an animal model for the activation of the CNS 5-HT(2A) receptors in mice. Tramadol attenuated 5-HTP-induced HTR in a dose-dependent manner as morphine. Furthermore, the nonselective opioid receptor antagonists, naloxone and diprenorphine (M5050), reversed the effect of tramadol on 5-HTP-induced HTR dose-dependently. Interestingly, in contrast to the selective delta opioid receptor antagonist NTI, beta-FNA, a selective mu receptor antagonist, and nor-BNI, a selective kappa opioid receptor antagonist, antagonized the attenuation of 5-HTP-induced HTR by tramadol. In conclusion, administration of tramadol systemically inhibits 5-HTP-induced HTR in mice by activating opiatergic system in the CNS. Our findings show that mu and kappa opioid receptors, but not delta opioid receptor, play an important role in the regulation of serotonergic function in the CNS.