CTLA-4 appears to be a negative regulator of T cell activation and is implicated in T cell-mediated autoimmune diseases. Experimental autoimmune myasthenia gravis (EAMG), induced by immunization of C57BL/6 mice with acetylcholine receptor (AChR) in adjuvant, is an autoantibody-mediated disease model for human myasthenia gravis (MG). The production of anti-AChR Abs in MG and EAMG is T cell dependent. In the present study, we demonstrate that anti-CTLA-4 Ab treatment enhances T cell responses to AChR, increases anti-AChR Ab production, and provokes a rapid onset and severe EAMG. To address possible mechanisms underlying the enhanced autoreactive T cell responses after anti-CTLA-4 Ab treatment, mice were immunized with the immunodominant peptide alpha(146-162) representing an extracellular sequence of the ACHR: Anti-CTLA-4 Ab, but not control Ab, treatment subsequent to peptide immunization results in clinical EAMG with diversification of the autoantibody repertoire as well as enhanced T cell proliferation against not only the immunizing alpha(146-162) peptide, but also against other subdominant epitopes. Thus, treatment with anti-CTLA-4 Ab appears to induce determinant spreading, diversify the autoantibody repertoire, and enhance B cell-mediated autoimmune disease in this murine model of MG. (+info)
Early Vlambda diversification in sheep.
(26/350)
This study examined a number of tissues during early gestation in foetal sheep to determine the earliest site of Vlambda expression and time of generation of the Vlambda repertoire. Tissues, including spleen, liver, gut, blood and bone marrow, were obtained from 48, 55, 60 and 63 gestational day (g.d.) ovine foetuses and cDNA libraries were prepared from them by reverse transcription-polymerase chain reaction. Clones were randomly selected from cDNA libraries and subjected to sequencing. Analysis of these sequences and comparison with a pool of germline genes led to the following conclusions. The expression of Vlambda occurs earlier in spleen (48 g.d.) than in all of the other tissues examined. Also, diversity is seen earlier and at higher levels in early foetal spleen than in all of the other tissues examined. In this regard, it is notable that splenic Vlambda expression is readily apparent even before such gut-associated lymphoid tissue as the ileal Peyer's patch (IPP) has developed. Two germline Vlambda genes, 5.1 and 5.3 predominate in early immunoglobulin lambda light-chain gene rearrangement. Examination of Jlambda usage revealed the existence of a new Jlambda gene and its utilization during the early phases of the development of the ovine antibody repertoire. This study indicates that sites other than the IPP contribute to the diversification of the Vlambda repertoire in sheep. We suggest that it is likely that foetal spleen may provide a partially diversified B-cell repertoire before the IPP becomes active as a major site for massive clonal expansion and extensive diversification of B cells. (+info)
The clonal analysis of anticardiolipin antibodies in a single patient with primary antiphospholipid syndrome reveals an extreme antibody heterogeneity.
(27/350)
The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) the cloning of immunoglobulin (Ig) variable (V) region genes originating from individual IgG anticardiolipin-specific B cells by a single-cell polymerase chain reaction technique. The corresponding V regions were cloned in order to express human recombinant antibodies in insect cells by a baculovirus expression system. The molecular analysis, the fine specificity, and the protein cofactor dependency of the first 5 monoclonal IgG anticardiolipins are reported here. This clonal analysis reveals the extreme heterogeneity of these antibodies, which could account for the difficulties in the previous attempts to define the pathogenic antiphospholipid response. This approach should help to unravel the complex antiphospholipid immune response and the mechanism of the prothrombotic state associated with these antibodies, but it could also shed some light on their possible origins. (Blood. 2001;97:3820-3828) (+info)
Third complementarity-determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non-mutated genes.
(28/350)
The third complementarity-determining region (CDR3) of immunoglobulin variable genes for the heavy chain (VH) has been shown to be shorter in length in hypermutated antibodies than in non-hypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of VH6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (JH) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non-mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter JH4 segments and decreased use of longer JH6 segments compared to the non-mutated proteins. The overall length of CDR3 did not change with age within the mutated and non-mutated categories, but was significantly shorter by three amino acids in the mutated clones compared to the non-mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of VH, D, and JH gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy. (+info)
Epigenetic reprogramming of the genome--from the germ line to the embryo and back again.
(29/350)
Mammalian parental genomes are not functionally equivalent, and both a maternal and paternal contribution is required for normal development. The differences between the parental genomes are the result of genomic imprinting--a form of gene regulation that results in monoallelic expression of imprinted genes. Cis-regulatory elements at imprinted loci are responsible for directing allele-specific epigenetic marks required for correct gene expression. This cis information must be interpreted at various points in development, including in the germline where existing imprints are erased and reset. Imprints must also be maintained during preimplantation development, when the genome undergoes dramatic global epigenetic changes. (+info)
Unequal VH gene rearrangement frequency within the large VH7183 gene family is not due to recombination signal sequence variation, and mapping of the genes shows a bias of rearrangement based on chromosomal location.
(30/350)
Much of the nonrandom usage of V, D, and J genes in the Ab repertoire is due to different frequencies with which gene segments undergo V(D)J rearrangement. The recombination signal sequences flanking each segment are seldom identical with consensus sequences, and this natural variation in recombination signal sequence (RSS) accounts for some differences in rearrangement frequencies in vivo. Here, we have sequenced the RSS of 19 individual V(H)7183 genes, revealing that the majority have one of two closely related RSS. One group has a consensus heptamer, and the other has a nonconsensus heptamer. In vitro recombination substrate studies show that the RSS with the nonconsensus heptamer, which include the frequently rearranging 81X, rearrange less well than the RSS with the consensus heptamer. Although 81X differs from the other 7183-I genes at three positions in the spacer, this does not significantly increase its recombination potency in vitro. The rearrangement frequency of all members of the family was determined in microMT mice, and there was no correlation between the in vitro recombination potential and V(H) gene rearrangement frequency in vivo. Furthermore, genes with identical RSS rearrange at different frequencies in vivo. This demonstrates that other factors can override differences in RSS potency in vivo. We have also determined the gene order of all V(H)7183 genes in a bacterial artificial chromosome contig and show that most of the frequently rearranging genes are in the 3' half of the region. This suggests that chromosomal location plays an important role in nonrandom rearrangement of the V(H)7183 genes. (+info)
Functional heterogeneity of anti-endothelial cell antibodies.
(31/350)
While it has been claimed that some anti-endothelial cell antibodies (AECA) activate EC, there is also evidence that others trigger apoptosis. To address the issue of whether activation is a prerequisite for AECA-mediated apoptosis of EC, 23 AECA-positive sera were evaluated for their ability to induce activation and/or apoptosis. Activation was defined as an over-expression of E-selectin and intercellular adhesion molecule 1. Optical microscopy, annexin V binding, hypoploid cell enumeration, and determination of poly (ADP-ribose) polymerase cleavage-related products were used to assess apoptosis. Four functional profiles were defined: 10 sera promoted activation and apoptosis (act+/apo+), one was act+/apo-, six act-/apo+, and the remaining six act-/apo-. The reduced membrane expression of thrombomodulin was associated with apoptosis, rather than activation. Caspase-3 was implicated in the two models of apoptosis, the ratios of several survival proteins to Bax decreased, regardless of the ability of apo+ AECA to activate the cells, while radical oxygen species did not appear to be involved. Furthermore, it occurred that macrophages engulfed EC treated with apoptosis-promoting AECA, but not those incubated with AECA that did not induce apoptosis. Hence, AECA represent an extremely heterogeneous family of autoantibodies, not only because of the variety of their target antigens, but also the subsequent diversity of their effects. (+info)
Circulating human B cells that express surrogate light chains display a unique antibody repertoire.
(32/350)
Circulating human B cells that coexpress V-preB and conventional L chains (V-preB+L+ B cells) are a recently described subset of B cells that express Abs with features of self-reactivity. Initial analysis of V-preB+L+ B cells was limited to Ig-kappa and to the small, underused VH5 family. To determine whether Abs commonly expressed by V-preB+L+ B cells show similar features, we analyzed Ig H chains from three highly expressed VH families, VH1, VH3, and VH4, and Ig-lambda. We find that VH1 and VH3 Abs expressed by V-preB+L+ B cells resemble VH5 in that they display increased JH6 use, long CDR3s, and an increased frequency of D-D fusions. Abs in all three of these VH families also show skewed D reading frame use resulting in predominance of hydrophobic amino acids, which are counterselected in conventional B cells. Like Ig-kappa genes, the Ig-lambda genes in V-preB+L+ B cells show long CDR3s, but they differ from Ig-kappa genes in that they display no evidence of receptor editing. We conclude that a large number of H and L chain Abs expressed by V-preB+L+ B cells display features associated with self-reactive Abs. (+info)