[Show abstract][Hide abstract] ABSTRACT: The surrogate light chain (SLC) is a key regulator of B cell development in the bone marrow, resulting in mature B cells that produce antibodies that are capable of interacting with antigens. The SLC comprises two noncovalently interacting proteins: VpreB and 14.1. We engineered a construct to represent the complete immunoglobulin-like domain of the SLC variable domain in a single protein chain that could be bacterially expressed. In this construct, the incomplete immunoglobulin domain of VpreB (residues 1-102) was linked to the J-segment of 14.1 (residues 40-53), which provided one beta-strand to complete the V-like domain (VpreBJ). Because VpreBJ has the interface to VH chains, but lacks the unique region of 14.1, which is important for SLC signaling, we predict that a properly folded VpreBJ would have the potential to act as a dominant negative mutant of the surrogate light chain. X-ray crystallography of VpreBJ at 2.0 A resolution showed that the engineering was successful. With its two beta-pleated sheets, packed face-to-face, the single chain VpreBJ resembles a mature light chain immunoglobulin V-domain (VL). The surface that would normally interact with the VH chain interacts with a crystallographically related VpreBJ molecule. The presence of dimeric species in solution was verified by analytical ultracentrifugation. VpreBJ is easily overexpressed in bacteria, while retaining the native conformation of an immunoglobulin domain, and thus may serve as an important reagent for future studies in B-cell development.
Protein Science 04/2008; 17(3):458-65. · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peripheral blood memory B cells latently infected with EBV bear somatic mutations and are typically isotype switched consistent with being classical Ag-selected memory B cells. In this work, we performed a comparative analysis of the expressed Ig genes between large sets of EBV-infected and uninfected peripheral blood B cells, isolated from the same infectious mononucleosis patients, to determine whether differences exist that could reveal the influence of EBV on the production and maintenance of these cells. We observed that EBV(+) cells on average accumulated more somatic hypermutations than EBV(-) cells. In addition, they had more replacement mutations and a higher replacement-silent ratio of mutations in their CDRs. We also found that EBV occupies a skewed niche within the memory compartment, due to its exclusion from the CD27(+)IgD(+)IgM(+) subset, but this skewing does not affect the overall structure of the compartment. These results indicate that EBV impacts the mutation and selection process of infected cells but that once they enter memory they cannot be distinguished from uninfected cells by host homeostasis mechanisms.
The Journal of Immunology 10/2007; 179(5):3153-60. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although the causative agent of Lyme disease is definitively known to be the tick-borne spirochete, Borrelia burgdorferi, the etiology of chronic joint inflammation that ensues in a subset of patients remains less well understood. Persistence of arthritis after apparent eradication of the spirochete suggests an autoimmune reaction downstream of the original bacterial infection. We have generated recombinant Ab probes from synovial lesions within affected arthritic joints in an attempt to recapitulate disease-relevant Ag-binding specificities at the site of injury. Using this panel of intra-articular probes, as well as Ab fragments derived from patient peripheral blood, we have identified cytokeratin 10, present in synovial microvascular endothelium, as a target ligand and a putative autoantigen in chronic, antibiotic treatment-resistant Lyme arthritis. Furthermore, there is cross-reactivity between cytokeratin 10 and a prominent B. burgdorferi Ag, outer surface protein A. Release of the self protein in the context of inflammation-induced tissue injury and the resulting in situ response to it could set in motion a feed-forward loop, which amplifies the inflammatory process, thereby rendering it chronic and self-perpetuating, even in the absence of the inciting pathogen.
The Journal of Immunology 09/2006; 177(4):2486-94. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) establishes a lifelong persistent infection within peripheral blood B cells with the surface phenotype of memory cells. To date there is no proof that these cells have the genotype of true germinal-center-derived memory B cells. It is critical to understand the relative contribution of viral mimicry versus antigen signaling to the production of these cells because EBV encodes proteins that can affect the surface phenotype of infected cells and provide both T cell help and B cell receptor signals in the absence of cognate antigen. To address these questions we have developed a technique to identify single EBV-infected cells in the peripheral blood and examine their expressed Ig genes. The genes were all isotype-switched and somatically mutated. Furthermore, the mutations do not cause stop codons and display the pattern expected for antigen-selected memory cells based on their frequency, type, and location within the Ig gene. We conclude that latently infected peripheral blood B cells display the molecular hallmarks of classical antigen-selected memory B cells. Therefore, EBV does not disrupt the normal processing of latently infected cells into memory, and deviations from normal B cell biology are not tolerated in the infected cells. This article provides definitive evidence that EBV in the peripheral blood persists in true memory B cells.
Proceedings of the National Academy of Sciences 01/2006; 102(50):18093-8. · 9.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To test the fate of developing B cells with autoreactive receptor components, we studied mice homozygous for a knock-in transgene coding the VH domain of an IgM ssDNA-binding antibody. The transgene has unmutated C57 BL/6 V gene segments. Homozygous knock-in mice developed normal numbers of spleen and bone marrow B cells and normal serum Ig concentrations, and had the same low level of serum anti-ssDNA antibody as non-transgenic mice. Mature B cells expressed the transgene, and it underwent mutation and class switching. In young knock-in animals, nearly all IgM and some IgG cDNA clones from bone marrow and spleen contained the transgene V(H)D(H)J(H), with few or no mutations. In many IgM clones from older animals, however, and many IgG clones from both young and old mice, VH domains were revised by productive replacement with a new V(H)D(H) segment. VL segments were diverse. Immunized homozygous knock-in mice produced serum antibodies to polysaccharide, nucleic acid and protein antigens. Monoclonal IgM and IgG antibodies to nucleic acids used either transgenic or revised VH domains; but all of 20 IgG monoclonal antibodies to thyroglobulin used revised VH domain genes. Thus, B cells expressing an autoreactive (ssDNA-binding) VH domain did progress through development and were precursors for cells producing IgM and IgG, but underwent extensive VH gene revision in diversification of antibody responses.
[Show abstract][Hide abstract] ABSTRACT: Lyme arthritis is initiated by the tick-borne spirochete, Borrelia burgdorferi. In a subset of patients, symptoms do not resolve in response to standard courses of antibiotics. Chronic joint inflammation may persist despite spirochetal killing, suggesting an autoimmune etiology. The pathogenic mechanisms that sustain chronic Lyme arthritis have not been fully elucidated, although T cells are believed to play a role. The synovial lesion contains elements of a peripheral lymph node, with lymphoid aggregates, plasma cells and follicular dendritic cells. An analysis of activated cells at the site of injury could yield clues regarding the nature of the response and the identity of potential autoantigens. Using laser-capture microdissection, we have isolated plasma cells from the joint tissue of chronic Lyme arthritis patients who underwent synovectomy. Expressed Ig V regions were amplified by RT-PCR. A majority of isolated cells expressed gamma H chains, which is indicative of a class-switched response. There were a large number of nucleotide substitutions from germline, with a higher fraction of replacement mutations in the CDRs, suggesting a process of Ag-driven selection. We have recovered clonal clusters of cells containing identical junctions and V(D)J rearrangements. Sequence analysis reveals a hierarchy of shared somatic mutations between members of a given clone. Intraclonal diversity among plasma cells of close physical proximity points toward an ongoing process of diversification and affinity maturation, possibly driven by the chronic presence of an autoantigen.
The Journal of Immunology 04/2005; 174(5):2860-9. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The carboxyl-terminal domain of murine H1° histone was compared with that of human H1°, bovine H1° and other H1 and H5 histones. Two sets of antibodies were induced by murine H1°. One set reacted with only the carboxyl-terminal domain of murine H1° and preferred the murine over the bovine and human proteins. The second set of antibodies reacted with the globular domain of murine H1° and did not distinguish among murine, bovine and human H1° species. There were five positions in the first 60 residues of the carboxyl-terminal domain in which the murine H1° differed from the human H1°. In this region, the murine H1° had no more than 49% overall homology with other H1 and H5 histones; however, short sequences in the domain were very similar to short sequences that occur in rabbit H1.3, trout H1 and goose or chicken H5. In comparisons based on these and other published data, the carboxyl-terminal domain of H1° is found to be more variable among species than is the globular domain; the first two-thirds of the H1° carboxyl-terminal domain is largely unique and does not show great overall homology with H1 or H5, whereas the last third is again more conserved. As the first two-thirds of the domain is the only portion where the homology with H5 is less than 50%, it may be responsible for functional differences between H1° and H5.
European Journal of Biochemistry. 03/2005; 168(1):161 - 167.
[Show abstract][Hide abstract] ABSTRACT: At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x<y. S-DNAs formed many different species of slipped isomers, as indicated by its multiple electrophoretic species. In contrast, SI-DNAs formed distinct structures, as indicated by the limited electrophoretic species for all possible repeat length pairings. Sister SI-DNAs with an excess of CAG repeats always migrated slower than their sister SI-DNAs with an excess of CTG repeats. Strikingly, both the propensity to form slipped structures and the pattern of S-DNAs, but not SI-DNAs, varied for similar lengths of CTG/CAG repeats between the DM1 and SCA1 loci, highlighting a role for flanking cis-elements in S-DNA but not SI-DNA formation. Slipped structures bound structure and nucleotide-specific anti-DNA antibodies. Binding of anti-B-DNA antibodies was reduced for both S-DNAs and SI-DNAs relative to their linear forms. SI-DNAs bound anti-Z-DNA antibodies, while both S and SI-DNAs bound anti-cruciform antibodies, revealing shared characteristics between the corresponding DNA structures and slipped DNAs. Such features of the repeats may be recognized by cellular proteins known to bind such structures.
Journal of Molecular Biology 10/2003; 332(3):585-600. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anti-DNA antibodies contribute to the pathology of systemic lupus erythematosus. Their depositon in tissue lesions could result from localization of preformed immune complexes of antibodies with DNA or nucleosomes, or from cross-reaction of anti-DNA antibodies directly with tissue proteins. Structural analyses contribute to understanding their pathogenic potential. Primary structures of lupus immunoglobulin G double-stranded DNA-binding autoantibodies are determined by immunoglobulin genes with mutated variable region segments, indicative of selection by immunizing antigen. Arginine, lysine and asparagine residues in complementarity-determining region favor DNA binding. Heavy-chain variable regions make major contributions to DNA binding; affinity and specificity of binding are modulated or can be abrogated by the light-chain variable domain. Crytallographic structure is known for a few antibody-DNA complexes and several ligand-free Fab fragments. Computer modeling supplements this limited information. Structural information of lupus antibody interactions with both DNA and cross-reacting molecules will support use of ligands to inhibit tissue deposition of the antibodies and prevent lesion formation in lupus.
Cellular and Molecular Life Sciences CMLS 03/2003; 60(2):309-20. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies of age-associated immune system changes revealed alterations in expressed immunoglobulin heavy chain variable domain repertoires, and variability in the fraction of expressed heavy chain variable domain genes with mutations. To test whether the latter finding reflected a variation in memory B-cell numbers, we measured circulating memory B cells of 11 healthy elderly subjects, 173 nursing-home residents, and 34 healthy young adults. A large fraction of old adults have low values for memory cells both as a percentage of all B cells and as an absolute memory B-cell concentration. The range of both values is much wider in old adults than in young adults, and it is much wider than the range of T-cell concentrations. Memory B-cell concentration, which was positively correlated with memory T-cell concentrations but inversely related to in vitro T-cell responses to mitogens, may reflect highly individual rates of immune senescence, and it may serve as an amplified marker of underlying T-cell function.
The Journals of Gerontology Series A Biological Sciences and Medical Sciences 09/2002; 57(8):B304-11. · 4.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Drs Berden and Koutouzov presented evidence that nucleosomes are antigens in lupus pathogenesis and that apoptotic cells are the source of nucleosomes. Berden's group measured persistence of circulating nucleosomes and nucleosome-antibody complexes in autoimmune mice and demonstrated nucleosomal deposition in skin of SLE patients as well as in renal lesions. Koutouzov reported that anti-nucleosomes are among the earliest autoantibodies in MRL+/+ mice, appearing several weeks before anti-DNA antibodies. Treatment of the mice with a pro-apoptotic drug, taxotere, accelerated autoantibody production and development of lesions. Herrmann proposed that persistent immunization results from reduced dead cell clearance and reduced production of immunosuppressive cytokines by defective scavenger macrophages. He also described accumulation of apoptotic cells in germinal follicles in SLE patients and attachment of nuclear antigens that are produced in apoptosis to the surface of follicular dendritic cells. Apoptosis-derived nucleosomes may be important in both the immunizing and effector arms of pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.
The Journal of Immunology 11/2001; 167(7):3746-55. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.
Journal of Molecular Recognition 01/2001; 14(1):18-28. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This protocol describes application of single cell reverse transcription polymerase chain reaction (RT-PCR) to the study of human immunoglobulin V region usage. The procedure begins with separation of peripheral blood mononuclear cells (PBMC) from human blood. The PBMC are stained with the B cell selective marker, anti-CD19. Stained B cells are sorted by flow cytometry and deposited, consecutively, one cell into each of an array of tubes. cDNA for one or more antibody variable regions (VH and/or VL) is synthesized with a primer (or primers) complementary to sequence(s) within the constant region (Cmu, Cgamma, Ckappa and/or Clambda). The cDNA is used as template for PCR amplification with gene or gene family specific primers. A second PCR is then performed with two nested primers to increase both the specificity and quantity of V region PCR products. The purified PCR products are sequenced directly and aligned to V region germline database and the Genbank database. Single cell RT-PCR is a fast and convenient way to analyze V region gene expression. It avoids the bias that may be introduced into V region cDNA library construction by the presence of highly variable levels of mRNA in different cells. The PCR products are obtained in quantities that can be cloned into bacterial expression vectors for production of recombinant V region protein domains.
Journal of Immunological Methods 11/2000; 244(1-2):217-25. · 2.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immune responses change in aging humans, but it is not known whether there is an age-associated change in the expressed B cell repertoire. We compared Ig VH cDNA libraries from circulating B cells of five elderly and three young human adults. As in young persons, nearly two-thirds of the cDNA clones from older subjects had zero to three V(H) mutations, although there was more individual variation among the elderly. V(H)4 family expression increased in older subjects, both in unmutated and in mutated cDNA clones, whereas V(H)3 family expression predominated in young adults. To test for bias toward activated cells in the cDNA libraries, we studied two older persons by both cDNA library analysis and single-cell RT-PCR. In one subject, more than 85% of VH segments were unmutated by either analysis. In the second, mutated Ig segments were much more frequent in cDNA clones than in consecutive single cells; however, V(H) family usage and high representation of particular genes were similar in both analyses. While aging humans continue to produce naive B cells with unmutated Ig genes, a shift to greater use of the V(H)4 family members and expression of particular genes may reflect changes in selection of developing B cells before affinity maturation toward reactivity with foreign antigen.