Ig Gene Usage Research Articles

Gingival tissue antibody gene utilization in aging and periodontitis.

This study used a nonhuman primate model of ligature-induced periodontitis to document the characteristics of immunoglobulin (Ig) gene usage in gingival tissues with disease and affected by age. Adaptive immune responses to an array of oral bacteria are routinely detected in local gingival tissues and the systemic circulation across the human population. The level and diversity of antibody increases with periodontitis, reflecting the increased quantity of B cells and plasmacytes in the tissues at sites of periodontal lesions. Macaca mulatta (n=36) in four groups (young - ≤3 years; adolescent >3-7 years; adult - 12-15 years; aged - 17-23 years) were used in this study. Gingival tissues were sampled at baseline (health), 2 weeks (initiation), 1 and 3months (progression), and 5months (resolution) of the lesion development and transcriptomic analysis included 78 Ig-related genes. The results demonstrated extensive variation in Ig gene usage patterns and changes with the disease process that was substantially affected by the age of the animal. Of note was that the aged animals generally demonstrated elevated expression on multiple Ig genes even in the baseline/healthy gingival tissues. The expression levels revealed 5 aggregates of Ig gene change profiles across the age groups. The number of gene changes were greatly increased in adult animals with the initiation of disease, while the young and adolescent animals showed extensive changes with disease progression. Elevated Ig gene transcripts remained with disease resolution except in the aged animals. The response profiles demonstrated selective heavy/light change gene transcripts that differed with age and clustering of the transcript expression was dominated by the age of the animals. The results suggested potential critical variations in the molecular aspects of Ig gene expression in gingival tissues that can contribute to understanding the kinetics of periodontal lesions, as well as the variation in episodes, rapidity of progression, and role in resolution that are impacted by age.

CLL-Specific B Cell Receptor Immunoglobulin Stereotypes Are Very Infrequent in Low Count Monoclonal B Cell Lymphocytosis: Implications for Disease Ontogeny

In chronic lymphocytic leukemia (CLL), the existence of subsets of patients with stereotyped B cell receptor immunoglobulin (BcR IG), collectively accounting for ~30% of all CLL, strongly implicates antigen selection in disease ontogeny. That said, it is still inconclusive if and how frequently “CLL-specific” BcR IG gene rearrangements occur in the repertoire of unaffected individuals. This is especially relevant when also considering monoclonal B-cell lymphocytosis (MBL), detected in 3-12% of the general population and characterized by the presence of CLL-like clonal B cells in lower numbers compared to CLL with no evidence of disease. MBL is distinguished into high and low count (HC-MBL and LC-MBL, respectively) with the former considered as a pre-leukemic condition, while the latter having an unclear, if any, link to CLL. Our previous immunogenetic studies have revealed similarities between HC-MBL and CLL, thus setting them apart from LC-MBL that displayed distinct IG gene usage and low incidence of stereotypy. However, these studies were Sanger-based, thus inherently limited regarding sensitivity and accurate quantification.Here we sought to overcome these limitations and obtain a comprehensive view into the composition of the IG repertoire in LC-MBL by NGS immunoprofiling, searching particularly for major CLL IG stereotypes. The study group comprised 23 individuals with CLL-like (CD5+) LC-MBL identified by a standardized flow cytometry protocol. PBMCs were the starting material in 11 cases, whereas sorted MBL cells were studied in the remaining 12 cases; in 9 cases, the normal B cell compartment was also flow sorted. Included in the analysis were also 6 individuals without LC-MBL in whom we sorted different B cell subpopulations, including naïve B cells (n=6) and memory B cells (n=5). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified with leader primers and sequencing libraries were prepared with the TruSeq DNA LT Kit and run on the MiSeq Sequencer using the MiSeq Reagent Kit v3. NGS reads were subjected to an in-house bioinformatics pipeline for clonotype characterization and the identification of CLL-specific BcR IG stereotypes.Overall, 16,857,788 productive filtered-in sequence reads were obtained corresponding to an average of 366,474 reads/sample (range: 67,175-662,994). We identified very few reads (2,470/16,857,788; 0.15%) corresponding to 17/19 major CLL BcR IG stereotypes. Their incidence in LC-MBL did not follow the CLL trend: for instance, the stereotypic IGHV3-21 gene rearrangement defining subset #2, the most frequent in CLL, accounted for only 27/2,470 reads (1.1%) corresponding to major subset stereotypes. Overall, stereotypes were identified in 26/47 samples (55%) from 13/23 LC-MBL and 6/6 non LC-MBL cases. In LC-MBL cases bearing stereotypes the breakdown according to sample type was as follows: (i) PBMCs, n=3 (ii) normal B cell compartment, n=7; (iii) sorted MBL cells, n=5: the stereotypes detected in this particular sample type were present at very low frequencies and concerned rearrangements with unmutated IGHV genes. In individuals without LC-MBL, stereotypes were detected in all 6 samples from naïve B cells versus only 2/5 samples from memory B cells. In general, we did not identify significant associations between specific B cell subpopulations and particular stereotypes, except for the IGHV4-34 rearrangement defining CLL subset #4, an indolent CLL variant with features of BcR-anergized clones, which was found only within naïve B cells.In conclusion, we demonstrate that LC-MBL is immunogenetically distinct from CLL. Major CLL BCR IG stereotypes are very scarce in individuals with or without LC-MBL. This could be taken to imply either that these stereotypes are indeed scarce in the blood of individuals unaffected by CLL or, alternatively, that they might reside within other B cell compartments not assessed in this study. DisclosuresAgathangelidis:Gilead Sciences: Research Funding. Stamatopoulos:Janssen Pharmaceuticals: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Novartis SA: Research Funding. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Research Funding.

IgGeneUsage: differential gene usage in immune repertoires.

Decoding the properties of immune repertoires is key to understanding the adaptive immune response to challenges such as viral infection. One important quantitative property is differential usage of Ig genes between biological conditions. Yet, most analyses for differential Ig gene usage are performed qualitatively or with inadequate statistical methods. Here we introduce IgGeneUsage, a computational tool for the analysis of differential Ig gene usage. IgGeneUsage employs Bayesian inference with hierarchical models to analyze complex gene usage data from high-throughput sequencing experiments of immune repertoires. It quantifies differential Ig gene usage probabilistically and avoids some common problems related to the current practice of null-hypothesis significance testing. IgGeneUsage is an R-package freely available as part of Bioconductor at: https://bioconductor.org/packages/IgGeneUsage/. simo.kitanovski@uni-due.de. Supplementary data are available at Bioinformatics online.

Chystallographic Evidence of Autologous Recognition By a Clonotypic B Cell Receptor in Chronic Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL) leukemic cells express B-cell receptor immunoglobulin (BcR IG) whose signaling is of paramount importance throughout the natural history of the disease. Indeed, signaling pathways downstream of the BcR are constitutively active in all cases of CLL and inhibitors of the Bruton's tyrosine kinase BTK (Ibrutinib) or PI3Kδ (Idelalisib), two downstream signaling effectors, are clinically effective. This functional evidence complements earlier molecular observations supporting antigen drive in CLL ontogeny, including the distinction of CLL into cases with somatically hypermutated BcR IG (M-CLL) that have a significantly better outcome compared to those with unmutated, germline-like receptors (U-CLL). CLL also displays a remarkably skewed BcR IG gene repertoire, culminating in the existence of highly homologous, stereotyped BcR IG in >30% of cases, indicating selection by a limited set of antigenis. A number of potential antigenic elements have been described, being recognized by the monoclonal receptors and able to deliver intracellular signals. More recently, it has been reported that CLL cells are endowed with the apparently unique property of autonomous signaling, since individual CLL-derived BcR IG can promote Ca2+ influx and NF-κB target gene transcription in a reconstituted B cell system upon self-recognition of common BcR-intrinsic epitopes. However, the precise molecular details of such process are unknown. In order to gain insight into the molecular interactions, particularly to further understand the role played by autonomous signaling, we determined the crystal structures of two BcR IG of CLL cases assigned to subset #4. This is a CLL subset expressing stereotyped, G(κ)-switched BcR IG encoded by the IGHV4-34/IGKV2-30 gene combination. Subset #4 accounts for ~1% of all CLL and is the largest within M-CLL, distinctive for a particularly indolent clinical course. BcR IG derived from two subset #4 cases were found to bind autologously via their VH CDR3 loops to a composite surface spanning the variable and constant regions of the heavy chain; the relevant epitope is conserved in all cases belonging to subset #4 and differs from other non-subset #4 BcR IG. This specific self-recognition was identified as dependent on the individual IG gene usage in the BcR, and is functionally relevant as it occurs in solution and leads to intracellular signalling in B cells. Analysis of epitope and paratope mutants revealed that the interactions observed in the crystal structures are mediated by a few critical amino acid residues. Indeed, the distinctively conserved amino acid residues in the VH CDR3 loop of the BcR IG both dictate a specific VH-VK pairing and shape the combining site for autologous recognition. Moreover, the epitope comprises specific amino acids from the CH1 domain that restrict the autologous recognition to IgG molecules. Finally, we found persisting long-lived interaction occurring between subset #4 BcR IGs, thus recalling high affinity receptor-cognate antigen interactions associated with the induction of anergy. This scenario well fits with the anergic phenotype of the subset #4 leukemic cells, and thus provides a biochemical explanation for the indolent clinical course of this subset. In conclusion, though focusing on a particular CLL subset, the structural and biochemical analysis here presented describes a general model for autologous recognition that may epitomize the molecular events leading to the expansion of CLL B lymphocytes at large. It is conceivable that CLL-associated BcR IGs can each bind to a distinct internal epitope with the specific nature of the interaction dictated by diverse factors e.g. VDJ recombination, heavy and light chain pairing, SHM, and isotype switch. The strength and persistence of the autologous recognition can then lead to a specific outcome in the intracellular signaling process, ranging from proliferation to anergy. The structural diversity thus produced in the BcR IG development may be linked to and underlie the heterogeneity characterizing CLL at the biological and clinical level. Disclosures Stamatopoulos: Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding. Ghia:Pharmacyclics: Honoraria; Gilead: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria; Roche: Research Funding; GSK: Research Funding; AbbVie: Honoraria; Celgene: Honoraria; Adaptive Biotechnologies: Consultancy.

Single-Cell and Deep Sequencing of IgG-Switched Macaque B Cells Reveal a Diverse Ig Repertoire following Immunization

The nonhuman primate model is important for preclinical evaluation of prophylactic and therapeutic intervention strategies. The recent description of the rhesus macaque germline Ig loci and establishment of a database of germline gene segments offer improved opportunities to delineate Ig gene usage in the overall B cell repertoire as well as in response to vaccination. We applied 454-pyrosequencing and single-cell RT-PCR of bulk and sorted memory B cells, respectively, to investigate IGHV gene segment expression in rhesus macaques. The two methods gave remarkably concordant results and identified groups of gene segments that are frequently or rarely used. We further examined the VH repertoire of Ag-specific memory B cells induced by immunization with recombinant HIV-1 envelope glycoproteins, an important vaccine component. We demonstrate that HIV-1 envelope glycoprotein immunization activates a highly polyclonal response composed of most of the expressed VH gene segments, illustrating the considerable genetic diversity of responding B cells following vaccination.

Different Pattern of Immunoglobulin Gene Usage by HIV-1 Compared to Non-HIV-1 Antibodies Derived from the Same Infected Subject

A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis.

Rituximab induces sustained reduction of pathogenic B cells in patients with peripheral nervous system autoimmunity

The B cell-depleting IgG1 monoclonal antibody rituximab can persistently suppress disease progression in some patients with autoimmune diseases. However, the mechanism underlying these long-term beneficial effects has remained unclear. Here, we evaluated Ig gene usage in patients with anti-myelin-associated glycoprotein (anti-MAG) neuropathy, an autoimmune disease of the peripheral nervous system that is mediated by IgM autoantibodies binding to MAG antigen. Patients with anti-MAG neuropathy showed substantial clonal expansions of blood IgM memory B cells that recognized MAG antigen. The group of patients showing no clinical improvement after rituximab therapy were distinguished from clinical responders by a higher load of clonal IgM memory B cell expansions before and after therapy, by persistence of clonal expansions despite efficient peripheral B cell depletion, and by a lack of substantial changes in somatic hypermutation frequencies of IgM memory B cells. We infer from these data that the effectiveness of rituximab therapy depends on efficient depletion of noncirculating B cells and is associated with qualitative immunological changes that indicate reconfiguration of B cell memory through sustained reduction of autoreactive clonal expansions. These findings support the continued development of B cell-depleting therapies for autoimmune diseases.

Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1

Human anti-V3 monoclonal antibodies (mAbs) generated from HIV-1 infected individuals display diversity in the range of their cross-neutralization that may be related to their immunogenetic background. The study of the immunoglobulin (Ig) variable region gene usage of heavy chains have shown a preferential usage of the VH5-51 gene segment which was detected in 35% of 51 human anti-V3 mAbs. In contrast, human mAbs against other envelope regions of HIV-1 (anti-Env), including the CD4-binding domain, the CD4-induced epitope, and gp41 preferentially used the VH1-69 gene segment, and none of them used the VH5-51 gene. Furthermore, the usage of the VH4 family by anti-V3 mAbs was restricted to only one gene segment, VH4-59, while the VH3 gene family was used at a significantly lower frequency by all of the analyzed anti-HIV-1 mAbs. Multivariate analysis showed that usage of VH gene segments was significantly different between anti-V3 and anti-Env mAbs, and compared to antibodies from healthy subjects. In addition, the anti-V3 mAbs preferentially used the JH3 and D2-15 gene segments. The preferential usage of selected Ig gene segments and the characteristic pattern of Ig gene usage by anti-V3 mAbs can be related to the conserved structure of the V3 region.

Evidence for preferential Ig gene usage and differential TdT and exonuclease activities in human naïve and memory B cells

Memory B cells and the antibodies they encode are important for protective immunity against infectious pathogens. Characterization of naïve and memory B cell antibody repertoires will elucidate the molecular basis for the generation of antibody diversity in human B cells and the optimization of antibody structures that bind microbial antigens. In this study we aimed to investigate the influence of antigenic selection on the antibody genes of the two CD27 + memory B cell subsets, comparing them with the naïve repertoire in CD27 − cells. We analyzed and compared the Ig heavy chain gene transcripts in three recently defined circulating naïve and memory B cell subsets (CD19 +IgD +CD27 − [naïve], CD19 +IgD +CD27 + [un-class-switched memory] or CD19 +IgD − CD27 + [class-switched memory]) at the single cell level. We found similar biased patterns of variable, diversity and joining heavy chain gene usages in all three groups of cells. CD19 +IgD +CD27 + memory B cells harbored as diverse an antibody gene repertoire as CD19 +IgD −CD27 + memory B cells. Interestingly, CD19 +IgD +CD27 + memory B cells possessed a lower frequency of somatic mutations, a higher incidence of exonuclease activity at the 3′ end of D regions, and a lower frequency of N and P nucleotide additions at both V H–D and D–J H junctions of CDR3 regions compared to CD19 +IgD −CD27 + memory B cells. These data suggest distinct functional mechanisms underlying selection of this unique subset of un-class-switched memory B cells.

The First Dose of aHaemophilus influenzaeType b Conjugate Vaccine Reactivates Memory B Cells: Evidence for Extensive Clonal Selection, Intraclonal Affinity Maturation, and Multiple Isotype Switches to IgA2

AbstractThe Ab response of a healthy adult to the first dose of a Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugate vaccine was studied at the level of Ig gene usage by circulating Ab-secreting cells. Forty-one IgA and 17 IgG mRNA sequences were obtained. The major part of the response was confined to IgA Ab-secreting cells, and 72% of the IgA sequences were derived from the progeny of a single rearranged B cell. These sequences could be arranged in a genealogical tree showing multiple somatic mutations and at least two intraclonal isotype switches to IgA2. Fourteen somatic mutations were shared by this clonal progeny, indicating that extreme clonal selection had occurred early in the clonal development. Taking into account the frequency of somatic mutations and the clone size, it was evident that the responding cell population must have originated from a mutated, highly selected, and expanded population of cells existing before vaccination, i.e., memory B cells. The dominating heavy and light chains of the response were combined in a Fab that bound HibCP. It was shown that the shared heavy and light chain mutations increased the affinity for HibCP considerably, indicating that the clonal selection had been driven by affinity. Pre-existing memory cells in unvaccinated adults may explain several features of Ab responses to polysaccharide vaccines and may play a role in acquiring the ability to respond to pure polysaccharides during infancy.

Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

Antimitochondrial Antibodies in Primary Biliary Cirrhosis

In the last decade, the cloning and biochemical identification of mitochondrial autoantigens in primary biliary cirrhosis (PBC) as members of the 2-oxoacid dehydrogenase complex has greatly advanced the detection of antimitochondrial antibodies (AMA) and the understanding of the immunobiology of the disease. Here, we discuss the methods of detecting AMA and its isotypes, methods of epitope mapping, and using these methods in PBC liver immunohistochemistry and Ig gene usage. Increasing evidence, including the specific association of AMA with PBC, the unique similar but noncross-reactive conformational epitope of the lipoyl domains of the mitochondrial autoantigens, the specific binding of anti-PDC-E2 monoclonal antibodies and human combinatorial antibodies derived from PBC patients to the apical area of bile duct epithelial cells in PBC livers, and Ig gene usage of AMA, suggests that AMA is not an epiphenomenon of the disease but plays a significant role in the pathogenesis of PBC.

Evidence for the overexpression of the VH4-34 (VH4.21) Ig gene segment in the normal adult human peripheral blood B cell repertoire.

The VH4-34 (VH4.21) gene has been repeatedly found to encode monoclonal anti-i/l cold agglutinins and is occasionally used by other autoantibodies with anti-DNA and rheumatoid factor specificity. To understand the basis for the frequent expression of the VH4-34 gene in autoimmunity, we prepared linker-based, amplified C mu and C gamma V gene (cDNA) libraries from the peripheral blood of two healthy adults (PBL-3 and PBL-4). The frequency of VH4 family gene-containing clones was determined with VH family-specific oligonucleotide probes, and by random sequencing we examined the frequency of VH4-34 gene expression among the VH4+ C mu clones. Collectively, these studies suggested that VH4-34 gene expression accounted for 7.8 and 10.3% of all IgM clones in the cDNA libraries of PBL-3 and PBL-4, respectively. In VH4 family-specific libraries prepared from genomic rearranged Ig DNA, the VH4-34 gene was also overrepresented relative to other VH4 family genes. In both the mu-cDNA as well as the rearranged Ig DNA libraries, the VH4-34-expressing clones were 95 to 100% homologous to the germline sequence and contained different CDR3 sequences. By contrast, the VH4-34-expressing C gamma clones showed somatic diversification from germline; in addition, the C gamma clones often had the same CDR3 sequences, suggesting that these clones were derived from either activated B cells or expanded, clonally related B cells. Multiparameter flow cytometric analysis of peripheral blood lymphocytes indicated that 3.2 to 6.2% of mu+CD20+ cells express the VH4-34-related Id 9G4. These current findings are relevant to the interpretation of restricted Ig gene usage reported in monoclonal cold agglutinin disease and other autoimmune disorders.

B-1 Cells in Systemic Autoimmune Responses: IGM+, FCϵRDULLB Cells Are Lost During Chronic Graft-Versus-Host Disease But Not in Murine AIDS or Collagen-Induced Arthritis

The potential role of B-1 cells (i.e. the CD5+ B cell and "sister" B cell subsets) in autoimmunity is controversial. CD5+ B cells have been shown to secrete antibodies of similar specificity as those found in many systemic autoimmune diseases; in addition, increases in CD5+ B cell frequency have been reported in patients suffering from rheumatoid arthritis, Sjögren's syndrome, myasthenia gravis, insulin-dependent diabetes mellitus and Hashimoto's thyroiditis. Whether these increases are due to expansion of B-1 lineage cells in the human or due to activation-induced expression of CD5 by conventional B cells is unclear. In the present study, we used three murine models of systemic autoimmunity: murine acquired immunodeficiency syndrome (MAIDS), chronic graft-versus-host disease (cGvHD), and collagen-induced arthritis (CIA) to determine whether increases in B-1 cell frequency are universally seen in models of autoimmunity which are mechanistically distinct. In contrast to the aforementioned human systemic autoimmune diseases which exhibit an increase in CD5+ B cell frequency, the percentage of CD5+ B cells declined in all three murine models of systemic autoimmune disease. Even though there was a decrease in the frequency of CD5+ B cells there was no change in the actual number of CD5+ B cells. Thus, the apparent decline in CD5+ B cell frequency was due to increases in either T cells, conventional Fc epsilon R+ B cells, or both. The only actual decline in a B cell subset was the loss of IgM+, Fc epsilon Rdull cells in both the spleen and peritoneal cavity of mice undergoing a chronic graft-versus-host reaction. Therefore, our data suggests that expansion of the B-1 subset does not occur as a general feature of murine systemic autoimmune disease. These observations, consistent with previous studies of Ig gene usage in autoreactive antibodies, support the view that expansion and differentiation of the CD5+ B cell subset is not a central event leading to autoantibody production.

Immunoglobulin genes in autoimmunity.

The contribution of V germline genes and somatic mutation as well as the mechanism governing expression of the various V family genes in response to self-antigens are still unknown. Thus, we are still far from understanding the contribution and role of the B cell repertoire in human autoimmunity. Much of our current data on autoantibody gene repertoire are derived from laboratory generated hybridomas or animal model of autoimmune diseases. These may not reflect the human situation. In contrast, very few human autoantibodies with defined specificities have been structurally and genetically analyzed. In the future, meaningful data, perhaps from direct cloning and sequencing of autoantibodies derived from patients with autoimmune diseases, will be necessary to resolve issues in autoantibody repertoire usage. In this article, the prominent features of the human Ig repertoire, the usage of germline genes in autoantibodies, identifications of somatic mutations among autoantibodies and current data supporting either restricted or nonrestricted Ig gene usage and idiotypic expression among autoantibodies in several well-studied autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis are discussed.