Faculty Bibliography

B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development.

Background/Purpose: Malondialdehyde (MDA) is a naturally occurring reactive aldehyde that arises during apoptosis or as a consequence of elevated reactive oxygen species and lipid peroxidation. Free MDA can posttranslationally modify lysine residues in a protein carbonylation process that generates neo-epitopes that can be recognized by autoantibodies. Methods: Serum IgG anti-MDA levels were compared in 71 healthy controls, 30 OA, and 283 SLE and 162 RA patients, identified by ACR criteria. IgG anti-MDA was measured by sandwich ELISA using MDA-modified BSA. After flow cytometry sorting of RA synovial memory B cells, and single cell Ab-gene PCR, 114 mAbs were expressed and tested for MDA-reactivity. Analyses used the 2-sided Mann-Whitney test or Spearman correlation. Results: In sera, IgG anti-MDA was significantly increased in SLE (17+/-21 RU/ml, p<0.0001) and RA patients (13+/-11 RU/ml, p<0.0001), compared to controls (5+/-3 RU/ml). In SLE, IgG anti-MDA correlated with disease activity by SELENA-SLEDAI (p<0.0001, R=0.34, n=219). Levels were also significantly higher in SLE patients with active disease (SLEDAI>6, 18.9+/-17.3 RU/ml, p=0.001) than with low disease activity (SLEDAI<6, 11.5+/-16.6). In RA patients, IgG anti-MDA correlated with DAS28-ESR (p<0.0001, R=0.35, n=157). Compared to RA patients with low disease activity (DAS28<3.2, 6+/-3), levels were significantly increased in RA with moderate activity (DAS28 3.2-5.1, 12+/-11 RU/ml, p=0.005) and high activity (DAS28>5.1, 15+/-12 RU/ml, p=0.001). In DMARD naive RA (n=62), IgG anti-MDA also correlated with serum TNFalpha (p=0.002, R=0.39), IL-6 (p=0.03, R=0.27), and CRP levels (p=0.003, R=0.37). In RA synovial mAbs, we identified four clones (3.5%) that recognized MDA-modified epitopes. The most reactive clone, 1276:01F04, showed high specific binding to MDA but was non-reactive with carbamylated or 4-HNE-modifications. Specificity was confirmed in antigen-competition studies with MDA or MDA acetaldehyde (MAA) protein adducts. This mAb also bound MDA-modified human fibrinogen and albumin. No cross-reactivity with citrullinated epitopes was detected, and mAbs with ACPA or RF reactivity did not bind MDA. Notably, 1276:01F04 originated from an IgG1-bearing B cell that was encoded by near germline variable genes (VH4-39, 1 R-mutation in HCDR3; VL1-51, 2 S-mutations in FR1). Conclusion: IgM binding to MDA-adducts may be common in health from birth and are part of the natural antibody pool. Yet, IgG anti-MDA are associated with inflammatory conditions including increased disease activity in autoimmune patients. Importantly, MDA-autoreactive B cells could also be isolated from RA synovium, the site of disease. Further studies are merited to investigate the potential pathogenic properties of IgG anti-MDA B-cells/autoantibodies

Background/Purpose: SLE is a complex multifactorial systemic autoimmune disease, which has been attributed to poorly understood interactions between genetic and environmental factors. Recent reports have begun to elucidate how imbalances within intestinal communities of commensal bacteria may lead to triggering of inflammatory and autoimmune conditions. Our studies are designed to shed light on the interactions of the immune system and the gut microbiome that may contribute to lupus pathogenesis. Methods: We have assembled a cohort of 60 female SLE patients and matched 20 healthy controls, and biobanked blood and stool samples. DNA was then extracted from fecal bacterial samples, and from sorted endogenous IgA-coated and non-coated bacterial fractions. 16S bacterial rRNA gene sequencing was then performed by illumina NGS technology. Fecal and serum total Igs and autoantibodies were measured by ELISA and by multiplex-bead autoantigen assays. Results: Our analyses showed that SLE patients have significantly reduced diversity (i.e., number of different taxa) in their gut microbiomes compared to controls (p=0.038). This dysbiosis was treatment independent, with more marked contractions in patients with high disease activity, based on SLEDAI (p=0.002). The distribution of microbiome taxa was more heterogeneous among SLE patients than healthy individuals (p=0.002). Interestingly, patients with the most active disease commonly displayed expansions of genus and species with putative pathobiont properties, with reciprocal contractions of others associated with protective properties (e.g. R. gnavus (p= 0.001) vs. F. prausnitzii (p= 0.022) and B. uniformis (p=0.016). In immunologic surveys, we found that IgA (the most highly produced Ig isotype in the body), was significantly elevated in SLE patients compared to healthy subjects (p<0.002). Only a limited proportion of bacterial taxa are specifically coated by endogenous intestinal IgA. Yet, SLE patients with high disease activity displayed an increased abundance among IgA-coated taxa of Prevotella copri (p= 0.018), a species which has recently been linked to new-onset RA. In addition, intestinal IgA in SLE patients included high levels of antibodies to lupus autoantigens, with the same IgA autoantibody profiles in the matched sera of individual patients. Conclusion: Our studies document that SLE is associated with a dysbiosis in the gut microbiome with expansions of specific pathobiont bacteria and reciprocal contractions that may contribute to immune dysregulation. This imbalance was more significant in patients with high disease activity. Certain microbial taxa/species are preferentially recognized by the adaptive immune system of SLE patients and coated in vivo by intestinal IgA, and this correlated with elevated overall levels of fecal and serum IgA. Taken together, these data support the hypothesis that the pathogenesis of SLE may arise from imbalances in the gut microbiome and immune recognition of certain bacterial taxa

Background/Purpose: Natural IgM autoantibodies have been proposed to have protective properties, and decreased levels of IgM to phosphorylcholine (PC) in SLE are associated with higher risk of atherosclerotic plaques and cardiovascular events. In the current study we investigated levels of total IgM and IgM anti-PC levels in different SLE subgroups. Methods: Serum IgM levels were compared in 322 population controls and 351 SLE patients meeting ACR criteria. SLE patients were subgrouped based on antibody profiles into antiphospholipid (APS)-like (n=54, with >2 positive tests among anti-CL/beta2GPI; LA), Sjogren's syndrome (SS)-like (n= 63, with >2 positive tests among anti-Ro52/Ro60/SSB), or SLE only with no "secondary syndrome" (n=234). A majority, but not all, of the APS-like and SS-like fulfilled clinical criteria for antiphospholipid syndrome or Sjogren's syndrome. Total IgM levels were determined in the clinical laboratory, and IgM anti-PC measured using PC-BSA by sandwich ELISA. Analyses used the 2-sided Mann-Whitney test or Spearman correlation. Results: IgM anti-PC levels were moderately but significantly decreased in the SLE patients (41.3+/-45.3 RU/ml) compared to matched controls (48.9+/-43.15 RU/ml) (n=316, p=0.002). Similarly, the total IgM levels were only slightly lower in SLE than controls (n=310, 1.22+/-1.1; 1.26+/-0.68 mg/ml, p=0.0002). There was a weak inverse correlation with age and disease duration for IgM anti-PC (p=0.02, R=-0.13; p=0.0003, R=-0.19). Importantly, APS-like patients did not have significantly altered total IgM (1.51+/-1.1 mg/ml) or IgM anti- PC levels (58.7+/-58.7 RU/ml) compared to controls (IgM 1.26+/-0.7 mg/ml; IgM anti-PC 48.7+/-43.2 RU/ml), while the SLE only group had moderately decreased IgM levels (IgM 1.21+/-1.3 mg/ml, p<0.0001; IgM anti-PC 39.5+/-43.0 RU/ml; p=0.002), and the SS-like patients had considerably impaired IgM (IgM 0.96+/-0.6 mg/ml, p<0.0001; IgM anti-PC 23.4+/-24.2 RU/ml, p<0.0001). There were no statistically significant differences based on age, disease duration, female sex, or anti-dsDNA positivity. In all SLE patients, total IgG levels were significantly increased compared to controls, and were highest in the SS-like group. Conclusion: Alterations in IgM levels between SLE subgroups further emphasize the immune heterogeneity of patients diagnosed with SLE. Patients have different clinical manifestations, risk of co-morbidities, and which also have distinct immunological features. Future studies are needed to investigate the clinical relevance of depressed IgM levels, and if the moderate IgM impairment in certain SLE patients reflects underlying immunological differences and/or is due to consumption of natural IgM or others causes resulting in skewing. of the B-cell repertoire

At birth, the human immune system already contains substantial levels of polymeric IgM, that include autoantibodies to neo-epitopes on apoptotic cells (ACs) that are proposed to play homeostatic and anti-inflammatory roles. Yet the biologic origins and developmental regulation of these naturally arising antibodies remain poorly understood. Herein, we report that levels of IgM-antibodies to malondialdehyde (MDA) protein adducts, a common type of in vivo generated oxidative stress-related neoepitope, directly correlate with the relative binding of neonatal-IgM to ACs. Levels of IgM to phosphorylcholine (PC), a natural antibody prevalent in adults, were relatively scant in cord blood, while there was significantly greater relative representation of IgM anti-MDA antibodies in newborns compared to adults. To investigate the potential interrelationships between neonatal IgM with pathogenic IgG-autoantibodies, we studied 103 newborns born to autoimmune mothers with IgG anti-Ro (i.e., 70 with neonatal lupus and 33 without neonatal lupus). In these subjects the mean levels of IgM anti-Ro60 were significantly higher than in the newborns from non-autoimmune mothers. In contrast, levels of IgM anti-MDA in IgG anti-Ro exposed neonates were significantly lower than in neonates from non-autoimmune mothers. The presence or absence of neonatal lupus did not appear to influence the total levels of IgM in the anti-Ro exposed newborns. Taken together, our studies provide evidence that the immune development of the natural IgM-repertoire may be affected, and become imprinted by, the transfer of maternal IgG into the fetus.

Background SLE is an archetypical systemic autoimmune disease,which has been attributed to interactions between genetic andenvironmental factors that are currently not well understood. Yetrecent reports have begun to elucidate how intestinal bacteriainfluence the development of physiologic B-cell/T-cell responses,and can affect the pathogenesis of inflammatory and autoimmuneconditions. Our studies are designed to shed light on the potential roles of the gut microbiome in SLE pathogenesis.Methods We have assembled and characterised a cohort of 60female SLE patients and 20 healthy controls. DNA from theunfractionated bacteria in faecal samples, and from the sortedendogenous IgA-coated and non-coated bacterial fractions, wasthen extracted. 16 S bacterial rRNA genes were then barcodedand amplified, and over 20,000 reads were determined per sample using illumina NGS technology. Faecal and serum total Ig andautoantibodies were measured by ELISA.Results Our analysis showed less microbiome diversity in SLEthan healthy controls (p = 0.002). This dysbiosis was treatmentindependent, with more severe intestinal dysbiosis and decreasedbacterial diversity in patients with high disease activity, based onSLEDAI. In addition, SLE patients had increased representationof certain bacterial families, genus's and species, based on 16 SrRNA assignments of operational taxonomic units (OTUs).Patients with active disease displayed contractions of bacterialtaxa with reported protective properties and reciprocal expansions of taxa with putative pathobiont properties. We alsoassessed IgA, which is the most prevalent antibody isotype madeby the human body, and found evidence of exuberant levels inboth intestinal and blood samples of SLE patients. While only aminority of bacterial taxa are specifically coated by endogenousintestinal IgA, IgA-coated bacteria in SLE patients had differentialrepresentation with recurrent taxa-specific expansion in SLEpatients. Strikingly, Prevotella copri, which has recently beenlinked to new-onset RA, was significantly over-representedamong the IgA-coated taxa only in SLE patients with high diseaseactivity, and was not detected in healthy controls.Conclusion Our studies provide the first evidence that SLE isassociated with gut microbiome dysbiosis with expansions of specific bacterial taxa that may contribute to immune dysregulation.This imbalance was more significant in patients with high diseaseactivity Characterisation of in vivo IgA-coated bacteria demonstrated that certain microbes taxa/species are preferentially recognised by the adaptive immune system of SLE patients, and thesediffer significantly from healthy adults. We are now studyingthese candidate pathobionts in longitudinal studies to addresswhether the microbiome predicts and/or tracks flares

BACKGROUND: Rheumatic heart disease (RHD) and HIV are prevalent diseases in sub-Saharan Africa, but little is known about their potential interrelationships. The objective of this study was to assess the prevalence of protective natural autoantibodies among patients with RHD in Uganda, and to determine whether the levels of these autoantibodies are affected by HIV status. METHODS: Participants were grouped according to RHD and HIV status. The three control groups (R

OBJECTIVE: The presence of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) indicates a breach in immune tolerance. Recent studies indicate that this breach extends to homocitrullination of lysines with the formation of anti-carbamylated protein (anti-CarP) antibodies. We analyzed the clinical and serologic relationships of anti-CarP in 2 RA cohorts. METHODS: Circulating levels of immunoglobulin G anti-CarP antibodies were determined by ELISA in established (Dartmouth-Hitchcock Medical Center) and early (Sherbrooke University Hospital Center) cohorts and evaluated for anticyclic citrullinated peptide antibodies (anti-CCP), specific ACPA, and rheumatoid factor (RF) levels using the Student t test and correlation analysis. RESULTS: We identified elevated anti-CarP antibodies titers in 47.0% of seropositive patients (Dartmouth, n = 164), with relationships to anti-CCP (p < 0.0001) and IgM-RF (p = 0.001). Similarly, 38.2% of seropositive patients from the Sherbrooke cohort (n = 171) had elevated anti-CarP antibodies; titers correlated to anti-CCP (p = 0.01) but not IgM-RF (p = 0.09). A strong correlation with anti-Sa was observed: 47.9% anti-Sa+ patients were anti-CarP antibodies+ versus only 25.4% anti-Sa- in the Sherbrooke cohort (p = 0.0002), and 62.6% anti-Sa+ patients versus 26.9% anti-Sa- were anti-CarP antibodies+ in Dartmouth (p < 0.0001). We found a more variable response for reactivity to citrullinated fibrinogen or to citrullinated peptides from fibrinogen and alpha enolase. CONCLUSION: In 2 North American RA cohorts, we observed a high prevalence of anti-CarP antibody positivity. We also describe a surprising and unexpected association of anti-CarP with anti-Sa antibodies that could not be explained by cross-reactivity. Further, considerable heterogeneity exists between anti-CarP reactivity and other citrullinated peptide reactivity, raising the question of how the pathogenesis of antibody responses for carbamylated proteins and citrullinated proteins may be linked in vivo.

During murine immune development, recurrent B cell clones arise in a predictable fashion. Among these B cells, an archetypical clonotypic set that recognizes phosphorylcholine (PC) antigens and produces anti-PC IgM, first implicated for roles in microbial protection, was later found to become expanded in hyperlipidemic mice and in response to an increased in vivo burden of apoptotic cells. These IgM natural antibodies can enhance clearance of damaged cells and induce intracellular blockade of inflammatory signaling cascades. In clinical populations, raised levels of anti-PC IgM correlate with protection from atherosclerosis and may also downmodulate the severity of autoimmune disease. Human anti-PC-producing clones without hypermutation have been isolated that can similarly discriminate apoptotic from healthy cells. An independent report on unrelated adults has described anti-PC-producing B cells with IgM genes that have conserved CDR3 motifs, similar to stereotypic clonal sets of B cell chronic lymphocytic leukemia. Taken together, emerging evidence suggests that, despite the capacity to form an effectively limitless range of Ig receptors, the human immune system may often recurrently generate lymphocytes expressing structurally convergent B cell receptors with protective and homeostatic roles.

Identifying the germline genes involved in immunoglobulin rearrangements is an essential first step in the analysis of antibody repertoires. Based on our prior work in analysing diverse recombinant viruses, we present IgSCUEAL (Immunoglobulin Subtype Classification Using Evolutionary ALgorithms), a phylogenetic approach to assign V and J regions of immunoglobulin sequences to their corresponding germline alleles, with D regions assigned using a simple pairwise alignment algorithm. We also develop an interactive web application for viewing the results, allowing the user to explore the frequency distribution of sequence assignments and CDR3 region length statistics, which is useful for summarizing repertoires, as well as a detailed viewer of rearrangements and region alignments for individual query sequences. We demonstrate the accuracy and utility of our method compared with sequence similarity-based approaches and other non-phylogenetic model-based approaches, using both simulated data and a set of evaluation datasets of human immunoglobulin heavy chain sequences. IgSCUEAL demonstrates the highest accuracy of V and J assignment amongst existing approaches, even when the reassorted sequence is highly mutated, and can successfully cluster sequences on the basis of shared V/J germline alleles.