Lisa K. Blum

Barcode-enabled sequencing of plasmablast antibody repertoires in rheumatoid arthritis.

A hallmark of rheumatoid arthritis (RA) is the production of autoantibodies, including anti–citrullinated protein antibodies (ACPAs). Nevertheless, the specific targets of these autoantibodies remain incompletely defined. During an immune response, B cells specific for the inciting antigen(s) are activated and differentiate into plasmablasts, which are released into the blood. We undertook this study to sequence the plasmablast antibody repertoire to define the targets of the active immune response in RA.

Mass Cytometry Analysis Shows That a Novel Memory Phenotype B Cell Is Expanded in Multiple Myeloma

Hansmann and colleagues used cytometry by time-of-flight (CyTOF) and next-generation sequencing to detect cancer-associated immune phenotypes in human peripheral blood on a single-cell level, leading to identification of an expanded novel memory B-cell phenotype in multiple myeloma. It would be very beneficial if the status of cancers could be determined from a blood specimen. However, peripheral blood leukocytes are very heterogeneous between individuals, and thus high-resolution technologies are likely required. We used cytometry by time-of-flight and next-generation sequencing to ask whether a plasma cell cancer (multiple myeloma) and related precancerous states had any consistent effect on the peripheral blood mononuclear cell phenotypes of patients. Analysis of peripheral blood samples from 13 cancer patients, 9 precancer patients, and 9 healthy individuals revealed significant differences in the frequencies of the T-cell, B-cell, and natural killer–cell compartments. Most strikingly, we identified a novel B-cell population that normally accounts for 4.0% ± 0.7% (mean ± SD) of total B cells and is up to 13-fold expanded in multiple myeloma patients with active disease. This population expressed markers previously associated with both memory (CD27+) and naïve (CD24loCD38+) phenotypes. Single-cell immunoglobulin gene sequencing showed polyclonality, indicating that these cells are not precursors to the myeloma, and somatic mutations, a characteristic of memory cells. SYK, ERK, and p38 phosphorylation responses, and the fact that most of these cells expressed isotypes other than IgM or IgD, confirmed the memory character of this population, defining it as a novel type of memory B cells. Cancer Immunol Res; 3(6); 650–60. ©2015 AACR.

Elevated IgA Plasmablast Levels in Subjects at Risk of Developing Rheumatoid Arthritis.

The disease process in rheumatoid arthritis (RA) starts years before the clinical diagnosis is made, and elevated levels of disease‐specific autoantibodies can be detected during this period. Early responses to known or novel autoantigens likely drive the eventual production of pathogenic autoimmunity. Importantly, the presence of disease‐specific autoantibodies can identify individuals who are at high risk of developing RA but who do not currently have arthritis. The goal of the current study was to characterize plasmablasts from individuals at risk of developing RA.

IgA Plasmablasts are Elevated in Subjects At Risk for Future Rheumatoid Arthritis

The disease process in rheumatoid arthritis (RA) starts years before the clinical diagnosis is made, and elevated levels of disease‐specific autoantibodies can be detected during this period. Early responses to known or novel autoantigens likely drive the eventual production of pathogenic autoimmunity. Importantly, the presence of disease‐specific autoantibodies can identify individuals who are at high risk of developing RA but who do not currently have arthritis. The goal of the current study was to characterize plasmablasts from individuals at risk of developing RA.

VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans

Individual human VP4- and VP7-binding monoclonal antibodies mediate serotype cross-reactive neutralizing immunity to rotaviruses. Investigating heterotypic immunity to rotavirus There are many serotypes of rotavirus, and currently available vaccines include only one or a handful of different strains. However, these vaccines are generally able to induce cross-reactive immunity that prevents diarrheal disease in young children. To get a better understanding of heterotypic immunity, Nair et al. isolated rotavirus-specific B cells from human intestinal tissue and cloned the antibodies for functional analyses. They discovered that heterotypic immunity was often derived from antibodies targeting the VP5* stalk epitope. Although only a small number of antibodies were studied, these results suggest that vaccines focused on the VP5* region may be better able to induce broadly protective immunity to rotavirus. Human rotaviruses (RVs) are the leading cause of severe diarrhea in young children worldwide. The molecular mechanisms underlying the rapid induction of heterotypic protective immunity to RV, which provides the basis for the efficacy of licensed monovalent RV vaccines, have remained unknown for more than 30 years. We used RV-specific single cell–sorted intestinal B cells from human adults, barcode-based deep sequencing of antibody repertoires, monoclonal antibody expression, and serologic and functional characterization to demonstrate that infection-induced heterotypic immunoglobulins (Igs) primarily directed to VP5*, the stalk region of the RV attachment protein, VP4, are able to mediate heterotypic protective immunity. Heterotypic protective Igs against VP7, the capsid glycoprotein, and VP8*, the cell-binding region of VP4, are also generated after infection; however, our data suggest that homotypic anti-VP7 and non-neutralizing VP8* responses occur more commonly in people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination.

Robust B Cell Responses Predict Rapid Resolution of Lyme Disease

Lyme disease (Borrelia burgdorferi infection) is increasingly recognized as a significant source of morbidity worldwide. Here, we show that blood plasmablasts and CD27− memory B cells are elevated in untreated Lyme disease, with higher plasmablast levels associated with more rapid resolution of clinical symptoms. Stronger serum reactivity to surface proteins and peptides from B. burgdorferi was also associated with faster resolution of clinical symptoms. Through molecular identifier-enabled antibody heavy-chain sequencing of bulk B cells and single-cell paired-chain antibody sequencing of blood plasmablasts, we characterized immunoglobulin gene usage patterns specific to B. burgdorferi infection. Recombinantly expressed antibodies from expanded lineages bound B. burgdorferi antigens, confirming that these clones are driven by the infection. Furthermore, recombinant sequence-derived antibodies were functional, inhibiting growth of B. burgdorferi in vitro. Elevations and clonal expansion of blood plasmablasts were associated with rapid return to health, while poor plasmablast responses were associated with a longer duration of symptoms following treatment. Plasmablasts induced by B. burgdorferi infection showed preferential antibody gene segment usage, while bulk sequencing of total B cells revealed convergent CDR3 motifs specific to B. burgdorferi-infected patients. Our results show that robust plasmablast responses encoding Bb-static antibodies are associated with more rapid resolution of Lyme disease, and these antibodies could provide the basis for next-generation therapeutics for Lyme disease.

Circulating plasmablasts are elevated and produce pathogenic anti-endothelial cell autoantibodies in idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a devastating pulmonary vascular disease in which autoimmune and inflammatory phenomena are implicated. B cells and autoantibodies have been associated with IPAH and identified as potential therapeutic targets. However, the specific populations of B cells involved and their roles in disease pathogenesis are not clearly defined. We aimed to assess the levels of activated B cells (plasmablasts) in IPAH, and to characterize recombinant antibodies derived from these plasmablasts. Blood plasmablasts are elevated in IPAH, remain elevated over time, and produce IgA autoantibodies. Single‐cell sequencing of plasmablasts in IPAH revealed repertoires of affinity‐matured antibodies with small clonal expansions, consistent with an ongoing autoimmune response. Recombinant antibodies representative of these clonal lineages bound known autoantigen targets and displayed an unexpectedly high degree of polyreactivity. Representative IPAH plasmablast recombinant antibodies stimulated human umbilical vein endothelial cells to produce cytokines and overexpress the adhesion molecule ICAM‐1. Together, our results demonstrate an ongoing adaptive autoimmune response involving IgA plasmablasts that produce anti‐endothelial cell autoantibodies in IPAH. These antibodies stimulate endothelial cell production of cytokines and adhesion molecules, which may contribute to disease pathogenesis. These findings suggest a role for mucosally‐driven autoimmunity and autoimmune injury in the pathogenesis of IPAH.

Plasmablast antibody repertoires in elderly influenza vaccine responders exhibit restricted diversity but increased breadth of binding across influenza strains

Seasonal influenza vaccines elicit antibody responses that can prevent infection, but their efficacy is reduced in the elderly. While a subset of elderly individuals can still mount sufficient vaccine-induced antibody responses, little is known about the properties of the vaccine-induced antibody repertoires in elderly as compared to young responders. To gain insights into the effects of aging on influenza vaccine-induced antibody responses, we used flow cytometry and a cell-barcoding method to sequence antibody heavy and light chain gene pairs expressed by individual blood plasmablasts generated in response to influenza vaccination in elderly (aged 70-89) and young (aged 20-29) responders. We found similar blood plasmablast levels in the elderly and young responders seven days post vaccination. Informatics analysis revealed increased clonality, but similar heavy chain V(D)J gene usage in the elderly as compared to young vaccine responders. Although the elderly responders exhibited decreased antibody sequence diversity and fewer consequential mutations relative to young responders, recombinant antibodies from elderly responders bound a broader range of influenza strain HAs. Thus elderly influenza vaccine responders mount plasmablast responses with restricted diversity but with an increased breadth of binding across influenza strains. Our results suggest that the ability to generate plasmablast responses encoding cross-strain binding antibodies likely represents a mechanism important to vaccine responses in the elderly.

Affinity Maturation Drives Epitope Spreading and Generation of Proinflammatory Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is characterized by the presence of anti–citrullinated protein antibodies (ACPAs); nevertheless, the origin, specificity, and functional properties of ACPAs remain poorly understood. The aim of this study was to characterize the evolution of ACPAs by sequencing the plasmablast antibody repertoire at serial time points in patients with established RA.