To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regu- lation ("Big Biology"), necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identify- ing correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodefi- ciency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort.
by
Geoffrey Hart;
Munir Akkaya;
Asiya Chida;
Chungwen Wei;
Scott Jenks;
Christopher Tipton;
Chenfeng He;
Ben S. Wendel;
Jeff Skinner;
Gunjan Arora;
Kassoum Kayentao;
Aissata Ongoiba;
Ogobara Doumbo;
Boubacar Traore;
David L. Narum;
Ning Jiang;
Peter D. Crompton;
Ignacio Sanz;
Susan K. Pierce
Plasmodium falciparum malaria is a deadly infectious disease in which Abs play a critical role in naturally acquired immunity. However, the specificity and nature of Abs elicited in response to malaria are only partially understood. Autoreactivity and polyreactivity are common features of Ab responses in several infections and were suggested to contribute to effective pathogen-specific Ab responses. In this article, we report on the regulation of B cells expressing the inherently autoreactive VH4-34 H chain (identified by the 9G4 mAb) and 9G4 + plasma IgG in adults and children living in a P. falciparum malaria-endemic area in West Africa. The frequency of 9G4 + peripheral blood CD19 + B cells was similar in United States adults and African adults and children; however, more 9G4 + B cells appeared in classical and atypical memory B cell compartments in African children and adults compared with United States adults. The levels of 9G4 + IgG increased following acute febrile malaria but did not increase with age as humoral immunity is acquired or correlate with protection from acute disease. This was the case, even though a portion of 9G4 + B cells acquired phenotypes of atypical and classical memory B cells and 9G4 + IgG contained equivalent numbers of somatic hypermutations compared with all other VHs, a characteristic of secondary Ab repertoire diversification in response to Ag stimulation. Determining the origin and function of 9G4 + B cells and 9G4 + IgG in malaria may contribute to a better understanding of the varied roles of autoreactivity in infectious diseases.
Acute systemic lupus erythematosus (SLE) courses with surges of antibody-secreting cells (ASCs) whose origin, diversity and contribution to serum autoantibodies remain unknown. Here, deep sequencing, proteomic profiling of autoantibodies and single-cell analysis demonstrated highly diversified ASCs punctuated by clones expressing the variable heavy-chain region V<inf>H</inf>4-34 that produced dominant serum autoantibodies. A fraction of ASC clones contained autoantibodies without mutation, a finding consistent with differentiation outside the germinal centers. A substantial ASC segment was derived from a distinct subset of newly activated naive cells of considerable clonality that persisted in the circulation for several months. Thus, selection of SLE autoreactivities occurred during polyclonal activation, with prolonged recruitment of recently activated naive B cells. Our findings shed light on the pathogenesis of SLE, help explain the benefit of agents that target B cells and should facilitate the design of future therapies.
Systemic lupus erythematosus is a chronic autoimmune disease of complex clinical presentation and etiology and is likely influenced by numerous genetic and environmental factors. While a large number of susceptibility genes have been identified, the production of antibodies against a distinct subset of nuclear proteins remains a primary distinguishing characteristic in disease diagnosis. However, the utility of autoantibody biomarkers for disease sub-classification and grouping remains elusive, in part, because of the difficulty in large scale profiling using a uniform, quantitative platform. In the present study serological profiles of several known SLE antigens, including Sm-D3, RNP-A, RNP-70k, Ro52, Ro60, and La, as well as other cytokine and neuronal antigens were obtained using the luciferase immunoprecipitation systems (LIPS) approach. The resulting autoantibody profiles revealed that 88% of a pilot cohort and 98% of a second independent cohort segregated into one of two distinct clusters defined by autoantibodies against Sm/anti-RNP or Ro/La autoantigens, proteins often involved in RNA binding activities. The Sm/RNP cluster was associated with a higher prevalence of serositis in comparison to the Ro/La cluster (P = 0.0022). However, from the available clinical information, no other clinical characteristics were associated with either cluster. In contrast, evaluation of autoantibodies on an individual basis revealed an association between anti-Sm (P = 0.006), RNP-A (P = 0.018) and RNP-70k (P = 0.010) autoantibodies and mucocutaneous symptoms and between anti-RNP-70k and musculoskeletal manifestations (P = 0.059). Serologically active, but clinically quiescent disease also had a higher prevalence of anti-IFN-α autoantibodies. Based on our findings that most SLE patients belong to either a Sm/RNP or Ro/La autoantigen cluster, these results suggest the possibility that alterations in RNA-RNA-binding protein interactions may play a critical role in triggering and/or the pathogenesis of SLE.
In preclinical studies of fructose-induced NAFLD, endotoxin appears to play an important role. We retrospectively examined samples from three pediatric cohorts (1) to investigate whether endotoxemia is associated with the presence of hepatic steatosis; (2) to evaluate postprandial endotoxin levels in response to fructose beverage in an acute 24-hour feeding challenge, and (3) to determine the change of fasting endotoxin amounts in a 4-week randomized controlled trial comparing fructose to glucose beverages in NAFLD. We found that adolescents with hepatic steatosis had elevated endotoxin levels compared to obese controls and that the endotoxin level correlated with insulin resistance and several inflammatory cytokines. In a 24-hour feeding study, endotoxin levels in NAFLD adolescents increased after fructose beverages (consumed with meals) as compared to healthy children. Similarly, endotoxin was significantly increased after adolescents consumed fructose beverages for 2 weeks and remained high although not significantly at 4 weeks. In conclusion, these data provide support for the concept of low level endotoxemia contributing to pediatric NAFLD and the possible role of fructose in this process. Further studies are needed to determine if manipulation of the microbiome or other methods of endotoxin reduction would be useful as a therapy for pediatric NAFLD.
A discovery study was carried out where serum samples from 22 systemic lupus erythematosus (SLE) patients and matched healthy controls were hybridized to antibody-coated glass slide arrays that interrogated the level of 274 human proteins. On the basis of these screens, 48 proteins were selected for ELISA-based validation in an independent cohort of 28 SLE patients. Whereas AXL, ferritin, and sTNFRII were significantly elevated in patients with active lupus nephritis (LN) relative to SLE patients who were quiescent, other molecules such as OPN, sTNFRI, sTNFRII, IGFBP2, SIGLEC5, FAS, and MMP10 exhibited the capacity to distinguish SLE from healthy controls with ROC AUC exceeding 90%, all with p < 0.001 significance. These serum markers were next tested in a cohort of 45 LN patients, where serum was obtained at the time of renal biopsy. In these patients, sTNFRII exhibited the strongest correlation with eGFR (r = −0.50, p = 0.0014) and serum creatinine (r = 0.57, p = 0.0001), although AXL, FAS, and IGFBP2 also correlated with these clinical measures of renal function. When concurrent renal biopsies from these patients were examined, serum FAS, IGFBP2, and TNFRII showed significant positive correlations with renal pathology activity index, while sTNFRII displayed the highest correlation with concurrently scored renal pathology chronicity index (r = 0.57, p = 0.001). Finally, in a longitudinal cohort of seven SLE patients examined at ∼3 month intervals, AXL, ICAM-1, IGFBP2, SIGLEC5, sTNFRII, and VCAM-1 demonstrated the ability to track with concurrent disease flare, with significant subject to subject variation. In summary, serum proteins have the capacity to identify patients with active nephritis, flares, and renal pathology activity or chronicity changes, although larger longitudinal cohort studies are warranted.
The development of endemic Burkitt's lymphoma (eBL) is closely associated with Epstein-Barr virus (EBV) infection and holoendemic malaria infections. The role of EBV in the development of malignancy has been studied in depth, but there is still little known about the mechanisms by which malaria affects Burkitt's lymphomagenesis. Activation induced cytidine deaminase (AID) expression is necessary for the introduction of c-myc translocations that are characteristic of BL, but a link between AID and EBV or malaria is unclear. To determine whether frequency of malaria exposure leads to increased AID expression in peripheral blood mononuclear cells (PBMC) we examined two cohorts of children in western Kenya with endemic and sporadic malaria transmission dynamics. High frequency of malaria exposure led to increased expression of AID, which coincided with decreases in the IgM+ memory B cells. In the children from the malaria endemic region, the presence of a detectible EBV viral load was associated with higher AID expression compared to children with undetectable EBV, but this effect was not seen in children with sporadic exposure to malaria. This study demonstrates that intensity of malaria transmission correlates with AID expression levels in the presence of EBV suggesting that malaria and EBV infection have a synergistic effect on the development of c-myc translocations and BL.
Many hepatic functions including lipid metabolism, drug metabolism, and inflammatory responses are regulated in a sex-specific manner due to distinct patterns of hepatic gene expression between males and females. Regulation for the majority of these genes is under control of Nuclear Receptors (NRs). Retinoid X Receptor alpha (RXRα) is an obligate partner for multiple NRs and considered a master regulator of hepatic gene expression, yet the full extent of RXRα chromatin binding in male and female livers is unclear. ChIP-Seq analysis of RXRα and RNA Polymerase2 (Pol2) binding was performed livers of both genders and combined with microarray analysis. Mice were gavage-fed with the RXR ligand LG268 for 5 days (30 mg/kg/day) and RXRα-binding and RNA levels were determined by ChIP-qPCR and qPCR, respectively. ChIP-Seq revealed 47,845 (male) and 46,877 (female) RXRα binding sites (BS), associated with ~12,700 unique genes in livers of both genders, with 91% shared between sexes. RXRα-binding showed significant enrichment for 2227 and 1498 unique genes in male and female livers, respectively. Correlating RXRα binding strength with Pol2-binding revealed 44 genes being male-dominant and 43 female-dominant, many previously unknown to be sexually-dimorphic. Surprisingly, genes fundamental to lipid metabolism, including Scd1, Fasn, Elovl6, and Pnpla3-implicated in Fatty Liver Disease pathogenesis, were predominant in females. RXRα activation using LG268 confirmed RXRα-binding was 2–3 fold increased in female livers at multiple newly identified RXRα BS including for Pnpla3 and Elovl6, with corresponding ~10-fold and ~2-fold increases in Pnpla3 and Elovl6 RNA respectively in LG268-treated female livers, supporting a role for RXRα regulation of sexually-dimorphic responses for these genes. RXRα appears to be one of the most widely distributed transcriptional regulators in mouse liver and is engaged in determining sexually-dimorphic expression of key lipid-processing genes, suggesting novel gender- and gene-specific responses to NR-based treatments for lipid-related liver diseases.
by
Silvia Portugal;
Christopher M. Tipton;
Haewon Sohn;
Younoussou Kone;
Jing Wang;
Shanping Li;
Jeff Skinner;
Kimmo Virtaneva;
Daniel E Sturdevant;
Stephen F Porcella;
Ogobara K Doumbo;
Safiatou Doumbo;
Kassoum Kayentao;
Aissata Ongoiba;
Boubacar Traore;
Ignacio Sanz;
Susan K Pierce;
Peter D Crompton
Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity.