Advances in Human B Cell Phenotypic Profiling

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 regulation (“Big Biology”), necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, 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

Two Major Autoantibody Clusters in Systemic Lupus Erythematosus

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

Comparison of LIPS mixture test with the sum of the individual tests for SLE diagnosis.

<p>Antibody titer data from the LIPS mixture test was plotted against the sum of the titer of the individual Ro52, Ro60, La, Sm-D3, RNP-A and RNP-70k autoantibody titers. Each circle represents an individual patient. Using the Spearman rank test, the correlation between the two tests was <i>R</i> = 0.95.</p

Percentage of seropositive patients by ethnicity.

<p><b>C</b>: Caucasian; <b>AA</b>: African American; <b>A</b>: Asian. Significant differences in the frequency of autoantibody titers:</p><p>*White v. African American;</p><p>**African American v Asian.</p

CpG DNA activation and plasma-cell differentiation of CD27(−) naive human B cells

Unmethylated CpG DNA activation of naive CD27(−) B cells has been reported to require B-cell–receptor (BCR) cross-linking. We describe a culture system using CpG DNA with sequential steps for T-cell–independent activation of naive CD19(+)CD27(−) human peripheral blood B cells that induces efficient CD138(+) plasma-cell differentiation. CD27(+) and CD27(−) B cells were cultured in a 3-step system: (1) days 0 to 4: CpG, IL-2/10/15; (2) days 4 to 7: IL-2/6/10/15 and anti-CD40L; (3) days 7 to 10: IL-6/15, IFN-α, hepatocyte growth factor, and hyaluronic acid. Both CD27(+) and CD27(−) B cells up-regulated intracytoplasmic TLR-9 following CpG DNA activation. CD27(−) B-cell activation required cell-cell contact. Both naive and memory B cells progressed to a plasma-cell phenotype: CD19(low)CD20(low)CD27(+)CD38(+)HLA-DR(low). Seventy percent of the CD27(−)-derived CD138(+) cells demonstrated productive V chain rearrangements without somatic mutations, confirming their origin from naive precursors. Plasma cells derived from CD27(+) B cells were primarily IgG(+), while those from CD27(−) B cells were IgM(+). Our results indicate that under certain conditions, naive B cells increase TLR-9 expression and proliferate to CpG DNA stimulation without BCR signaling. In addition to its immunologic significance, this system should be a valuable method to interrogate the antigenic specificity of naive B cells

SLE autoantibody clusters in the pilot cohort.

<p>A colored heat map was used to visualize the autoantibody titers in the SLE patients. Autoantibody titers were transformed to <i>Z</i> scores as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032001#s2" target="_blank">Materials and Methods</a> and color coded as indicated by the scale at right, in which signal intensities from green to black indicate high and low titers, respectively. To segregate the SLE patients into clusters, the relative ratio (RR) of the sum titer of Sm-D3, RNP-A and RNP-70k divided by the sum titer of Ro52, Ro60 and La autoantibodies was calculated for each patient. Patients with a RR≥1 were assigned to the Sm/RNP cluster (top panel) while patients with a RR<1 were assigned to the Ro/La cluster (bottom panel). Patients with a pure Sm/RNP or pure Ro/La phenotype are indicated.</p