F. Eun-Hyung Lee

Diversity, cellular origin and autoreactivity of antibody-secreting cell population expansions in acute systemic lupus erythematosus

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 VH4-34 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.

Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing

Antigen-specific T cells demonstrate several potent effector functions during immune responses. Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but it has been difficult to measure the frequency of cytolytic cells. We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli β-galactosidase in mouse or human target cells, and an Elispot to detect release of β-galactosidase from killed target cells. This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-γ secretion are regulated independently. The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.

Potent High-Affinity Antibodies for Treatment and Prophylaxis of Respiratory Syncytial Virus Derived from B Cells of Infected Patients

Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million. Several of the newly cloned Abs bind to the RSV G protein central conserved motif with very high affinity (Kd 1–24 pM). Two of the Abs were characterized in detail and compared with palivizumab, a humanized mAb against the RSV F protein. Relative to palivizumab, the anti-G Abs showed improved viral neutralization potency in vitro and enhanced reduction of infectious virus in a prophylaxis mouse model. Furthermore, in a mouse model for postinfection treatment, both anti-G Abs were significantly more effective than palivizumab at reducing viral load. The combination of activity in mouse models for both prophylaxis and treatment makes these high-affinity human-derived Abs promising candidates for human clinical testing.

Frequencies of Human Influenza-specific Antibody Secreting Cells or Plasmablasts post Vaccination from Fresh and Frozen Peripheral Blood Mononuclear Cells

The rise in influenza-specific neutralizing antibody levels is proceeded by a burst of antigen-specific antibody secreting cells (ASC) or plasmablasts identified in peripheral blood approximately 5-10 days post immunization. Blood antigen-specific ASC may function as an immune marker of vaccine responses in comparison to the pre- and post-neutralizing titers; however, some have questioned whether there is adequate survival of ASC isolated from peripheral blood after freezing, making multi-center vaccine trials difficult. Here, we demonstrate similar frequencies of influenza-specific ASC from fresh and frozen peripheral blood mononuclear cells (PBMC). Influenza Hemagglutinin (HA) H1, H3, and H7-specific ASC IgG ELISpots frequencies were compared from the same fresh and frozen PBMC 7 days after 2006 Trivalent Influenza Vaccine (TIV) in 10 young healthy subjects. H1-, H3-, and H7-specific IgG ASC spots/10(6) from fresh PBMC on day 7 were 229+/-341, 98+/-90, and 6+/-11 respectively. Total IgG ASC spots/million PBMC pre- and 7-day post-vaccination were 290+/-188 (0.029% PBMC) and 1691+/-836 (0.17% PBMC) respectively. There was no difference in the H1 -H3-, and total specific ASC IgG ELISpot frequencies from the fresh versus frozen PBMC on day 7 (p=0.43, 0.28, 0.28 respectively). These results demonstrate feasibility of testing whether antigen-specific ASC from frozen PBMC are an early biomarker of long-term antibody responses in multi-center vaccine trials.

Human Rhinovirus Induced Cytokine/Chemokine Responses in Human Airway Epithelial and Immune Cells

Infections with human rhinovirus (HRV) are commonly associated with acute upper and lower respiratory tract disease and asthma exacerbations. The role that HRVs play in these diseases suggests it is important to understand host-specific or virus-specific factors that contribute to pathogenesis. Since species A HRVs are often associated with more serious HRV disease than species B HRVs, differences in immune responses they induce should inform disease pathogenesis. To identify species differences in induced responses, we evaluated 3 species A viruses, HRV 25, 31 and 36 and 3 species B viruses, HRV 4, 35 and 48 by exposing human PBMCs to HRV infected Calu-3 cells. To evaluate the potential effect of memory induced by previous HRV infection on study responses, we tested cord blood mononuclear cells that should be HRV naïve. There were HRV-associated increases (significant increase compared to mock-infected cells) for one or more HRVs for IP-10 and IL-15 that was unaffected by addition of PBMCs, for MIP-1α, MIP-1β, IFN-α, and HGF only with addition of PBMCs, and for ENA-78 only without addition of PBMCs. All three species B HRVs induced higher levels, compared to A HRVs, of MIP-1α and MIP-1β with PBMCs and ENA-78 without PBMCs. In contrast, addition of CBMCs had less effect and did not induce MIP-1α, MIP-1β, or IFN-α nor block ENA-78 production. Addition of CBMCs did, however, increase IP-10 levels for HRV 35 and HRV 36 infection. The presence of an effect with PBMCs and no effect with CBMCs for some responses suggest differences between the two types of cells possibly because of the presence of HRV memory responses in PBMCs and not CBMCs or limited response capacity for the immature CBMCs relative to PBMCs. Thus, our results indicate that different HRV strains can induce different patterns of cytokines and chemokines; some of these differences may be due to differences in memory responses induced by past HRV infections, and other differences related to virus factors that can inform disease pathogenesis.

Competitive SWIFT cluster templates enhance detection of aging changes

Clustering‐based algorithms for automated analysis of flow cytometry datasets have achieved more efficient and objective analysis than manual processing. Clustering organizes flow cytometry data into subpopulations with substantially homogenous characteristics but does not directly address the important problem of identifying the salient differences in subpopulations between subjects and groups. Here, we address this problem by augmenting SWIFT—a mixture model based clustering algorithm reported previously. First, we show that SWIFT clustering using a “template” mixture model, in which all subpopulations are represented, identifies small differences in cell numbers per subpopulation between samples. Second, we demonstrate that resolution of inter‐sample differences is increased by “competition” wherein a joint model is formed by combining the mixture model templates obtained from different groups. In the joint model, clusters from individual groups compete for the assignment of cells, sharpening differences between samples, particularly differences representing subpopulation shifts that are masked under clustering with a single template model. The benefit of competition was demonstrated first with a semisynthetic dataset obtained by deliberately shifting a known subpopulation within an actual flow cytometry sample. Single templates correctly identified changes in the number of cells in the subpopulation, but only the competition method detected small changes in median fluorescence. In further validation studies, competition identified a larger number of significantly altered subpopulations between young and elderly subjects. This enrichment was specific, because competition between templates from consensus male and female samples did not improve the detection of age‐related differences. Several changes between the young and elderly identified by SWIFT template competition were consistent with known alterations in the elderly, and additional altered subpopulations were also identified. Alternative algorithms detected far fewer significantly altered clusters. Thus SWIFT template competition is a powerful approach to sharpen comparisons between selected groups in flow cytometry datasets.

Identification of human plasma cells with a lamprey monoclonal antibody

Ab-producing plasma cells (PCs) serve as key participants in countering pathogenic challenges as well as being contributors to autoimmune and malignant disorders. Thus far, only a limited number of PC-specific markers have been identified. The characterization of the unique variable lymphocyte receptor (VLR) Abs that are made by evolutionarily distant jawless vertebrates prompted us to investigate whether VLR Abs could detect novel PC antigens that have not been recognized by conventional Abs. Here, we describe a monoclonal lamprey Ab, VLRB MM3, that was raised against primary multiple myeloma cells. VLRB MM3 recognizes a unique epitope of the CD38 ectoenzyme that is present on plasmablasts and PCs from healthy individuals and on most, but not all, multiple myelomas. Binding by the VLRB MM3 Ab coincides with CD38 dimerization and NAD glycohydrolase activity. Our data demonstrate that the lamprey VLRB MM3 Ab is a unique reagent for the identification of plasmablasts and PCs, with potential applications in the diagnosis and therapeutic intervention of PC or autoimmune disorders.

Extracellular vesicles from bone marrow-derived mesenchymal stromal cells support ex vivo survival of human antibody secreting cells

ABSTRACT Extracellular vesicles (EVs) from bone marrow (BM)-derived mesenchymal stromal cells (BM-MSC) are novel mechanisms of cell-cell communication over short and long distances. BM-MSC have been shown to support human antibody secreting cells (ASC) survival ex vivo, but whether the crosstalk between the MSC-ASC interaction can occur via EVs is not known. Thus, we evaluated the role of EVs in ASC survival and IgG secretion. EVs were isolated from irradiated and non-irradiated primary BM-MSC and were quantified. They were further characterized by electron microscopy (EM) and CD63 and CD81 immuno-gold EM staining. Human ASC were isolated via fluorescence-activated cell sorting (FACS) and cultured ex vivo with the EV fractions, the EV-reduced fractions, or conventional media. IgG Elispots were used to measure the survival and functionality of the ASC. Contents of the EV fractions were evaluated by proteomics. We saw that both irradiated and non-irradiated MSC secretome preparations afforded vesicles of a size consistent with EVs. Both preparations appeared comparable in EM morphology and CD63 and CD81 immuno-gold EM. Both irradiated and non-irradiated EV fractions supported ASC function, at 88% and 90%, respectively, by day 3. In contrast, conventional media maintained only 4% ASC survival by day 3. To identify the specific factors that provided in vitro ASC support, we compared proteomes of the irradiated and non-irradiated EV fractions with conventional media. Pathway analysis of these proteins identified factors involved in the vesicle-mediated delivery of integrin signalling proteins. These findings indicate that BM-MSC EVs provide an effective support system for ASC survival and IgG secretion.

BALDR: a computational pipeline for paired heavy and light chain immunoglobulin reconstruction in single-cell RNA-seq data

B cells play a critical role in the immune response by producing antibodies, which display remarkable diversity. Here we describe a bioinformatic pipeline, BALDR (BCR Assignment of Lineage using De novo Reconstruction) that accurately reconstructs the paired heavy and light chain immunoglobulin gene sequences from Illumina single-cell RNA-seq data. BALDR was accurate for clonotype identification in human and rhesus macaque influenza vaccine and simian immunodeficiency virus vaccine induced vaccine-induced plasmablasts and naïve and antigen-specific memory B cells. BALDR enables matching of clonotype identity with single-cell transcriptional information in B cell lineages and will have broad application in the fields of vaccines, human immunodeficiency virus broadly neutralizing antibody development, and cancer.BALDR is available at https://github.com/BosingerLab/BALDR.

Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus

Graphical Abstract Figure. No caption available. SUMMARY Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5‐ CD11c+ cells (DN2) representing pre‐plasma cells (PC). DN2 cells predominated in African‐American patients with active disease and nephritis, anti‐Smith and anti‐RNA autoantibodies. They expressed a T‐bet transcriptional network; increased Toll‐like receptor‐7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper‐responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin‐21 (IL‐21)‐mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper‐responsiveness to innate stimuli, as well as establishes prominence of extra‐follicular B cell activation in SLE, and identifies therapeutic targets. HIGHLIGHTSAutoreactive CD27‐ IgD‐ CXCR5‐ CD11c+ (DN2) B cells expand in lupus patientsDN2 cells derive from naive cells and are poised to generate plasmablastsDN2 B cells are hyper‐responsive to Toll‐like receptor‐7 signalingThe properties of SLE DN2 B cells stem from distinct transcriptional networks &NA; The role of extrafollicular B cells in human systemic lupus is unknown. Jenks et al. define the main components of this pathway and its prominence in severe disease. Its activation is mediated by hyper‐responsiveness to Toll‐like receptor‐7 and leads to the generation of autoreactive antibody‐secreting plasmablasts.