A nanowell platform to identify, sort and expand high antibody-producing cells.

Increased use of therapeutic monoclonal antibodies and the relatively high manufacturing costs fuel the need for more efficient production methods. Here we introduce a novel, fast, robust, and safe isolation platform for screening and isolating antibody-producing cell lines using a nanowell chip and an innovative single-cell isolation method. An anti-Her2 antibody producing CHO cell pool was used as a model. The platform; (1) Assures the single-cell origin of the production clone, (2) Detects the antibody production of individual cells and (3) Isolates and expands the individual cells based on their antibody production. Using the nanowell platform we demonstrated an 1.8-4.5 increase in anti-Her2 production by CHO cells that were screened and isolated with the nanowell platform compared to CHO cells that were not screened. This increase was also shown in Fed-Batch cultures where selected high production clones showed titers of 19-100 mg/L on harvest day, while the low producer cells did not show any detectable anti-Her2 IgG production. The screening of thousands of single cells is performed under sterile conditions and the individual cells were cultured in buffers and reagents without animal components. The time required from seeding a single cell and measuring the antibody production to fully expanded clones with increased Her-2 production was 4-6 weeks.

Nasal polyp antibody-secreting cells display proliferation signature in aspirin-exacerbated respiratory disease.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) causes nasal obstruction and olfactory dysfunction. Aspirin-exacerbated respiratory disease (AERD) is the triad of CRSwNP, asthma, and respiratory reactions to COX-1 inhibitors. Patients with AERD have elevated nasal IL-5 levels and high numbers of antibody-secreting cells (ASCs), including plasma cells and plasmablasts, in their polyp tissue; in addition, their nasal polyp (NP) IgE levels are correlated with disease severity and recurrence of nasal polyposis. OBJECTIVE: We sought to explore differences in the transcriptomic profile, activation markers, and IL-5Rα expression and function of NP ASCs from patients with AERD and CRSwNP. METHODS: NP tissue was collected from patients with AERD and CRSwNP and digested into single-cell suspensions. NP cells were analyzed for protein expression by mass cytometry. For IL-5Rα functional studies, plasma cells were purified and cultured in vitro with or without IL-5 and analyzed by bulk RNA sequencing. RESULTS: Compared with polyp tissue from patients with CRSwNP, polyp tissue from patients with AERD contained significantly more ASCs and had increased ASC expression of IL-5Rα. ASCs from patients with AERD expressed higher protein levels of B-cell activation and regulatory markers (CD40, CD19, CD32, and CD38) and the proliferation marker Ki-67. ASCs from patients with AERD also expressed more IL5RA, IGHE, and cell cycle- and proliferation-related transcripts (CCND2, MKI67, CDC25A, and CDC25B) than did ASCs from patients with CRSwNP. Stimulation of plasma cells from patients with AERD with IL-5 induced key cell cycle genes (CCND2 and PTP4A3), whereas IL-5 stimulation of ASCs from patients with CRSwNP induced few transcriptomic changes. CONCLUSION: NP tissue ASCs from patients with AERD express higher levels of functional IL-5Rα and markers associated with cell cycling and proliferation than do ASCs from patients with aspirin-tolerant CRSwNP.

Understanding heterogeneity of human bone marrow plasma cell maturation and survival pathways by single-cell analyses.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASCs) to long-lived plasma cells (LLPCs). We provide single-cell transcriptional resolution of 17,347 BM ASCs from five healthy adults. Fifteen clusters are identified ranging from newly minted ASCs (cluster 1) expressing MKI67 and high major histocompatibility complex (MHC) class II that progress to late clusters 5-8 through intermediate clusters 2-4. Additional ASC clusters include the following: immunoglobulin (Ig) M predominant (likely of extra-follicular origin), interferon responsive, and high mitochondrial activity. Late ASCs are distinguished by G2M checkpoints, mammalian target of rapamycin (mTOR) signaling, distinct metabolic pathways, CD38 expression, utilization of tumor necrosis factor (TNF)-receptor superfamily members, and two distinct maturation pathways involving TNF signaling through nuclear factor κB (NF-κB). This study provides a single-cell atlas and molecular roadmap of LLPC maturation trajectories essential in the BM microniche. Altogether, understanding BM ASC heterogeneity in health and disease enables development of new strategies to enhance protective ASCs and to deplete pathogenic ones.

Selective emergence of antibody-secreting cells in the multiple sclerosis brain.

BACKGROUND: Although distinct brain-homing B cells have been identified in multiple sclerosis (MS), it is unknown how these further evolve to contribute to local pathology. We explored B-cell maturation in the central nervous system (CNS) of MS patients and determined their association with immunoglobulin (Ig) production, T-cell presence, and lesion formation. METHODS: Ex vivo flow cytometry was performed on post-mortem blood, cerebrospinal fluid (CSF), meninges and white matter from 28 MS and 10 control brain donors to characterize B cells and antibody-secreting cells (ASCs). MS brain tissue sections were analysed with immunostainings and microarrays. IgG index and CSF oligoclonal bands were measured with nephelometry, isoelectric focusing, and immunoblotting. Blood-derived B cells were cocultured under T follicular helper-like conditions to evaluate their ASC-differentiating capacity in vitro. FINDINGS: ASC versus B-cell ratios were increased in post-mortem CNS compartments of MS but not control donors. Local presence of ASCs associated with a mature CD45low phenotype, focal MS lesional activity, lesional Ig gene expression, and CSF IgG levels as well as clonality. In vitro B-cell maturation into ASCs did not differ between MS and control donors. Notably, lesional CD4+ memory T cells positively correlated with ASC presence, reflected by local interplay with T cells. INTERPRETATION: These findings provide evidence that local B cells at least in late-stage MS preferentially mature into ASCs, which are largely responsible for intrathecal and local Ig production. This is especially seen in active MS white matter lesions and likely depends on the interaction with CD4+ memory T cells. FUNDING: Stichting MS Research (19-1057 MS; 20-490f MS), National MS Fonds (OZ2018-003).

Integrated Single-Cell (Phospho-)Protein and RNA Detection Uncovers Phenotypic Characteristics and Active Signal Transduction of Human Antibody-Secreting Cells.

Single-cell technologies are currently widely applied to obtain a deeper understanding of the phenotype of single-cells in heterogenous mixtures. However, integrated multilayer approaches including simultaneous detection of mRNA, protein expression, and intracellular phospho-proteins are still challenging. Here, we combined an adapted method to in vitro-differentiate peripheral B-cells into antibody-secreting cells (ASCs) (i.e., plasmablasts and plasma cells) with integrated multi-omic single-cell sequencing technologies to detect and quantify immunoglobulin subclass-specific surface markers, transcriptional profiles, and signaling transduction pathway components. Using a common set of surface proteins, we integrated two multimodal datasets to combine mRNA, protein expression, and phospho-protein detection in one integrated dataset. Next, we tested whether ASCs that only seem to differ in its ability to secrete different IgM, IgA, or IgG antibodies exhibit other differences that characterize these different ASCs. Our approach detected differential expression of plasmablast and plasma cell markers, homing receptors, and TNF receptors. In addition, differential sensitivity was observed for the different cytokine stimulations that were applied during in vitro differentiation. For example, IgM ASCs were more sensitive to IL-15, while IgG ASC responded more to IL-6 and IFN addition. Furthermore, tonic BCR activity was detected in IgA and IgM ASCs, while IgG ASC exhibited active BCR-independent SYK activity and NF-κB and mTOR signaling. We confirmed these findings using flow cytometry and small molecules inhibitors, demonstrating the importance of SYK, NF-κB, and mTOR activity for plasmablast/plasma cell differentiation/survival and/or IgG secretion. Taken together, our integrated multi-omics approach allowed high-resolution phenotypic characterization of single cells in a heterogenous sample of in vitro-differentiated human ASCs. Our strategy is expected to further our understanding of human ASCs in healthy and diseased samples and provide a valuable tool to identify novel biomarkers and potential drug targets.

Association of Circulating Antibody-Secreting Cell Maturity With Disease Features in Primary Sjögren's Syndrome.

OBJECTIVE: B cell hyperactivity plays an important role in primary Sjögren's syndrome (SS). We undertook this study to better understand the B cell effector branch, namely antibody-secreting cells (ASCs) in primary SS, and to examine the quantity, maturity, and inflammatory properties of ASCs in primary SS patients. METHODS: Circulating ASCs, defined as CD3-CD14-CD27+CD38++ cells, from 21 primary SS patients and 10 healthy controls were assessed using spectral flow cytometry. Expression levels of relevant ASC markers relating to maturity, survival, and inflammatory status were analyzed using a t-distributed stochastic neighbor embedding approach. Correlation of ASC properties with primary SS disease parameters was assessed. RESULTS: ASCs were more abundant in peripheral blood from primary SS patients than from healthy controls (mean ± SD 3.1 ± 5.1 cells/µl versus 1.1 ± 1.0 cells/µl, respectively; P = 0.048) and displayed a more mature phenotype (mean ± SD CD19- ASCs 0.37 ± 1.21 cells/µl versus 0.06 ± 0.11 cells/µl, respectively; P = 0.005). An inflammatory CXCR3+ phenotype of ASCs correlated positively with our newly developed ASC maturity index (r = 0.568, P = 0.007) but correlated negatively with antiinflammatory interleukin-10 expression (r = -0.769, P < 0.001). ASCs with a higher maturity index also demonstrated higher levels of the pro-survival protein myeloid cell leukemia 1 (r = 0.567, P = 0.007). Frequency and/or maturity of ASCs correlated with several primary SS disease parameters, such as antinuclear antibody and anti-La/SSB titers, salivary gland focus scores, and ocular staining scores. CONCLUSION: Quantity and maturity of ASCs in primary SS patients are increased and correlate with disease parameters. A higher maturity index of ASCs marks a pro-survival and proinflammatory phenotype. Altogether, B cell hyperactivity in primary SS extends to the peripheral ASC compartment, raising potential for ASCs as future biomarkers or targets for primary SS treatment.

Extrafollicular IgD+ B cells generate IgE antibody secreting cells in the nasal mucosa.

Increased IgE is a typical feature of allergic rhinitis. Local class-switch recombination has been intimated but B cell precursors and mechanisms remain elusive. Here we describe the dynamics underlying the generation of IgE-antibody secreting cells (ASC) in human nasal polyps (NP), mucosal tissues rich in ASC without germinal centers (GC). Using VH next generation sequencing, we identified an extrafollicular (EF) mucosal IgD+ naïve-like intermediate B cell population with high connectivity to the mucosal IgE ASC. Mucosal IgD+ B cells, express germline epsilon transcripts and predominantly co-express IgM. However, a small but significant fraction co-express IgG or IgA instead which also show connectivity to ASC IgE. Phenotypically, NP IgD+ B cells display an activated profile and molecular evidence of BCR engagement. Transcriptionally, mucosal IgD+ B cells reveal an intermediate profile between naïve B cells and ASC. Single cell IgE ASC analysis demonstrates lower mutational frequencies relative to IgG, IgA, and IgD ASC consistent with IgE ASC derivation from mucosal IgD+ B cell with low mutational load. In conclusion, we describe a novel mechanism of GC-independent, extrafollicular IgE ASC formation at the nasal mucosa whereby activated IgD+ naïve B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.

Molecular Signatures of Kidney Antibody-Secreting Cells in Lupus Patients With Active Nephritis Upon Immunosuppressive Therapy.

OBJECTIVE: This study was undertaken to characterize kidney and urine antibody-secreting cells (ASCs) from patients with active lupus nephritis, before and after induction therapy. METHODS: We included patients with biopsy-proven active lupus nephritis and performed anti-CD138 staining of kidney biopsy samples to visualize ASCs. We performed single-cell gene expression profiling on sorted ASCs from fresh biopsy samples using multiplex reverse transcriptase-polymerase chain reaction. We used a gene set that allowed for the study of ASC maturation from plasmablasts to long-lived plasma cells. We quantified urine ASCs from untreated patients with lupus nephritis at diagnosis and after 6 months of prospective follow-up during induction therapy. RESULTS: The number of kidney CD138+ ASCs in 46 untreated patients with lupus nephritis was correlated with a low estimated glomerular filtration rate and with tubulointerstitial damage. Most kidney ASCs from 3 untreated patients had a plasmablast molecular signature; in contrast, in 4 patients with refractory lupus nephritis, the kidney ASCs were mainly long-lived plasma cells, representing an ASC transcriptional profile similar to that in the bone marrow of 2 healthy donors. Some urine ASCs with a plasmablast signature were detected in patients with untreated active lupus nephritis. The presence of urine ASCs at 6 months was associated with treatment failure. CONCLUSION: Our results suggest potential for ASC-directed therapy in refractory lupus nephritis.

Single-Cell Technologies for the Study of Antibody-Secreting Cells.

Antibody-secreting cells (ASC), plasmablasts and plasma cells, are terminally differentiated B cells responsible for large-scale production and secretion of antibodies. ASC are derived from activated B cells, which may differentiate extrafollicularly or form germinal center (GC) reactions within secondary lymphoid organs. ASC therefore consist of short-lived, poorly matured plasmablasts that generally secrete lower-affinity antibodies, or long-lived, highly matured plasma cells that generally secrete higher-affinity antibodies. The ASC population is responsible for producing an immediate humoral B cell response, the polyclonal antibody repertoire, as well as in parallel building effective humoral memory and immunity, or potentially driving pathology in the case of autoimmunity. ASC are phenotypically and transcriptionally distinct from other B cells and further distinguishable by morphology, varied lifespans, and anatomical localization. Single cell analyses are required to interrogate the functional and transcriptional diversity of ASC and their secreted antibody repertoire and understand the contribution of individual ASC responses to the polyclonal humoral response. Here we summarize the current and emerging functional and molecular techniques for high-throughput characterization of ASC with single cell resolution, including flow and mass cytometry, spot-based and microfluidic-based assays, focusing on functional approaches of the secreted antibodies: specificity, affinity, and secretion rate.

Efficient homing of antibody-secreting cells to the bone marrow requires RNA-binding protein ZFP36L1.

Cell migration relies on coordinated activity of chemotactic and guidance receptors. Here, we report a specific role for the RNA-binding protein ZFP36L1 in limiting the abundance of molecules involved in the homing of antibody-secreting cells (ASCs) to the bone marrow (BM). In the absence of ZFP36L1, ASCs build up in the spleen and the liver and show diminished accumulation in the BM. ZFP36L1 facilitates migration by directly regulating G protein-coupled receptor kinase 2 (GRK2) and the integrin chains α4 and ß1 in splenic ASCs. Expression of CXCR4 and of the integrins α4 and ß1 is differentially regulated on ASCs produced at the early and late stages of the immune response. Consequently, deletion of the Zfp36l1 gene has a stronger effect on BM accumulation of high-affinity ASCs formed late in the response. Thus, ZFP36L1 is an integral part of the regulatory network controlling gene expression during ASC homing.

Detection of allergen-specific antibody-secreting cells in dogs by ELISPOT.

Current laboratory tests are unable to distinguish healthy from allergic dogs. Unlike serum antibody responses, circulating antibody-secreting cells (ASC) are temporarily induced after each contact with the antigen. These ASC can be identified using ELISPOT and the observation of allergen-specific ASC might correlate with the causative allergens in dogs with an allergic dermatitis. In this study, blood was sampled from six privately-owned allergic dogs and six non-allergic laboratory beagles to determine the frequency of circulating allergen-specific ASC for common allergens. Blood IgE+, IgA + and IgG + cells were magnetically isolated to determine the number of allergen-specific ASC with ELISPOT for Dermatophagoides farinae, Dermatophagoides pteronyssinus, Alternaria alternata, birch, timothy grass, wheat, cow's milk, bovine, chicken and lamb meat. For IgA and IgG, allergen-specific spots were observed, however for IgE, no spots were detected for any of the allergens. ELISPOT could not differentiate allergic from non-allergic dogs. When the responses to the different allergens were compared, more IgA ASC for D. pteronyssinus were observed compared to some of the other allergens which was statistically significant for the non-allergic dogs and approached significance in the allergic dogs. These findings indicate that ELISPOT can be used to identify circulating allergen-specific IgA- and IgG-secreting cells. The technique did however not detect allergen-specific IgE ASC and was unable to distinguish allergic from non-allergic dogs. Only a small number of studies have studied allergen-specific IgA in dogs. The finding that dogs have higher numbers of D. pteronyssinus-specific IgA ASC points out that apart from IgE and IgG, it might be interesting to include IgA measurements for certain allergens to analyse the complete spectrum of both the protective and pro-allergic antibody responses.

Antibody-secreting cell destiny emerges during the initial stages of B-cell activation.

Upon stimulation, B cells assume heterogeneous cell fates, with only a fraction differentiating into antibody-secreting cells (ASC). Here we investigate B cell fate programming and heterogeneity during ASC differentiation using T cell-independent models. We find that maximal ASC induction requires at least eight cell divisions in vivo, with BLIMP-1 being required for differentiation at division eight. Single cell RNA-sequencing of activated B cells and construction of differentiation trajectories reveal an early cell fate bifurcation. The ASC-destined branch requires induction of IRF4, MYC-target genes, and oxidative phosphorylation, with the loss of CD62L expression serving as a potential early marker of ASC fate commitment. Meanwhile, the non-ASC branch expresses an inflammatory signature, and maintains B cell fate programming. Finally, ASC can be further subseted based on their differential responses to ER-stress, indicating multiple development branch points. Our data thus define the cell division kinetics of B cell differentiation in vivo, and identify the molecular trajectories of B cell fate and ASC formation.

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.

Lentiviral-mediated delivery of classical swine fever virus Erns gene into porcine kidney-15 cells for production of recombinant ELISA diagnostic antigen.

Classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family causes contagious fatal disease in swine. Antibodies against E2, Erns and NS3 proteins of virus can be detected in infected animals. Development of an ELISA coating antigen to improve the sensitivity of detecting Erns-specific antibodies in pig sera is always desirable for diagnosis as well as for differentiation of infected from vaccinated animals. In present study, a lentivirus-based gene delivery system was used to develop a stable PK-15 cell line expressing Erns (PK-Erns) for production of diagnostic antigen. The Lenti-Erns virus was purified from the supernatant of co-transfected 293LTV cells and used to transduce PK-15 cells. The homogenous PK-Erns cell line was produced by single cell cloning by monitoring eGFP expression. The Erns gene in the genomic DNA and RNA transcripts in total RNA isolated from PK-Erns cells were detected by PCR and RT-PCR, respectively. Expression of 45 kDa Erns glycoprotein was detected in western blot using CSFV-specific hyperimmune sera. The use of PK-Erns cell lysate as antigen in serial dilution and single dilution ELISAs with known positive and negative pig sera was investigated. The PK-Erns ELISA revealed sensitivity equivalent to commercial HerdChek ELISA kit. The sensitivity, specificity and accuracy of the PK-Erns ELISA was 95%, 100% and 96.66%, respectively compared to ELISA using purified CSFV as coating antigen. When field pig sera (n = 69) were tested in PK-Erns ELISA, a significant correlation between the titers from serial dilution and single dilution ELISA was observed. This indicated that PK-Erns cell line can serve as continuous source of ELISA diagnostic antigen for detection of CSFV-specific antibodies in pig sera.

Isolation of Antigen-Specific, Antibody-Secreting Cells Using a Chip-Based Immunospot Array.

Antigen-specific monoclonal antibodies are useful tools to detect very small amounts of antigenic materials and are applicable for antibody therapeutics. To produce mouse monoclonal antibodies, a hybridoma between B lymphocytes and myeloma cells is used to produce antigen-specific monoclonal antibodies. However, a good hybridoma system is not available to obtain human monoclonal antibodies. To produce antigen-specific human monoclonal antibodies, transformation of B lymphocytes with Epstein-Barr viruses or a phage-display system is used. Here, we describe the screening of antigen-specific, antibody-secreting cells using microwell array chips to obtain antigen-specific human monoclonal antibodies. The system can be applied to screen antigen-specific, antibody-secreting cells from any animal species.

CD19-positive antibody-secreting cells provide immune memory.

Long-lived antibody-secreting cells (ASCs) are critical for the maintenance of humoral immunity through the continued production of antibodies specific for previously encountered pathogen or vaccine antigens. Recent reports describing humoral immune memory have suggested the importance of long-lived CD19- bone marrow (BM) ASCs, which secrete antibodies recognizing previously encountered vaccine antigens. However, these reports do not agree upon the unique contribution of the CD19+ BM ASC subset toward humoral immunity. Here, we found both CD19+ and negative ASCs from human BM were similar in functional capacity to react to a number of vaccine antigens via ELISpot assays. The CD19+ cells were the predominant ASC population found in lymphoid tissues, and unlike the CD19- ASCs, which were found only in spleen and BM, the CD19+ ASCs were found in tonsil and blood. CD19+ ASCs from the BM, spleen, and tonsil were capable of recognizing polio vaccine antigens, indicating the CD19+ ASC cells play a novel role in long-lasting immune defense. Comparative gene expression analysis indicated CD19+ and negative BM ASCs differed significantly by only 14 distinct messenger RNAs and exhibited similar gene expression for cell cycle, autophagy, and apoptosis control necessary for long life. In addition, we show identical CDR-H3 sequences found on both BM ASC subsets, indicating a shared developmental path. Together, these results provide novel insight for the distribution, function, genetic regulation, and development of long-lived ASCs and may not only impact improved cell therapies but also enhance strategies for vaccine development.

Factors of the bone marrow microniche that support human plasma cell survival and immunoglobulin secretion.

Human antibody-secreting cells (ASC) in peripheral blood are found after vaccination or infection but rapidly apoptose unless they migrate to the bone marrow (BM). Yet, elements of the BM microenvironment required to sustain long-lived plasma cells (LLPC) remain elusive. Here, we identify BM factors that maintain human ASC > 50 days in vitro. The critical components of the cell-free in vitro BM mimic consist of products from primary BM mesenchymal stromal cells (MSC), a proliferation-inducing ligand (APRIL), and hypoxic conditions. Comparative analysis of protein-protein interactions between BM-MSC proteomics with differential RNA transcriptomics of blood ASC and BM LLPC identify two major survival factors, fibronectin and YWHAZ. The MSC secretome proteins and hypoxic conditions play a role in LLPC survival utilizing mechanisms that downregulate mTORC1 signaling and upregulate hypoxia signatures. In summary, we identify elements of the BM survival niche critical for maturation of blood ASC to BM LLPC.

Evidence of Alternative Modes of B Cell Activation Involving Acquired Fab Regions of N-Glycosylation in Antibody-Secreting Cells Infiltrating the Labial Salivary Glands of Patients With Sjögren's Syndrome.

OBJECTIVE: To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. METHODS: A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. RESULTS: V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. CONCLUSION: These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Circulating TFH cells, serological memory, and tissue compartmentalization shape human influenza-specific B cell immunity.

Immunization with the inactivated influenza vaccine (IIV) remains the most effective strategy to combat seasonal influenza infections. IIV activates B cells and T follicular helper (TFH) cells and thus engenders antibody-secreting cells and serum antibody titers. However, the cellular events preceding generation of protective immunity in humans are inadequately understood. We undertook an in-depth analysis of B cell and T cell immune responses to IIV in 35 healthy adults. Using recombinant hemagglutinin (rHA) probes to dissect the quantity, phenotype, and isotype of influenza-specific B cells against A/California09-H1N1, A/Switzerland-H3N2, and B/Phuket, we showed that vaccination induced a three-pronged B cell response comprising a transient CXCR5-CXCR3+ antibody-secreting B cell population, CD21hiCD27+ memory B cells, and CD21loCD27+ B cells. Activation of circulating TFH cells correlated with the development of both CD21lo and CD21hi memory B cells. However, preexisting antibodies could limit increases in serum antibody titers. IIV had no marked effect on CD8+, mucosal-associated invariant T, γδ T, and natural killer cell activation. In addition, vaccine-induced B cells were not maintained in peripheral blood at 1 year after vaccination. We provide a dissection of rHA-specific B cells across seven human tissue compartments, showing that influenza-specific memory (CD21hiCD27+) B cells primarily reside within secondary lymphoid tissues and the lungs. Our study suggests that a rational design of universal vaccines needs to consider circulating TFH cells, preexisting serological memory, and tissue compartmentalization for effective B cell immunity, as well as to improve targeting cellular T cell immunity.