High-throughput single-cell screening of viable hybridomas and patient-derived antibody-secreting cells using punchable microwells.

Monoclonal antibodies (mAbs) hold significant potential as therapeutic agents and are invaluable tools in biomedical research. However, the lack of efficient high-throughput screening methods for single antibody-secreting cells (ASCs) has limited the diversity of available antibodies. Here, we introduce a novel, integrated workflow employing self-seeding microwells and an automated microscope-puncher system for the swift, high-throughput screening and isolation of single ASCs. The system allows for the individual screening and isolation of up to 6,400 cells within approximately one day, with the opportunity for parallelization and efficient upscaling. We successfully applied this workflow to both hybridomas and human patient-derived B cells, enabling subsequent clonal expansion or antibody sequence analysis through an optimized, single-cell nested reverse transcription-polymerase chain reaction (RT-PCR) procedure. By providing a time-efficient and more streamlined single ASC screening and isolation process, our workflow holds promise for driving forward progress in mAb development.

PI3Kγ in B cells promotes antibody responses and generation of antibody-secreting cells.

The differentiation of naive and memory B cells into antibody-secreting cells (ASCs) is a key feature of adaptive immunity. The requirement for phosphoinositide 3-kinase-delta (PI3Kδ) to support B cell biology has been investigated intensively; however, specific functions of the related phosphoinositide 3-kinase-gamma (PI3Kγ) complex in B lineage cells have not. In the present study, we report that PI3Kγ promotes robust antibody responses induced by T cell-dependent antigens. The inborn error of immunity caused by human deficiency in PI3Kγ results in broad humoral defects, prompting our investigation of roles for this kinase in antibody responses. Using mouse immunization models, we found that PI3Kγ functions cell intrinsically within activated B cells in a kinase activity-dependent manner to transduce signals required for the transcriptional program supporting differentiation of ASCs. Furthermore, ASC fate choice coincides with upregulation of PIK3CG expression and is impaired in the context of PI3Kγ disruption in naive B cells on in vitro CD40-/cytokine-driven activation, in memory B cells on toll-like receptor activation, or in human tonsillar organoids. Taken together, our study uncovers a fundamental role for PI3Kγ in supporting humoral immunity by integrating signals instructing commitment to the ASC fate.

Unique and redundant roles of mouse BCMA, TACI, BAFF, APRIL, and IL-6 in supporting antibody-producing cells in different tissues.

Antibody-producing plasma cells fuel humoral immune responses. They also contribute to autoimmune diseases such as systemic lupus erythematosus or IgA nephropathy. Interleukin-6 and the tumor necrosis factor (TNF) family ligands BAFF (B cell-activating factor) and APRIL (a proliferation-inducing ligand) participate in plasma cell survival. BAFF binds to three receptors, BAFFR (BAFF receptor), TACI (transmembrane activator and CAML interactor), and BCMA (B cell maturation antigen), while APRIL binds to TACI, BCMA, and proteoglycans. However, which ligand-receptor pair(s) are required to maintain plasma cells in different body locations remains unknown. Here, by combining mouse genetic and pharmacological approaches, we found that plasma cells required BCMA and/or TACI but not BAFFR. BCMA responded exclusively to APRIL, while TACI responded to both BAFF and APRIL, identifying three self-sufficient ligand-receptor pairs for plasma cell maintenance: BAFF-TACI, APRIL-TACI, and APRIL-BCMA. Together, these actors accounted for 90% of circulating antibodies. In BAFF-ko mice, the reduction of plasma cells upon APRIL inhibition indicated that APRIL could function in the absence of BAFF-APRIL heteromers. No evidence was found that in the absence of BCMA and TACI, binding of APRIL to proteoglycans would help maintain plasma cells. IL-6, alone or together with BAFF and APRIL, supported mainly splenic plasmablasts and plasma cells and contributed to circulating IgG but not IgA levels. In conclusion, survival factors for plasma cells can vary with body location and with the antibody isotype that plasma cells produce. To efficiently target plasma cells, in particular IgA-producing ones, dual inhibition of BAFF and APRIL is required.

Antibody-secreting cells as a source of NR1-IgGs in N-methyl-D-aspartate receptor-antibody encephalitis.

BACKGROUND: The pathogenicity of NR1-IgGs in N-methyl-D-aspartate receptor (NMDAR)-antibody encephalitis is known, but the immunobiological mechanisms underlying their production remain unclear. METHODS: For the first time, we explore the origin of NR1-IgGs and evaluate the contribution of B-cells to serum NR1-IgGs levels. Peripheral blood mononuclear cells (PBMCs) were obtained from patients and healthy controls (HCs). Naïve, unswitched memory (USM), switched memory B cells (SM), antibody-secreting cells (ASCs), and PBMC depleted of ASCs were obtained by fluorescence-activated cell sorting and cultured in vitro. RESULTS: For some patients, PBMCs spontaneously produced NR1-IgGs. Compared to the patients in PBMC negative group, the positive group had higher NR1-IgG titers in cerebrospinal fluid and Modified Rankin scale scores. The proportions of NR1-IgG positive wells in PBMCs cultures were correlated with NR1-IgGs titers in serum and CSF. The purified ASCs, SM, USM B cells produced NR1-IgGs in vitro. Compared to the patients in ASCs negative group, the positive group exhibited a worse response to second-line IT at 3-month follow-up. Naïve B cells also produce NR1-IgGs, implicating that NR1-IgGs originate from naïve B cells and a pre-germinal centres defect in B cell tolerance checkpoint in some patients. For HCs, no NR1-IgG from cultures was observed. PBMC depleted of ASCs almost eliminated the production of NR1-IgGs. CONCLUSIONS: These collective findings suggested that ASCs might mainly contribute to the production of peripheral NR1-IgG in patients with NMDAR-antibody encephalitis in the acute phase. Our study reveals the pathogenesis and helps develop tailored treatments (eg, anti-CD38) for NMDAR-antibody encephalitis.

Age-dependent distribution of IgA and IgG antibody-secreting cells in the pharyngeal tonsil of the Bactrian camel.

The pharyngeal tonsil, located in the nasopharynx, can effectively defend against pathogens invading the body from the upper respiratory tract and play a crucial role in mucosal immunity of the respiratory tract. Immunoglobulin A (IgA) and Immunoglobulin G (IgG) serve as key effector molecules in mucosal immunity, exhibiting multiple immune functions. This study aimed to investigate the distribution patterns and age-related alterations of IgA and IgG antibody-secreting cells (ASCs) in the pharyngeal tonsils of Bactrian camels. Twelve Alashan Bactrian camels were categorized into four age groups: young (1-2 years, n=3), pubertal (3-5 years, n=3), middle-aged (6-16 years, n=3) and old (17-20 years, n=3). The distribution patterns of IgA and IgG ASCs in the pharyngeal tonsils of Bactrian camels of different ages were meticulously observed, analyzed and compared using immunohistochemical and statistical methods. The results revealed that IgA ASCs in the pharyngeal tonsils of all age groups were primarily clustered or diffusely distributed in the reticular epithelium and its subepithelial regions (region A) and around the glands (region C), scattered in the subepithelial regions of non-reticular epithelium (region B), and sporadically distributed in the interfollicular regions (region D). Interestingly, the distribution pattern of IgG ASCs in the pharyngeal tonsils closely mirrored that of IgA ASCs. The distribution densities of IgA and IgG ASCs in these four regions were significantly decreased in turn (P<0.05). However, IgA ASCs exhibited significantly higher densities than IgG ASCs in the same region (P<0.05). Age-related alterations indicated that the distribution densities of IgA and IgG ASCs in each region of the pharyngeal tonsils exhibited a trend of initially increasing and subsequently decreasing from young to old camels, reaching a peak in the pubertal group. As camels age, there was a significant decrease in the densities of IgA and IgG ASCs in all regions of the pharyngeal tonsils (P<0.05). The results demonstrate that the reticular epithelium and its subepithelial regions in the pharyngeal tonsils of Bactrian camels are the primary regions where IgA and IgG ASCs colonize and exert their immune functions. These regions play a pivotal role in inducing immune responses and defending against pathogen invasions in the pharyngeal tonsils. IgA ASCs may be the principal effector cells of the mucosal immune response in the pharyngeal tonsils of Bactrian camels. Aging significantly reduces the densities of IgA and IgG ASCs, while leaving their distribution patterns unaffected. These findings will provide valuable insights for further investigations into the immunomorphology, immunosenescence, and response mechanisms of the pharyngeal tonsils in Bactrian camels.

Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow.

Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19low-BMPC are derived from follicular, while CD19high-BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19low- and CD19high-BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19high-BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observable in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.

Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

Rapid generation of human recombinant monoclonal antibodies from antibody-secreting cells using ferrofluid-based technology.

Monoclonal antibodies (mAbs) are one of the most important classes of biologics with high therapeutic and diagnostic value, but traditional methods for mAbs generation, such as hybridoma screening and phage display, have limitations, including low efficiency and loss of natural chain pairing. To overcome these challenges, novel single B cell antibody technologies have emerged, but they also have limitations such as in vitro differentiation of memory B cells and expensive cell sorters. In this study, we present a rapid and efficient workflow for obtaining human recombinant monoclonal antibodies directly from single antigen-specific antibody secreting cells (ASCs) in the peripheral blood of convalescent COVID-19 patients using ferrofluid technology. This process allows the identification and expression of recombinant antigen-specific mAbs in less than 10 days, using RT-PCR to generate linear Ig heavy and light chain gene expression cassettes, called "minigenes", for rapid expression of recombinant antibodies without cloning procedures. This approach has several advantages. First, it saves time and resources by eliminating the need for in vitro differentiation. It also allows individual antigen-specific ASCs to be screened for effector function prior to recombinant antibody cloning, enabling the selection of mAbs with desired characteristics and functional activity. In addition, the method allows comprehensive analysis of variable region repertoires in combination with functional assays to evaluate the specificity and function of the generated antigen-specific antibodies. Our approach, which rapidly generates recombinant monoclonal antibodies from single antigen-specific ASCs, could help to identify functional antibodies and deepen our understanding of antibody dynamics in the immune response through combined antibody repertoire sequence analysis and functional reactivity testing.

Genomics analysis of three phosphorus-dissolving bacteria isolated from Torreya grandis soil

With the increasingly serious problem of phosphorus deficiency in the subtropical zone, chemical fertilizers are widely used. But it pollutes the environment. Phosphorus-solubilizing microorganisms (PSMs) are referred to as a new solution to this problem. We explored the phosphorus-dissolving characteristics of PSB strains isolated from the rhizosphere soil of Torreya grandis to provide a theoretical basis for selecting the strain for managing phosphorus deficiency in subtropical soils and also provides a more sufficient theoretical basis for the utilization of PSMs. From 84 strains, three strains exhibiting high phosphorus solubility and strong IAA producing capacity were selected through a series of experiments. The phosphate-solubilizing capacity of the three selected strains W1, W74, and W83 were 339.78 mg/L, 332.57 mg/L, and 358.61 mg/L, respectively. Furthermore, W1 showed the strongest IAA secreting capacity of 8.62 mg/L, followed by W74 (7.58 mg/L), and W83 (7.59 mg/L). Determination by metabolites, it was observed that these three strains dissolved phosphorus by secreting a large amount of lactic acid, aromatic acid, and succinic acid. The genome of these PSBs were sequenced and annotated in this study. Our results revealed that PSB primarily promotes their metabolic pathway, especially carbon metabolism, to secrete plenty organic acids for dissolving insoluble phosphorus. (AU)

Single-B cell analysis correlates high-lactate secretion with stress and increased apoptosis.

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSRhigh B cells in vivo, while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSRhigh cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.

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.

Establishment of novel cell lines that maintain the features of B cells derived from patients with neuromyelitis optica spectrum disorder.

B cells that produce anti-aquaporin-4 (AQP4) antibodies play a crucial role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis. We previously reported that naïve B (NB) cells from patients with NMOSD, unlike those from healthy controls, exhibit transcriptional changes suggesting the adoption of an antibody-secreting cell (ASCs) phenotype. CD25+ NB cells, whose numbers are increased in NMOSD patients, have a greater capacity to differentiate into ASCs than do CD25- NB cells. Here, we attempted to establish novel B cell subset cell lines from patients with NMOSD to enable molecular analysis of their abnormalities. We generated Epstein-Barr virus-immortalized lymphoblastoid cell lines (LCLs) from CD25+ NB, CD25- NB, and switched memory B (SMB) cells. All LCLs largely maintained the features of the original cell type in terms of cell surface marker expression and could differentiate into ASCs. Notably, CD25+ NB-LCLs derived from patients with NMOSD exhibited a greater capacity to differentiate into SMB-LCLs than did CD25- NB-LCLs derived from patients with NMOSD, suggesting that the established LCLs maintained the characteristics of cells isolated from patients. The LCLs established in this study are likely to be useful for elucidating the mechanism by which cells that produce anti-AQP4 antibodies develop in NMOSD.

Distinct transcriptomes and autocrine cytokines underpin maturation and survival of antibody-secreting cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibody types, some of which are produced by long-lived plasma cells (LLPC). Active SLE generates increased circulating antibody-secreting cells (ASC). Here, we examine the phenotypic, molecular, structural, and functional features of ASC in SLE. Relative to post-vaccination ASC in healthy controls, circulating blood ASC from patients with active SLE are enriched with newly generated mature CD19-CD138+ ASC, similar to bone marrow LLPC. ASC from patients with SLE displayed morphological features of premature maturation and a transcriptome epigenetically initiated in SLE B cells. ASC from patients with SLE exhibited elevated protein levels of CXCR4, CXCR3 and CD138, along with molecular programs that promote survival. Furthermore, they demonstrate autocrine production of APRIL and IL-10, which contributed to their prolonged in vitro survival. Our work provides insight into the mechanisms of generation, expansion, maturation and survival of SLE ASC.

Assessing the Affinity Spectrum of the Antigen-Specific B Cell Repertoire via ImmunoSpot®.

The affinity distribution of the antigen-specific memory B cell (Bmem) repertoire in the body is a critical variable that defines an individual's ability to rapidly generate high-affinity protective antibody specificities. Detailed measurement of antibody affinity so far has largely been confined to studies of monoclonal antibodies (mAbs) and are laborious since each individual mAb needs to be evaluated in isolation. Here, we introduce two variants of the B cell ImmunoSpot® assay that are suitable for simultaneously assessing the affinity distribution of hundreds of individual B cells within a test sample at single-cell resolution using relatively little labor and with high-throughput capacity. First, we experimentally validated that both ImmunoSpot® assay variants are suitable for establishing functional affinity hierarchies using B cell hybridoma lines as model antibody-secreting cells (ASC), each producing mAb with known affinity for a defined antigen. We then leveraged both ImmunoSpot® variants for characterizing the affinity distribution of SARS-CoV-2 Spike-specific ASC in PBMC following COVID-19 mRNA vaccination. Such ImmunoSpot® assays promise to offer tremendous value for future B cell immune monitoring efforts, owing to their ease of implementation, applicability to essentially any antigenic system, economy of PBMC utilization, high-throughput capacity, and suitability for regulated testing.

Four-Color ImmunoSpot® Assays Requiring Only 1-3 mL of Blood Permit Precise Frequency Measurements of Antigen-Specific B Cells-Secreting Immunoglobulins of All Four Classes and Subclasses.

The B lymphocyte response can encompass four immunoglobulin (Ig) classes and four IgG subclasses, each contributing fundamentally different effector functions. Production of the appropriate Ig class/subclass is critical for both successful host defense and avoidance of immunopathology. The assessment of an antigen-specific B cell response, including its magnitude and Ig class/subclass composition, is most often confined to the antibodies present in serum and other biological fluids and neglects monitoring of the memory B cell (Bmem) compartment capable of mounting a faster and more efficient antibody response following antigen reencounter. Here, we describe how the frequency and Ig class and IgG subclass use of an antigen-specific Bmem repertoire can be determined with relatively little labor and cost, requiring only 8 × 105 freshly isolated peripheral blood mononuclear cells (PBMC), or if additional cryopreservation and polyclonal stimulation is necessary, 3 × 106 PBMC per antigen. To experimentally validate such cell saving assays, we have documented that frequency measurements of antibody-secreting cells (ASC) yield results indistinguishable from those of enzymatic (ELISPOT) or fluorescent (FluoroSpot) versions of the ImmunoSpot® assay, including when the latter are detected in alternative fluorescent channels. Moreover, we have shown that frequency calculations that are based on linear regression analysis of serial PBMC dilutions using a single well per dilution step are as accurate as those performed using replicate wells. Collectively, our data highlight the capacity of multiplexed B cell FluoroSpot assays in conjunction with serial dilutions to significantly reduce the PBMC requirement for detailed assessment of antigen-specific B cells. The protocols presented here allow GLP-compliant high-throughput measurements which should help to introduce high-dimensional Bmem characterization into the standard immune monitoring repertoire.

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.

Role of affinity in plasma cell development in the germinal center light zone.

Protective immune responses to many pathogens depend on the development of high-affinity antibody-producing plasma cells (PC) in germinal centers (GCs). Transgenic models suggest that there is a stringent affinity-based barrier to PC development. Whether a similar high-affinity barrier regulates PC development under physiologic circumstances and the nature of the PC fate decision has not been defined precisely. Here, we use a fate-mapping approach to examine the relationship between GC B cells selected to undergo additional rounds of affinity maturation, GC pre-PC, and PC. The data show that initial PC selection overlaps with GC B cell selection, but that the PC compartment accumulates a less diverse and higher affinity collection of antibodies over time. Thus, whereas the GC continues to diversify over time, affinity-based pre-PC selection sieves the GC to enable the accumulation of a more restricted group of high-affinity antibody-secreting PC.

Circulating antibody-secreting cells are a biomarker for early diagnosis in patients with Lyme disease.

BACKGROUND: Diagnostic immunoassays for Lyme disease have several limitations including: 1) not all patients seroconvert; 2) seroconversion occurs later than symptom onset; and 3) serum antibody levels remain elevated long after resolution of the infection. INTRODUCTION: MENSA (Medium Enriched for Newly Synthesized Antibodies) is a novel diagnostic fluid that contains antibodies produced in vitro by circulating antibody-secreting cells (ASC). It enables measurement of the active humoral immune response. METHODS: In this observational, case-control study, we developed the MicroB-plex Anti-C6/Anti-pepC10 Immunoassay to measure antibodies specific for the Borrelia burgdorferi peptide antigens C6 and pepC10 and validated it using a CDC serum sample collection. Then we examined serum and MENSA samples from 36 uninfected Control subjects and 12 Newly Diagnosed Lyme Disease Patients. RESULTS: Among the CDC samples, antibodies against C6 and/or pepC10 were detected in all seropositive Lyme patients (8/8), but not in sera from seronegative patients or healthy controls (0/24). Serum antibodies against C6 and pepC10 were detected in one of 36 uninfected control subjects (1/36); none were detected in the corresponding MENSA samples (0/36). In samples from newly diagnosed patients, serum antibodies identified 8/12 patients; MENSA antibodies also detected 8/12 patients. The two measures agreed on six positive individuals and differed on four others. In combination, the serum and MENSA tests identified 10/12 early Lyme patients. Typically, serum antibodies persisted 80 days or longer while MENSA antibodies declined to baseline within 40 days of successful treatment. DISCUSSION: MENSA-based immunoassays present a promising complement to serum immunoassays for diagnosis and tracking therapeutic success in Lyme infections.

TRAPnSeq allows high-throughput profiling of antigen-specific antibody-secreting cells.

Following activation by cognate antigen, B cells undergo fine-tuning of their antigen receptors and may ultimately differentiate into antibody-secreting cells (ASCs). While antigen-specific B cells that express surface receptors (B cell receptors [BCRs]) can be readily cloned and sequenced following flow sorting, antigen-specific ASCs that lack surface BCRs cannot be easily profiled. Here, we report an approach, TRAPnSeq (antigen specificity mapping through immunoglobulin [Ig] secretion TRAP and Sequencing), that allows capture of secreted antibodies on the surface of ASCs, which in turn enables high-throughput screening of single ASCs against large antigen panels. This approach incorporates flow cytometry, standard microfluidic platforms, and DNA-barcoding technologies to characterize antigen-specific ASCs through single-cell V(D)J, RNA, and antigen barcode sequencing. We show the utility of TRAPnSeq by profiling antigen-specific IgG and IgE ASCs from both mice and humans and highlight its capacity to accelerate therapeutic antibody discovery from ASCs.