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Introduction: CD38 is an ectoenzyme receptor found on hematopoietic cells and its expression is used in the flow cytometric analysis of sub-populations of circulating B cells among peripheral blood mononuclear cells (PBMC) to aid in diagnosing patients with different antibody production defects (AbD). Monoclonal antibodies derived from the sea lamprey Variable Lymphocyte Receptor B (VLRB) are emerging as an alternative to conventional mammalian antibodies. We hypothesized that VLRB MM3 (V-CD38) which specifically recognizes CD38 in a manner correlating with its enzymatic activity could identify terminally differentiated B cells in human PBMC. Here we investigate the ability of V-CD38 as a tool to diagnose patients with diverse immune abnormalities including AbD., Methods: The expression of CD38 on CD3 - CD19 + CD27 + plasmablasts and CD3 - CD19 + IgM hi CD27 - transitional B cells in PBMC was analyzed by flow cytometry using V-CD38 and compared with a commercial conventional antibody to CD38 (C-CD38)., Results: A highly significant correlation (p<0.001, r=0.99) between the percentages of plasmablasts recognized by V-CD38 and C-CD38 was observed among 36 healthy controls (HC), 7 patients with AbD and 24 allergic individuals (AI). The use of V-CD38 enabled improved gating of the CD38 expressing cells (CD38+), aiding in the observation that patients with AbD had significantly lower (p=0.002) CD38+ plasmablasts (0.13%±0.13%) than HC (0.52%±0.57%). Only 61.3% of the transitional B cells detected by C-CD38 were also recognized by V-CD38 (r=0.95, p<0.001) among the 67 participants. AI had significantly reduced V-CD38 and C-CD38 transitional cells compared to HC (p=0.026 and p=0.012, respectively)., Conclusions: V-CD38 is a novel reagent that can assess B cells in human PBMC., Competing Interests: GS has received research support from Aimmune, Amgen, Astra-Zeneca, DBV technologies, Genentech, Leo Pharma Novartis, Sanofi, Regeneron and ALK; is a medical advisor for Novartis, CSL Behring, Pfizer, Abvie, Astra-Zeneca, Nuvo Pharmaceuticals and the Allergy Asthma and Immunology Society of Ontario. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Nair, Leon-Ponte, Kim, Sussman, Ehrhardt and Grunebaum.)

The leucine-rich repeat-based variable lymphocyte receptor B (VLRB) antibody system of jawless vertebrates is capable of generating an antibody repertoire equal to or exceeding the diversity of antibody repertoires of jawed vertebrates. Unlike immunoglobulin-based immune repertoires, the VLRB repertoire diversity is characterized by variable lengths of VLRB encoding transcripts, rendering conventional immunoreceptor repertoire sequencing approaches unsuitable for VLRB repertoire sequencing. Here we demonstrate that long-read single-molecule real-time (SMRT) sequencing (PacBio) approaches permit the efficient large-scale assessment of the VLRB repertoire. We present a computational pipeline for sequence data processing and provide the first repertoire-based analysis of VLRB protein characteristics including properties of its subunits and regions of diversity within each structural leucine-rich repeat subunit. Our study provides a template to explore changes in the VLRB repertoire during immune responses and to establish large scale VLRB repertoire databases for computational approaches aimed at isolating monoclonal VLRB reagents for biomedical research and clinical applications., Competing Interests: AZ is a founder of dYcode Inc. However, while he did contribute his time, he did not receive any funding for his contribution, nor any intellectual property assignments. dYcode did not contribute financially to this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Zia, Orozco, Fang, Tang, Mendoza Viruega, Dong, Leung, Devraj, Oludada and Ehrhardt.)

Although high titers of neutralizing Abs in human serum are associated with protection from reinfection by SARS-CoV-2, there is considerable heterogeneity in human serum-neutralizing Abs against SARS-CoV-2 during convalescence between individuals. Standard human serum live virus neutralization assays require inactivation of serum/plasma prior to testing. In this study, we report that the SARS-CoV-2 neutralization titers of human convalescent sera were relatively consistent across all disease states except for severe COVID-19, which yielded significantly higher neutralization titers. Furthermore, we show that heat inactivation of human serum significantly lowered neutralization activity in a live virus SARS-CoV-2 neutralization assay. Heat inactivation of human convalescent serum was shown to inactivate complement proteins, and the contribution of complement in SARS-CoV-2 neutralization was often >50% of the neutralizing activity of human sera without heat inactivation and could account for neutralizing activity when standard titers were zero after heat inactivation. This effect was also observed in COVID-19 vaccinees and could be abolished in individuals who were undergoing treatment with therapeutic anti-complement Abs. Complement activity was mainly dependent on the classical pathway with little contributions from mannose-binding lectin and alternative pathways. Our study demonstrates the importance of the complement pathway in significantly increasing viral neutralization activity against SARS-CoV-2 in spike seropositive individuals., (Copyright © 2024 by The American Association of Immunologists, Inc.)

SARS-CoV-2 is a respiratory pathogen that can cause severe disease in at-risk populations but results in asymptomatic infections or a mild course of disease in the majority of cases. We report the identification of SARS-CoV-2-reactive B cells in human tonsillar tissue obtained from children who were negative for coronavirus disease 2019 prior to the pandemic and the generation of mAbs recognizing the SARS-CoV-2 Spike protein from these B cells. These Abs showed reduced binding to Spike proteins of SARS-CoV-2 variants and did not recognize Spike proteins of endemic coronaviruses, but subsets reacted with commensal microbiota and exhibited SARS-CoV-2-neutralizing potential. Our study demonstrates pre-existing SARS-CoV-2-reactive Abs in various B cell populations in the upper respiratory tract lymphoid tissue that may lead to the rapid engagement of the pathogen and contribute to prevent manifestations of symptomatic or severe disease., (Copyright © 2021 by The American Association of Immunologists, Inc.)

SARS-CoV-2 is a newly emerged betacoronavirus and the causative agent for the COVID-19 pandemic. Antibodies recognizing the viral spike protein are instrumental in natural and vaccine-induced immune responses to the pathogen and in clinical diagnostic and therapeutic applications. Unlike conventional immunoglobulins, the variable lymphocyte receptor antibodies of jawless vertebrates are structurally distinct, indicating that they may recognize different epitopes. Here we report the isolation of monoclonal variable lymphocyte receptor antibodies from immunized sea lamprey larvae that recognize the spike protein of SARS-CoV-2 but not of other coronaviruses. We further demonstrate that these monoclonal variable lymphocyte receptor antibodies can efficiently neutralize the virus and form the basis of a rapid, single step SARS-CoV-2 detection system. This study provides evidence for monoclonal variable lymphocyte receptor antibodies as unique biomedical research and potential clinical diagnostic reagents targeting SARS-CoV-2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Leung, Khan, Budylowski, Li, Goroshko, Liu, Dong, Carlyle, Rini, Ostrowski and Ehrhardt.)

Fc receptor-like (FCRL) 4 is an immunoregulatory receptor expressed on a subpopulation of human memory B cells of mucosa-associated lymphoid tissue. Fc receptor function of FCRL4 was demonstrated by binding of IgA to FCRL4 following heat aggregation of the Ig. In this study, we demonstrate that FCRL4 recognizes J chain-linked systemic IgA in the absence of heat aggregation. We further demonstrate that mucosal secretory IgA is not recognized by FCRL4 and that systemic IgA binding can be competitively inhibited by recombinant secretory component protein. Finally, we provide evidence that primary FCRL4-bearing human memory B cells are constitutively bound to IgA. Our study provides a mechanism for the negative regulatory activity of FCRL4 on AgR-mediated B cell activation., (Copyright © 2020 by The American Association of Immunologists, Inc.)

CD38 is a multifunctional cell surface receptor expressed on multiple cell lineages of hematopoietic origin with high levels of expression on human plasma cells. Previously, we isolated the monoclonal variable lymphocyte receptor B (VLRB) MM3 antibody from the evolutionarily distant sea lamprey, which recognized the CD38 ectoenzyme exclusively on human plasma cells in a manner that correlated with CD38 enzymatic activity. The plasma cell-specific binding of VLRB MM3 contrasts with the broad pattern of expression of CD38-determined conventional antibodies specific for this antigen. In an effort to facilitate the application of this unique reagent in combination with conventional antibody panels, we explored a strategy to generate VLRB MM3 tetramers. The resulting reagent maintained the threshold-based recognition of CD38. Increased sensitivity achieved with VLRB MM3 tetramers also showed preferential recognition of germinal center centroblasts over centrocytes. VLRB MM3 tetramers thus provided a unique and versatile single-step staining reagent for the detection of human CD38 that is readily incorporated into multi-color flow cytometry panels.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Memory B cells and plasma cells are antigen-experienced cells tasked with the maintenance of humoral protection. Despite these prominent functions, definitive cell surface markers have not been identified for these cells. We report here the isolation and characterization of the monoclonal variable lymphocyte receptor B (VLRB) N8 antibody from the evolutionarily distant sea lamprey that specifically recognizes memory B cells and plasma cells in humans. Unexpectedly, we determined that VLRB N8 recognizes the human leukocyte antigen-I (HLA-I) antigen in a tyrosine sulfation-dependent manner. Furthermore, we observed increased binding of VLRB N8 to memory B cells in individuals with autoimmune disorders multiple sclerosis and systemic lupus erythematosus. Our study indicates that lamprey VLR antibodies uniquely recognize a memory B cell- and plasma cell-specific posttranslational modification of HLA-I, the expression of which is up-regulated during B cell activation.

FCRL4, a low-affinity IgA Ab receptor with strong immunoregulatory potential, is an identifying feature of a tissue-based population of memory B cells (Bmem). We used two independent approaches to perform a comparative analysis of the Ag receptor repertoires of FCRL4 + and FCRL4 - Bmem in human tonsils. We determined that FCRL4 + Bmem displayed lower levels of somatic mutations in their Ag receptors compared with FCRL4 - Bmem but had similar frequencies of variable gene family usage. Importantly, Abs with reactivity to commensal microbiota were enriched in FCRL4 + cells, a phenotype not due to polyreactive binding characteristics. Our study links expression of the immunoregulatory FCRL4 molecule with increased recognition of commensal microbial Ags., (Copyright © 2018 by The American Association of Immunologists, Inc.)