Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

Degauque, Nicolas; Ngono, Annie Elong; Akl, Ahmed; Lepetit, M.; Crochette, Romain; Giral, Magali; Lepourry, J.; Pallier, Annaïck; Castagnet, Sophie; Dugast, Emilie; Guillot-Gueguen, Cécile; Jacq-Foucher, Marylène; Saulquin, Xavier; Cesbron, Anne; Laplaud, David‐Axel; Nicot, Arnaud; Brouard, Sophie; Soulillou, Jean‐Paul

doi:10.1371/journal.pone.0084273

Cited by 14 publications

(19 citation statements)

References 35 publications

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“…Previous published studies report the number of detected B cells and the antigenic reactivity of antibodies secreted in supernatants of ex vivo B cell cultures without cloning, production, or testing of the derived antibody clones (16,32). A similar B cell identification process to the one reported in our study described the frequency of antigen-specific B cells detected using a modified bead-based method (31). With this tool, it was possible to monitor the frequencies of anti-HLA, anti-tetanus toxin-, and anti-EBNA1-committed B cells in different individuals.…”

Section: Discussionsupporting

confidence: 60%

“…For instance, antibodies used for detection of BCR on B cells may also interact not only with BCRs but also with Fc receptors on human B cells (30). Tetramer technologies may be applicable to the selection of antigen-specific B cells, but fluorophores have been known to be released from antigen complexes and to bind non-specifically to immune cells, yielding false positive events (31). Our protocol is also specifically designed to avoid the requirement for B cell culture and ex vivo expansion or for antibody selection from culture supernatants prior to sub-cloning, limiting dilution and isolation of antigen-specific B cell clones.…”

Section: Discussionmentioning

confidence: 99%

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Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

et al. 2018

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Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

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Section: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

et al. 2018

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“…More recently, the wish to isolate the antigen-specific B cells has led to development of complementary technologies based on flow cytometry. One setup is based on antigens being coupled to beads and then used to stain B cells [47], and in another setup large antigens could be directly coupled to fluorochromes and used as staining reagents [48, 49]; last, for peptide-antigens, tetramerization of biotinylated peptides before labeling to create so-called B cell tetramers has been utilized [50]. The parallel use of a decoy tetramer allows the purging of false-positive events making this technology robust, and this approach has already been employed to study cit-specific B cells in RA [51, 52].…”

Section: Discussionmentioning

confidence: 99%

Antibody responses to de novo identified citrullinated fibrinogen peptides in rheumatoid arthritis and visualization of the corresponding B cells

et al. 2016

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BackgroundAntibodies against citrullinated proteins (ACPA) are common in patients with rheumatoid arthritis (RA). ACPA can appear before disease onset and target many self-antigens. Citrullinated fibrin/fibrinogen represents a classical ACPA target antigen, and mass spectrometry of RA synovial fluid reveals elevated citrullinated (cit) fibrinogen (Fib) peptides compared to non-RA controls. We investigated the extent to which these less-studied peptides represent autoantibody targets and sought to visualize the corresponding cit-Fib-reactive B cells in RA patients.MethodsAn in-house ELISA was established against four cit-Fib α-subunit peptides (cit-Fib α-35; cit-Fib α-216,218; cit-Fib α-263,271 and cit-Fib α-425,426) and serum from patients with established RA (n = 347) and disease controls with psoriatic arthritis (PsA) or ankylosing spondylitis (AS) (n = 236) were analyzed. RA patients were genotyped for HLA-DR alleles, PTPN22 R620W and screened for anti-CCP2 and cit-Fib protein antibodies. The cit-Fib peptides were also used to assemble antigen tetramers to identify cit-Fib-reactive B cells in peripheral blood by flow cytometry.ResultsThe frequencies of autoantibodies against different cit-Fib epitopes in RA patients compared to PsA/AS patients were: cit-Fib α-35 (RA 20%, vs PsA/AS 1%); cit-Fib α-216,218 (13% vs 0.5%); cit-Fib α-263,271 (21% vs 0.5%) and cit-Fib α-425,426 (17% vs 1%). The presence of autoantibodies against these peptides was associated with presence of anti-CCP2 and anti-cit-Fib protein antibodies. No association was found between HLA-DR shared epitope and antibodies to the different cit-Fib peptides. However, association was observed between the PTPN22 risk allele and positivity to cit-Fib α-35 and cit-Fib α-263,271. B cells carrying surface Ig reactive to these cit-Fib peptides were found in RA peripheral blood and these tend to be more common in PTPN22 risk allele carriers.ConclusionsOur data show that several cit-Fib peptides are targeted by autoantibodies in RA, but not in PsA/AS, implicating that these are not due to arthritis but more specific for RA etiology. The RA-associated anti-cit protein response is broad with many parallel immune responses. The association between cit-Fib autoantibodies and the PTPN22 R620W risk allele supports the hypothesis of altered B cell regulation, such as autoreactive B cells evading tolerance checkpoints.

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“…More recently, a related approach to identify HLA-specific B cells using microspheres coated with HLA molecules was described 99 . In this assay, HLA-specific B cells bind to, and form HLA bead-B cell rosettes (BBR) that are identified by flow cytometry.…”

Section: Tracking Allospecific B Cellsmentioning

confidence: 99%

Evolving Approaches in the Identification of Allograft-Reactive T and B Cells in Mice and Humans

Young

McIntosh²,

Alegre³

et al. 2017

Transplantation

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Whether a transplanted allograft is stably accepted, rejected or achieves immunological tolerance is dependent on the frequency and function of alloreactive lymphocytes, making the identification and analysis of alloreactive T and B cells in transplant recipients critical for understanding mechanisms, and the prediction of allograft outcome. In animal models, tracking the fate of graft-reactive T and B cells allows investigators to uncover their biology and develop new therapeutic strategies to protect the graft. In the clinic, identification and quantification of graft-reactive T and B cells allows for the early diagnosis of immune reactivity and therapeutic intervention to prevent graft loss. In addition to rejection, probing of T and B cell fate in vivo provides insights into the underlying mechanisms of alloimmunity or tolerance that may lead to biomarkers predicting graft fate. In this review, we discuss existing and developing approaches to track and analyze alloreactive T and B cells in mice and humans and provide examples of discoveries made utilizing these techniques. These approaches include mixed lymphocyte reactions (MLRs), trans-vivo delayed-type hypersensitivity (DTH), enzyme-linked immunospot (ELISpot) assays, the use of antigen receptor transgenic lymphocytes, and utilization of peptide-MHC complex (pMHC) multimers, along with imaging techniques for static multiparameter analysis or dynamic in vivo tracking. Such approaches have already refined our understanding of the alloimmune response and are pointing to new ways to improve allograft outcomes in the clinic.

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Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

Cited by 14 publications

References 35 publications

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

Antibody responses to de novo identified citrullinated fibrinogen peptides in rheumatoid arthritis and visualization of the corresponding B cells

Evolving Approaches in the Identification of Allograft-Reactive T and B Cells in Mice and Humans

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