Profiling human antibody responses by integrated single-cell analysis

Abstract

Comprehensive characterization of the antigen-specific B cells induced during infections or following vaccination would facilitate the discovery of novel antibodies and inform how interventions shape protective humoral responses. The analysis of human B cells and their antibodies has been performed using flow cytometry to evaluate memory B cells and expanded plasmablasts, while microtechnologies have also provided a useful tool to examine plasmablasts/plasma cells after vaccination. Here we present an integrated analytical platform using arrays of subnanoliter wells (nanowells) for constructing detailed profiles for human B cells comprising the immunophenotypes of the cells, the distribution of isotypes of secreted antibodies, the specificity and relative affinity for defined antigens, and for a subset of cells, the genes encoding the heavy and light chains. The approach combines on-chip image cytometry, microengraving, and single-cell RT-PCR. Using clinical samples from HIV-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies, is compatible with both plasmablasts/plasma cells and activated memory B cells, and is well-suited for characterizing the limited numbers of B cells isolated from tissue biopsies (e.g., colonic biopsies). The technology should facilitate detailed analyses of human humoral responses for evaluating vaccines and their ability to raise protective antibody responses across multiple anatomical compartments.

Peanut oral immunotherapy transiently expands circulating Ara h 2–specific B cells with a homologous repertoire in unrelated subjects

“…The following nested RT-PCR protocol was adapted from previous papers. 17-19 The cells underwent heat lysis (65°C for 10 minutes, 25°C for 3 minutes, then 4°C) along with incubation with 3uL of NP40, and 150ng of random hexamers. The RT reaction was performed with first strand buffer (Invitrogen), 0.1mM DTT (Invitrogen), 2.5mM dNTP (Invitrogen), and SuperScript III Reverse Transcriptase (Invitrogen).…”

“…Competent E. coli NEB5α bacteria (New England Biolabs) were transformed at 42 °C (30 s) with the ligation produ ct, grown for 1.5 hours at 37°C, followed by selection on LB plates with 100ug/mL ampicillin. Ampicillin-resistant clones were selected and screened for vector insertion with colony PCR using forward and reverse primers 19 followed by gel electrophoresis of PCR products on a 1% agarose gel. Plasmid DNA from overnight liquid cultures (LB with 100ug/mL ampicillin) of selected colonies which contained vectors was obtained using QIAprep Spin Miniprep Kit (Qiagen).…”

Microscale Technologies for High-Throughput Analysis of Immune Cells

“…Using this technique Sendra et al were able to identify and isolate antibodies specifi c for anti-citrullinated protein, a diagnostic and disease marker for rheumatoid arthritis [ 51 ]. Additionally, the microengraving technique is easily multiplexed for probing specifi city to multiple antigens and classifi cation of antibody isotype [ 52 ]. This approach was followed up to identify antigenspecifi c neutralizing antibodies from HIV-infected individuals, from both blood mononuclear cells and cells isolated from the colon [ 52 ].…”

“…Additionally, the microengraving technique is easily multiplexed for probing specifi city to multiple antigens and classifi cation of antibody isotype [ 52 ]. This approach was followed up to identify antigenspecifi c neutralizing antibodies from HIV-infected individuals, from both blood mononuclear cells and cells isolated from the colon [ 52 ].…”

HIV-Specific Antibody Responses in HIV-Infected Patients: From a Monoclonal to a Polyclonal View

“…In addition, several techniques were developed to generate monoclonal antibodies and to study immune complexes (e.g. virion-bound antibodies) as well as antibody-secreting cells [14, [35][36][37][38][39][40][41][42][43][44] . For example, the neutralizing activity of antibodies and their specificity can be assessed by measuring the neutralization of different Env-pseudotyped viruses, i.e.…”