Combining Fluorescent Cell Sorting and Single B Cell Amplification to Screen the Monoclonal Antibody Gene against Human Glypican-1 in Pancreatic Cancer

In this report, one novel method has been developed to screen the monoclonal antibody against human pancreatic cancer biomarker glypican-1 (GPC1) through the combination of fluorescent cell sorting and single B cell amplification. GPC1-positive B cells were sorted out from the peripheral blood mononuclear cells (PBMCs) by fluorescent cell sorting after the GPC1 immunization to the New Zealand white rabbit. Then, total RNA was extracted and reversely transcribed into cDNA, which was used as the template, and the variable region sequences of both heavy and light chains were amplified from the same B cell. Next, their recombinant antibody was expressed and purified from the human 293T cell after the antibody gene amplification and expression vector construction. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays were used to determine the antibody affinity. The antibody named GPC-12 that we screened and obtained was proven to have natural heavy-light chain pairing information, and it was highly specific to the GPC1 antigen, and the affinity could reach 1 × 10−7 M. Overall, an effective and novel method has been successfully developed to screen the antibody by combining the fluorescent cell sorting and single-cell amplifying technologies, which was proved to be workable in our setting.

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.

An apoptosis-dependent checkpoint for autoimmunity in memory B and plasma cells

B lymphocytes acquire self-reactivity as an unavoidable byproduct of antibody gene diversification in the bone marrow and in germinal centers (GCs). Autoreactive B cells emerging from the bone marrow are silenced in a series of well-defined checkpoints, but less is known about how self-reactivity that develops by somatic mutation in GCs is controlled. Here, we report the existence of an apoptosis-dependent tolerance checkpoint in post-GC B cells. Whereas defective GC B cell apoptosis has no measurable effect on autoantibody development, disruption of post-GC apoptosis results in accumulation of autoreactive memory B cells and plasma cells, antinuclear antibody production, and autoimmunity. The data presented shed light on mechanisms that regulate immune tolerance and the development of autoantibodies.

Direct observation of RAG recombinase recruitment to chromatin and the IgH locus in live pro-B cells

The RAG1 and RAG2 proteins introduce double-strand DNA breaks at antigen-receptor loci in developing lymphocytes to initiate V(D)J recombination. How RAG proteins find the correct target locus in a vast excess of non-specific chromatin is not known. Here we measured dynamics of RAG1/RAG2 interactions with chromatin in living pro-B cells. We found that the majority of RAG1 or RAG1/RAG2 complex is in a fast 3D diffusive state, and the residual slow diffusive (bound) fraction was determined by a non-core portion of RAG1, and the PHD domain of RAG2. The RAG proteins exhibited distinct dynamics at the IgH locus. In particular, RAG2 increased the probability of RAG1 binding to IgH, a property that likely explains its non-catalytic role in V(D)J recombination. Our observations reveal how RAG finds its targets in developing B cells.One Sentence SummarySingle-molecule imaging of the RAG recombinase reveals its search strategy for chromatin, H3K4me3 and antibody gene loci in living cells.

PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification

The establishment of protective humoral immunity is dependent on the ability of mature B cells to undergo antibody gene diversification while adjusting to the physiological stressors induced by activation with the antigen. Mature B cells diversify their antibody genes by class switch recombination (CSR) and somatic hypermutation (SHM), which are both dependent on efficient induction of activation-induced cytidine deaminase (AID). Here, we identified PDGFA-associated protein 1 (Pdap1) as an essential regulator of cellular homeostasis in mature B cells. Pdap1 deficiency leads to sustained expression of the integrated stress response (ISR) effector activating transcription factor 4 (Atf4) and induction of the ISR transcriptional program, increased cell death, and defective AID expression. As a consequence, loss of Pdap1 reduces germinal center B cell formation and impairs CSR and SHM. Thus, Pdap1 protects mature B cells against chronic ISR activation and ensures efficient antibody diversification by promoting their survival and optimal function.