ABSTRACT
Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.
Subject(s)
Camelids, New World , Immunoglobulin Heavy Chains , Mice, Transgenic , Single-Domain Antibodies , Spike Glycoprotein, Coronavirus , Animals , Single-Domain Antibodies/genetics , Single-Domain Antibodies/immunology , Camelids, New World/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Mice , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/chemistry , Lectins, C-Type/metabolism , Lectins, C-Type/immunology , Lectins, C-Type/genetics , SARS-CoV-2/immunology , SARS-CoV-2/genetics , Immunoglobulin E/immunology , Humans , Dependovirus/genetics , Dependovirus/immunology , Immunoglobulin G/immunology , COVID-19/immunology , B-Lymphocytes/immunologyABSTRACT
Binding of BAFF to BAFFR activates in mature B cells PI3K/AKT signaling regulating protein synthesis, metabolic fitness, and survival. In humans, naive and memory B cells express the same levels of BAFFR, but only memory B cells seem to survive without BAFF. Here, we show that BAFF activates PI3K/AKT only in naive B cells and changes the expression of genes regulating migration, proliferation, growth, and survival. BAFF-induced PI3K/AKT activation requires direct interactions between BAFFR and the B cell antigen receptor (BCR) components CD79A and CD79B and is enhanced by the AKT coactivator TCL1A. Compared to memory B cells, naive B cells express more surface BCRs, which interact better with BAFFR than IgG or IgA, thus allowing stronger responses to BAFF. As ablation of BAFFR in naive and memory B cells causes cell death independent of BAFF-induced signaling, BAFFR seems to act also as an intrinsic factor for B cell survival.
