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
Polyamine synthesis represents one of the most profound metabolic changes during T cell activation, but the biological implications of this are scarcely known. Here, we show that polyamine metabolism is a fundamental process governing the ability of CD4+ helper T cells (TH) to polarize into different functional fates. Deficiency in ornithine decarboxylase, a crucial enzyme for polyamine synthesis, results in a severe failure of CD4+ T cells to adopt correct subset specification, underscored by ectopic expression of multiple cytokines and lineage-defining transcription factors across TH cell subsets. Polyamines control TH differentiation by providing substrates for deoxyhypusine synthase, which synthesizes the amino acid hypusine, and mice in which T cells are deficient for hypusine develop severe intestinal inflammatory disease. Polyamine-hypusine deficiency caused widespread epigenetic remodeling driven by alterations in histone acetylation and a re-wired tricarboxylic acid (TCA) cycle. Thus, polyamine metabolism is critical for maintaining the epigenome to focus TH cell subset fidelity.
