Production and use of antigen tetramers to study antigen-specific B cells

38 Pág.B cells generate antibodies that provide protection from infection, but also cause pathology in autoimmune and allergic conditions. Antigen-specific B cells can be detected by binding their surface antibody receptors with native antigens conjugated to fluorescent probes, a technique that has revealed substantial insight into B cell activation and function. This protocol describes the process of generating fluorescent antigen tetramer probes and delineates a process of enriching large samples based on antigen-specificity for high-resolution analyses of the antigen-specific B cell repertoire. Enrichment of tetramer-binding cells allows for detection of antigen-specific B cells as rare as 1 in 100 million cells, providing sufficient resolution to study naive B cells and IgE-expressing cells by flow cytometry. The generation of antigen tetramers involves antigen biotinylation, assessment of biotin:antigen ratio for optimal tetramer loading and polymerization around a streptavidin-fluorophore backbone. We also describe the construction of a control tetramer to exclude B cells binding to the tetramer backbone. We provide a framework to validate whether tetramer probes are detecting true antigen-specific B cells and discuss considerations for experimental design. This protocol can be performed by researchers trained in basic biomedical/immunological research techniques, using instrumentation commonly found in most laboratories. Constructing the antigen and control tetramers takes 9 h, though their specificity should be assessed before experimentation and may take weeks to months depending on the method of validation. Sample enrichment requires ~2 h but is generally time and cost neutral as fewer cells are run through the flow cytometer.We thank J. SoRelle (University of Texas Southwestern) for carefully reviewing and providing suggestions on the manuscript. We thank M. K. Jenkins (University of Minnesota) for supporting the initial development of these protocols and for the inclusion of Extended Data Fig. 1g. We thank J. Carter and D. Galloway (Fred Hutchinson Cancer Center) for providing supernatant containing GST. We thank the McMaster Flow Cytometry Core, H. Liang and M. Subapanditha for access to flow cytometers and experimental support. We thank M. S. Miller (McMaster University) and M. Larche (McMaster University) for providing recombinant RBD. The laboratories of M.J. and J.F.E.K. are funded by the Schroeder Foundation, Food Allergy Canada, ALK-Abello A/S, the Canadian Allergy Asthma and Immunology Foundation, the Zych family and the Satov family. A.P. is funded by an Ontario Graduate Scholarship and The Eva Eugenia Lillian Cope Scholarship. D.P.-C. was funded by Universidad Politécnica de Madrid and Banco Santander with predoctoral and travel Programa Propio grants. J.T.-A. was funded by Severo Ochoa Program (Production of Plant and Human health-relevant proteins in Super-Green Biofactories: PCD-UPM/7/2022). The Centre for Plant Biotechnology and Genomics was granted ‘Severo Ochoa’ Distinctions of Excellence by the Spanish Ministry of Science and Innovation (SEV-2016-0672 and CEX2020-000999-S). J.J.T. has been funded by the National Institutes of Health (R01AI122912, R01AI158728, R01AI167009), The Hartwell Foundation, Vir Biotechnology, Fred Hutch Cancer Center and generous donors. J.B. was supported by a Fast Grants award, and J.B. and J.J.T. were supported by a Fred Hutchinson Cancer Center COVID pilot award.Peer reviewe

Autophagy promotes cell and organismal survival by maintaining NAD(H) pools

Autophagy is an essential catabolic process that promotes clearance of surplus or damaged intracellular components 1 . As a recycling process, autophagy is also important for the maintenance of cellular metabolites during periods of starvation 2 . Loss of autophagy is sufficient to cause cell death in animal models and is likely to contribute to tissue degeneration in a number of human diseases including neurodegenerative and lysosomal storage disorders 3–7 . However, it remains unclear which of the many cellular functions of autophagy primarily underlies its role in cell survival. Here we have identified a critical role of autophagy in the maintenance of nicotinamide adenine dinucleotide (NAD + /NADH) levels. In respiring cells, loss of autophagy caused NAD(H) depletion resulting in mitochondrial membrane depolarisation and cell death. We also found that maintenance of NAD(H) is an evolutionary conserved function of autophagy from yeast to human cells. Importantly, cell death and reduced viability of autophagy-deficient animal models can be partially reversed by supplementation with an NAD(H) precursor. Our study provides a mechanistic link between autophagy and NAD(H) metabolism and suggests that boosting NAD(H) levels may be an effective intervention strategy to prevent cell death and tissue degeneration in human diseases associated with autophagy dysfunction