QUAS-R: Glutamine (Q) Uptake Assay with Single cell Resolution reveals metabolic heterogeneity with immune populations

Abstract

System level analysis of single cell data is rapidly transforming the field of immunometabolism. However, metabolic profiling of single cells and small populations by flow and mass cytometry is extremely limited by the availability of specific reagents such as antibodies and fluorescently nutrient analogues. Given the competitive demand for nutrients in pathogenic microenvironments including sites of infection, tumours and autoinflammation, there is a need to understand how and when immune cells access these nutrients. Fluorescent-tagging of nutrients is one approach to study nutrient transport but is extremely limited in its usefulness as tagging usually changes the transport characteristics and transporter specificity of the nutrient. Herein, we developed a completely new approach for single cell analysis of nutrient uptake where a fluorophore is attached to a functionalized amino acid after it has been transported across the plasma membrane and is within the cell. This in-cell biorthogonal labelling ensures that bona fide transport has been measured. System ASC transporter SLC1A5/ASCT2 transports multiple amino acids, most notably the crucial fuel glutamine, and has essential roles in supporting immune metabolism, signalling and function. This flow cytometry assay allows for rapid, sensitive, and quantitative measurement of SLC1A5-mediated uptake, which we used to interrogate the transport capacity of the complex immune subpopulations within the thymus, at a single cell resolution previously 'unreachable'. Taken together, our findings provide an easy procedure to assess which cells support their function via SLC1A5 mediated uptake of amino acids in a sensitive single cell assay. This assay is a significant addition to the single-cell metabolic toolbox required to decode the metabolic landscape of complex immune microenvironments

Asparagine availability controls B cell homeostasis

“…1D-E). We also performed immunostaining for the amino acid transporter ASCT2, the main transporter of Asn and which also transports L-AHA 13,26 , and found high expression in GC B cells, more pronounced in the DZ like ASNS (Fig. 1M and Extended Data 1F).…”

“…2A). HPG is then fluorescently labelled using Click chemistry, and its uptake measured by flow cytometry 26 . We found that there was clear dose-dependent competition between HPG and Asn in GC B cells and plasmablasts (Fig.…”

“…We followed the recently published QUAS-R protocol with certain modifications 26 . Briefly, spleens were harvested 8 days post SRBC immunisation and dissociated through 70μm cell strainers.…”

In-depth immunometabolic profiling by measuring cellular protein translation inhibition via bioorthogonal noncanonical amino acid tagging (CENCAT)

“…In BONCAT, cells are incubated with a non-canonical amino acid (ncAA) analog containing a minimal chemical modification, which can subsequently be targeted to fluorescently tag nascent proteins through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) (11), more commonly known as the 'click' reaction. Among others, BONCAT has been used for affinity purification of nascent proteins (12), to track and localize the newly synthesized proteins in cells (13), and to measure SLC1A5-mediated amino acid uptake (14). A significant advantage of employing BONCAT for measuring protein synthesis is that ncAAs are non-toxic (15).…”