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Interrogating in vivo T-cell metabolism in mice using stable isotope labeling metabolomics and rapid cell sorting

Abstract

T cells are integral players in the adaptive immune system that readily adapt their metabolism to meet their energetic and biosynthetic needs. A major hurdle to understand physiologic T-cell metabolism has been the differences between in vitro cell culture conditions and the complex in vivo milieu. To address this, we have developed a protocol that merges traditional immunology infection models with whole-body metabolite infusion and mass-spectrometry-based metabolomic profiling to assess T-cell metabolism in vivo. In this protocol, pathogen-infected mice are infused via the tail vein with an isotopically labeled metabolite (2–6 h), followed by rapid magnetic bead isolation to purify T-cell populations (<1 h) and then stable isotope labeling analysis conducted by mass spectrometry (~1–2 d). This procedure enables researchers to evaluate metabolic substrate utilization into central carbon metabolic pathways (i.e., glycolysis and the tricarboxylic acid cycle) by specific T-cell subpopulations in the context of physiological immune responses in vivo.

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Fig. 1: Overview of the procedure.
Fig. 2: Preparation of infusion lines.
Fig. 3: Bead isolation procedure.
Fig. 4: Blood glucose enrichment of 13C-glucose label in Listeria-infected mice.
Fig. 5: FACS analysis of T cells following magnetic bead isolation procedure.
Fig. 6: 13C-glucose labeling patterns in T cells isolated from Listeria-infected mice.
Fig. 7: Differentially enriched 13C-glucose-derived metabolites.
Fig. 8: Pathway analysis of 13C-glucose-depedent nucleotide metabolism in activated T cells from Listeria-infected mice.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Acknowledgements

We thank E. Levine, R. Sheridan and staff from the Van Andel Institute (VAI) Metabolomics and Bioenergetics and Flow Cytometry core facilities for technical assistance. We thank J. Wedberg and M. Minard for administrative assistance. Funding: The VAI Metabolomics and Bioenergetics Core Facility is supported by the Department of Metabolism and Nutritional Programming at VAI. This research has been supported by grants from the CIHR (MOP-142259 to RGJ) and funding from VAI and Agios Pharmaceuticals.

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Authors

Contributions

Conceptualization: R.D.S., E.H.M. and R.G.J.; validation: R.D.S. and E.H.M.; investigation: R.D.S., L.M.D. and E.H.M.; data curation: R.D.S. and E.H.M.; writing—original draft: R.D.S. and E.H.M.; writing—review and editing: R.D.S., E.H.M., K.S.W. and R.G.J.; visualization: K.S.W.; supervision: R.G.J.; funding acquisition: R.G.J.

Corresponding author

Correspondence to Russell G. Jones.

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Competing interests

R.G.J. is a consultant for Agios Pharmaceuticals and serves on the Scientific Advisory Board of ImmunoMet Therapeutics.

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Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Key reference using this protocol

Ma, E. H. et al. Immunity 51, 856–870.e5 (2019): https://doi.org/10.1016/j.immuni.2019.09.003

Supplementary information

Reporting Summary

Supplementary Table 1

Compound list using the described HILIC LC/MS method. Important: Retention times will vary from lab to lab and should be confirmed by chemical standards.

Supplementary Table 2

Compound list using the described GC/MS method. The quantifier ion is a molecular ion that can be used for isotope analysis. The qualifier ion should have the same retention and is used to improve confidence in compound identification. Important: Retention times will vary from lab to lab and should be confirmed by chemical standards.

Supplementary Table 3

This Excel document contains three tabs. The “Raw Tracing Data” tab contains raw peak areas and natural abundance corrected values and fractional enrichment for each compound. The “T cells total 13C-enrichment” contains the total fractional enrichment of 13C carbon in each compound. These data have been filtered to include only compounds with at least 5% enrichment on average in at least one group. The “Subset >5% enrichment” contains this filtered data. In each tab, sample identifiers are as follows: Phenformin group: S1-00, S1-01, S1-30; Vehicle group: S2-03, S2-10, S2-30.

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Sheldon, R.D., Ma, E.H., DeCamp, L.M. et al. Interrogating in vivo T-cell metabolism in mice using stable isotope labeling metabolomics and rapid cell sorting. Nat Protoc 16, 4494–4521 (2021). https://doi.org/10.1038/s41596-021-00586-2

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