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Age-associated impairment of T cell immunity is linked to sex-dimorphic elevation of N-glycan branching

Nature Aging volume 2, pages 231–242 (2022)Cite this article

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Abstract

Impaired T cell immunity with aging increases mortality from infectious disease. The branching of asparagine-linked glycans is a critical negative regulator of T cell immunity. Here we show that branching increases with age in females more than in males, in naive T cells (TN) more than in memory T cells, and in CD4+ more than in CD8+ T cells. Female sex hormones and thymic output of TN cells decrease with age; however, neither thymectomy nor ovariectomy altered branching. Interleukin-7 (IL-7) signaling was increased in old female more than male mouse TN cells, and triggered increased branching. N-acetylglucosamine, a rate-limiting metabolite for branching, increased with age in humans and synergized with IL-7 to raise branching. Reversing elevated branching rejuvenated T cell function and reduced severity of Salmonella infection in old female mice. These data suggest sex-dimorphic antagonistic pleiotropy, where IL-7 initially benefits immunity through TN maintenance but inhibits TN function by raising branching synergistically with age-dependent increases in N-acetylglucosamine.

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Fig. 1: Mouse T cells show a sex-dimorphic increase in N-glycan branching with age.
Fig. 2: Elevated IL-7 signaling increases N-glycan branching in old female CD4+ TN cells.
Fig. 3: Age-dependent increases in N-glycan branching suppress T cell function in female mice.
Fig. 4: Elevated N-glycan branching in T cells suppresses immune response to Salmonella in old female mice.
Fig. 5: N-glycan branching increases with age in human T cells.
Fig. 6: Serum N-acetylglucosamine increases with age and synergizes with IL-7 to raise N-glycan branching in human T cells.

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Data availability

RNA-seq data has been deposited with the Gene Expression Omnibus under accession number GSE184496. The mouse 10mm reference genome was obtained from https://hgdownload.soe.ucsc.edu/downloads.html#mouse. Statistical source data is provided for all main and Extended Data Figures in the supplementary information. Additional data that support the findings within this article are available from the corresponding author upon reasonable request.

Code availability

The code for differential analysis of RNA-seq data using R package DESeq2 has been uploaded to a github repository and can be found at https://github.com/ucightf/demetriou.

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Acknowledgements

We thank C. Kawas (UC Irvine) for access to subjects from the ‘90+ cohort’. Research was supported by the National Institute of Allergy and Infectious Disease (R01AI108917, M.D.; R01AI144403, M.D.; R01AI126277, M.R.; R01AI114625, M.R.), the National Center for Complementary and Integrative Health (R01AT007452, M.D.), the Burroughs Wellcome Fund (Investigator in the Pathogenesis of Infectious Disease Award, M.R.) and a predoctoral fellowship from the American Heart Association (S.K.). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Obtaining human blood was supported by a Clinical Translational Science Award to the Institute for Clinical and Translation Science, UC Irvine.

Author information

Author notes

  1. These authors contributed equally: Haik Mkhikian, Ken L. Hayama.

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, University of California, Irvine, Irvine, CA, USA

    Haik Mkhikian

  2. Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA, USA

    Ken L. Hayama, Suzi Klaus, Manuela Raffatellu & Michael Demetriou

  3. Department of Neurology, University of California, Irvine, Irvine, CA, USA

    Khachik Khachikyan, Carey Li, Raymond W. Zhou, Phuong Q. N. Tran, Kim M. Ly, Andrew D. Gong, Hayk Saryan, Jasper L. Hai, David Grigoryan, Philip L. Lee, Barbara L. Newton & Michael Demetriou

  4. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Judy Pawling & James W. Dennis

  5. Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA

    Manuela Raffatellu

  6. Center for Mucosal Immunology, Allergy, and Vaccines, Chiba University-UC San Diego, La Jolla, CA, USA

    Manuela Raffatellu

  7. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    James W. Dennis

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Contributions

H.M. and M.D. conceptualized the study. K.L.H., H.M., S.K., M.R., J.P., J.W.D. and M.D. developed the methodology. K.L.H., H.M., K.K., C.L., R.W.Z., J.P., S.K., P.Q.N.T, K.M.L., A.D.G., J.L.H., D.G., P.L.L., H.S. and B.L.N. performed the investigation. K.L.H, H.M. and M.D. wrote the original draft. H.M. and M.D. reviewed and edited the manuscript. K.L.H, H.M. and M.D. provided visualizations. H.M., M.R., J.W.D. and M.D. supervised the project. M.R. and M.D. acquired funding.

Corresponding author

Correspondence to Michael Demetriou.

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Competing interests

J.D. and M.D. are named as inventors on a patent application that describes GlcNAc as a biomarker for multiple sclerosis. J.D. and M.D. are named as inventors on a patent for use of GlcNAc in multiple sclerosis. The remaining authors declare no competing interests.

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Nature Aging thanks Jung-Hyun Park and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Mouse T cells display a sex-dimorphic increase in N-glycan branching with age.

a) Fructose 6-phosphate may be metabolized by glycolysis or enter the hexosamine pathway to supply UDP-GlcNAc to the Golgi branching enzymes Mgat1, 2, 4 and 5, which generate mono-, bi-, tri-, and tetra-antennary GlcNAc branched glycans, respectively. The branching enzymes utilize UDP-GlcNAc with declining efficiency such that both Mgat4 and Mgat5 are limited for branching by the metabolic production of UDP-GlcNAc. Small molecule inhibitor kifunensine (KIF) can be used to eliminate N-glycan branching. Plant lectin L-PHA (Phaseolus vulgaris, leukoagglutinin) and ConA (concanavalin A) binding sites are also shown. Abbreviations: OT, oligosaccharyltransferases; GI, glucosidase I; GII, glucosidase II; MI, mannosidase I; MII, mannosidase II; Mgat, N-acetylglucosaminyltransferase; GalT3, galactosyltransferase 3; iGnT, i-branching enzyme β1,3-N-acetylglucosaminyltransferase; KIF, kifunensine; GlcNAc, N-acetylglucosamine; UDP, uridine diphosphate; Km, Michaelis constant of the enzyme. b) The gating strategy is demonstrated for CD4+ TN cells. Lymphocytes were first gated on singlets, followed by gating on CD3+CD4+CD8−CD25−CD62L+CD44− cells by sequential steps. c, d) Splenocytes from five young and old mice were analyzed for L-PHA (c) or ConA (d) binding by flow cytometry, gating on the indicated CD4+ T cell subsets. Absolute geometric mean fluorescence intensity (MFI) is shown to allow direct comparison between naive and memory subsets. e-i) CD4+ TN cells (e-g) CD19+ B cells (h) or thymocytes (i) were obtained from the lymph node (e), spleen (f-h) or thymus (i) of female (e, g-i) or male (f-h) mice of the indicated ages, and analyzed for L-PHA binding by flow cytometry. Absolute or normalized geometric mean fluorescence intensity (MFI) are shown. Each symbol represents a single mouse. P-values by two-tailed Mann–Whitney (c, d) or two-tailed Wilcoxon (e, h, i). Error bars indicate mean ± s.e.m.

Source data

Extended Data Fig. 2 Elevated IL-7 signaling increases N-glycan branching in old female naiveT cells.

a) Representative flow cytometry plots of donor and recipient cells post-adoptive transfer. b) Negatively selected CD4+ T cells were FACS sorted for TN (CD62L+CD44−) and TEM (CD62L−CD44+) populations. Representative flow cytometry plots demonstrating purity of sorted cells used for RNA-seq. c) Principal component analysis (PCA) of RNA-seq data comparing gene expression in CD4+ TN and CD4+ TEM cells from young male (7-8 weeks old), young female (10-11 weeks old), old male (83-86 weeks old) and old female (85 weeks old) mice. Three biological replicates were performed for each group. d) Out of 24062 genes analyzed by RNA-seq, 158 DEGs were identified when comparing young and old CD4+ TN cells in females, 192 DEGs were identified in males, and 44 DEGs were shared. e) Young and old naive CD4+ T cell mRNA expression of N-glycan pathway genes by real-time qPCR. f) Flow cytometric analysis of IL7Rα in ex vivo CD4+ TN cells from young and old male mice. g) L-PHA versus IL7Rα expression in ex vivo CD4+ TN cells from old male and female mice. h, i) Flow cytometric analysis of IL7Rα in ex vivo young and old CD4+ TEM cells from female (h) and male (i) mice. j-l) C57BL/6 mice of the indicated ages were injected intraperitoneally with either isotype control (1.5 mg) or anti-IL-7 antibody (M25, 1.5 mg) three times per week for two (j) or four (k, l) weeks, and analyzed for L-PHA or IL7Rα expression in blood (k) or spleen (j, l). Each symbol represents a single mouse unless specified otherwise. P-values determined by one-tailed Wilcoxon (f), linear regression (g), two-tailed Wilcoxon (h, i), Kruskal–Wallis with Dunn’s multiple comparisons test (j), or one-tailed Mann–Whitney (k, l). Error bars indicate mean ± s.e.m.

Source data

Extended Data Fig. 3 Thymectomy and ovariectomy are insufficient to drive increases in N-glycan branching.

a-i) Female mice underwent thymectomy (a-c), ovariectomy (d-g), both surgeries (h, i), or corresponding sham procedures at the age of 9 weeks. Flow cytometry on blood at the indicated timepoints post-procedure was performed to detect percentage of CD4+ TN cells (a), IL7Rα expression (b, d, g, h), or L-PHA binding (c, e, f, i), gating on CD4+ TN cells. Absolute or normalized geometric mean fluorescence intensity (MFI) are shown. Each symbol at a particular timepoint represents a single mouse. P-values determined by one-tailed Mann–Whitney (b, d, g, h). Error bars indicate mean ± s.e.m.

Source data

Extended Data Fig. 4 Age-dependent increases in N-glycan branching suppress proinflammatory T cell function.

a) L-PHA binding of splenocytes from Mgat2fl/fl and Mgat2fl/fllck-cre female mice, gated on CD4+ TN cells. b) Splenocytes from an old mouse were treated with or without kifunensine for 24 hours, then analyzed for L-PHA binding by flow cytometry, gating on CD4+ TN cells. c) Splenocytes from female mice of the indicated ages and genotypes were activated with plate-bound anti-CD3 for 15 minutes. Following fixation and permeabilization, phospho-ERK1/2 induction was analyzed in CD4+ TN cells by flow cytometry, gating additionally on L-PHA negative cells in Mgat2fl/fl/lck-cre mice. d, e) Flow cytometric analysis of purified mouse splenic CD4+ T cells activated with anti-CD3 and anti-CD28 for 4 days with TH17 (TGFβ + IL-6+IL-23) or iTreg (TGFβ) inducing conditions, gating additionally on L-PHA negative cells in Mgat2fl/fl/lck-cre mice. f) Female Mgat2fl/fl and Mgat2fl/fllck-cre mice were inoculated with streptomycin (0.1 ml of a 200 mg/ml solution in sterile water) intragastrically one day prior to inoculation with S. Typhimurium (5×108 colony forming units, CFU per mouse) by oral gavage. CFU in the cecal content was determined 72 hours after infection. Data shown are representative of two (c), or at least three (a, b, d, e) independent experiments. P-values by one-tailed Mann–Whitney (f). Error bars indicate mean ± s.e.m (c, d, e) or geometric mean (f).

Source data

Extended Data Fig. 5 Age-dependent increases in N-glycan branching suppress T cell activity in human females.

a-d) Human PBMCs from healthy females (a, c) or males (b, d) as indicated were analyzed for L-PHA binding by flow cytometry gating on CD8+ T cells (a, b) or CD19+ B cells (c, d). e, f) CD4+ TN cells (CD45RA+CD45RO−) from healthy females (e) and males (f) under the age of 65 were analyzed for L-PHA binding by flow cytometry. g) Human PBMCs from young (22-38 years old) and old (90-94 years old) female subjects were analyzed for L-PHA binding on CD4+ TN cells (CD45RA+CD45RO−) before or after 96 hours of culture in complete media. Shown is the ratio or each old subject over the average of the young at the two timepoints. Each symbol represents a single individual. R2 and p-values by linear regression (a-f) or by paired one-tailed t test, following passage of Shapiro–Wilk normality test (g). Error bars indicate mean ± s.e.m.

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Extended Data Fig. 6 N-acetylglucosamine and IL-7 synergize to raise N-glycan branching in human T cells.

a, b) PBMCs from nine healthy female donors (28-45 years old) were cultured with or without rhIL-7 (50 ng/ml) and/or GlcNAc (10 mM or 40 mM) for 9 days, then analyzed for L-PHA binding by flow cytometry, gating on CD4+ TEM (CD45RA−CD45RO+CCR7−) cells (a) or CD8+ TEM (CD45RA−CD45RO+CCR7−) cells (b). c) Mouse plasma from female mice of the indicated ages was analyzed for HexNAc levels by LC-MS/MS. d, e) Flow cytometric analysis of human PBMCs stimulated by anti-CD3 in the presence or absence of kifunensine as indicated for 24 hours to analyze for activation marker CD69 (d) or 72 hours to assess proliferation by CFSE dilution (e), gating on CD4+ T cells. f) Female human PBMCs were treated in vitro with kifunensine for 24 hours, followed by analysis of L-PHA binding on CD4+ TN cells by flow cytometry. Data shown is representative of three independent experiments with different donors. g) Female human PBMCs were treated in vitro with kifunensine for up to four days, followed by analysis of L-PHA binding on CD4+ TN cells by flow cytometry. P-values by Kruskal–Wallis with Dunn’s multiple comparisons test (a, b), two-tailed Mann–Whitney (c), and one-tailed Mann–Whitney (d, e). Error bars indicate mean ± s.e.m.

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Mkhikian, H., Hayama, K.L., Khachikyan, K. et al. Age-associated impairment of T cell immunity is linked to sex-dimorphic elevation of N-glycan branching. Nat Aging 2, 231–242 (2022). https://doi.org/10.1038/s43587-022-00187-y

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