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. 2020 Jun 5;11(1):2857.
doi: 10.1038/s41467-020-16633-7.

Metabolic characteristics of CD8+ T cell subsets in young and aged individuals are not predictive of functionality

Kylie M Quinn  1   2 , Tabinda Hussain  3 , Felix Kraus  3 , Luke E Formosa  3 , Wai K Lam  3 , Michael J Dagley  4 , Eleanor C Saunders  4 , Lisa M Assmus  3   5 , Erica Wynne-Jones  6 , Liyen Loh  6 , Carolien E van de Sandt  6   7 , Lucy Cooper  3 , Kim L Good-Jacobson  3 , Katherine Kedzierska  6 , Laura K Mackay  6 , Malcolm J McConville  4 , Georg Ramm  4   8 , Michael T Ryan  3 , Nicole L La Gruta  9
Affiliations

Metabolic characteristics of CD8+ T cell subsets in young and aged individuals are not predictive of functionality

Kylie M Quinn et al. Nat Commun. .

Erratum in

Abstract

Virtual memory T (TVM) cells are antigen-naïve CD8+ T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (TMEM) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of TVM cells and their altered functionality with age, here we investigate TVM cell metabolism and its association with longevity and functionality. Elevated SRC is a feature of TVM, but not TMEM, cells and it increases with age in both subsets. The elevated SRC observed in aged mouse TVM cells and human CD8+ T cells from older individuals is associated with a heightened sensitivity to IL-15. We conclude that elevated SRC is a feature of TVM, but not TMEM, cells, is driven by physiological levels of IL-15, and is not indicative of enhanced functionality in CD8+ T cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. TVM cells have high SRC and CIV, which increases with age.
a Oxygen consumption rate (OCR) across time for sorted TN, TVM and TMEM cells from the spleens of naive young and aged SPF mice. Arrows indicate the addition of mitochondrial inhibitors (oligomycin; FCCP; antimycin A/rotenone) or timepoints for assessment of SRC (OCRBas, OCRMax) (n = 2–4, 5 experimental replicates). b Change in OCR from OCRBas to OCRMax (SRC) for each sorted subset (n = 2–4, 5 experimental replicates). c Electron microscope images of sorted cells directly ex vivo, scale bar indicates 0.2 µm (1 experimental replicate). d Confocal microscopy of sorted cells directly ex vivo, green fluorescence is Cytochrome C staining, scale bar indicates 2 µm, which was used to define (e), predominant mitochondrial morphology (fused, intermediate or fragmented) for 140 cells per subset and f average mitochondrial footprint per cell as calculated from confocal images (3 experimental replicates). g BN-PAGE and Blot for Cox5a from ETC CIV, with bands for CIV, CII, CIII2, CV and the CI/CIII2/CIV supercomplex indicated, alongside the Coomassie stained blot (3 experimental replicates). h Oxygen consumption rate (OCR) across time for sorted TVM cells from young mice, with high (200 µM) or low (5 µM) dose Etomoxir, (n = 3, 3 experimental replicates). Shown is mean ± standard error of the mean (SEM). NS indicates not significant, * indicates p ≤ 0.05, ** indicates p ≤ 0.01, unpaired t test.
Fig. 2
Fig. 2. TVM cells increase SRC with recent infection.
a OCR for sorted TN and TMEM cells from the spleens of uninfected young SPF mice or Tetramer+ TMEM (IAV) cells from IAV-infected mice (20 days post infection) and b change in OCR for each sorted subset (n = 4–2, 3 experimental replicates). c ECAR for sorted TN and TMEM cells from young uninfected mice or Tetramer+ TMEM (IAV) cells from IAV-infected mice (20 days post infection), with ECARBas and ECARMax indicated (n = 2–4, 3 experimental replicates). d OCR for sorted TN and TVM cells from young uninfected mice or TVM (IAV) cells from IAV-infected mice (20 days post infection) and e change in OCR for each sorted subset (n = 4–5, 3 experimental replicates). f ECAR for sorted TN and TVM cells from young uninfected mice or IAV-infected mice (20 days post infection) (n = 5, 3 experimental replicates). Shown is mean ± SEM. NS indicates not significant, * indicates p ≤ 0.05, ** indicates p ≤ 0.01, unpaired t test.
Fig. 3
Fig. 3. TVM cells comprise the majority of the CD44hiCD62Lhi TCM cell population.
a Histograms for CD49d expression on CD44hiCD62Lhi CD8+ T cells (TCM cells) from naive young mice, naive aged mice and young mice after infection with LCMV (40 days post infection), with bar graphs showing the proportion of TCM cells that are TVM cells (n = 4–5). b Overlays of CD44hiCD49dlo TVM cells on total CD8+ T cells with TEM/TCM cell gating (CD44hiCD62Llo and CD44hiCD62Lhi, respectively). c Overlays of CD44hiCD49dhi TMEM cells on total CD8+ T cells. d Representative dot plots identifying IAV-specific tetramer+ CD8+ T cells that are CD62Lhi (TCM cells) or CD62Llo (TEM cells) (60 days post infection), with bar graphs of the average frequency of tetramer+ CD8+ T cells that are in each subset (n = 5). e OCR for sorted TN and TVM cells from young uninfected mice and sorted TCM and TEM cells from LCMV-infected mice (60 days post infection) and f change in OCR for each sorted subset (n = 5–6, 3 experimental replicates). Data from a–d are representative of at least 2 individual experiments. Shown is mean ± SEM. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, unpaired t test.
Fig. 4
Fig. 4. SRC correlates with Bcl-2 expression but not with CD8+ T cell functionality.
a Percent lysis of OVA-loaded targets by sorted TN, TVM and TMEM cells from young or aged OT-I mice (n = 3–8). * Indicates p ≤ 0.05, ** indicates p ≤ 0.01, unpaired t test, data are representative of at least 3 individual experiments. Simple linear regression analyses of mean SRC from Fig. 1b against b the mean number of divisions of sorted CD8+ T cells following 60 h TCR stimulation from ref. , c the average percent lysis from (a), d the average proportion of sorted CD8+ T cells making IFN-γ at 36 h after TCR stimulation from ref. , and e the median fluorescence intensity (MFI) of Bcl-2 expression from ref. .
Fig. 5
Fig. 5. High IL-15 sensitivity in TVM cells increases with age and mediates increased SRC.
a IL-15Rβ MFI directly ex vivo from individual mice (n = 5–7) or b pSTAT5 MFI after 15 min of stimulation with IL-15 in vitro on sorted TN, TVM and TMEM cells from young or aged mice (n = 3). c Representative dot plots for CD8+ T cells gated on TN (CD44lo), TVM (CD44hiCD49dlo) and TMEM (CD44hiCD49dhi) cells (frequency of CD8+ T cells indicated) and d frequency of CD8+ T cells in each subset in WT or IL-15 KO mice (n = 5 for WT and 7 for KO). e OCR for sorted TN, TVM and TMEM cells from young uninfected, IAV-infected and IAV-infected/IL-15 neutralising mAb treated mice and f change in OCR for each sorted subset. Shown is mean ± SEM. * Indicates p ≤ 0.05, ** indicates p ≤ 0.01, unpaired t test, data for a, b, e, f are representative of at least 3 individual experiments.
Fig. 6
Fig. 6. Increased IL-15Rβ expression and SRC in older human CD8+ T cells.
a IL-15Rβ MFI directly ex vivo on CD8 T cell subsets from individual young (20–30 yo) or older (60–80 yo) adult human donors (n = 6–7) and b Representative histograms of IL-15Rβ expression, with expression on CD4+ T cells used as a control. c OCR for enriched CD8+ T cells from young or older human donors and d change in OCR for young or older human donors. This experiment was performed once. Bars or datapoints represent mean ± SEM. * Indicates p ≤ 0.05, ** indicates p ≤ 0.01, unpaired t test.
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