key: cord-0275825-e7dk3dhg
authors: Nicoli, Francesco; Cabral-Piccin, Mariela P.; Papagno, Laura; Gallerani, Eleonora; Folcher, Victor; Dubois, Marion; Clave, Emmanuel; Vallet, Hélène; Frere, Justin J.; Gostick, Emma; Llewellyn-Lacey, Sian; Price, David A.; Toubert, Antoine; Boddaert, Jacques; Caputo, Antonella; Gavioli, Riccardo; Appay, Victor
title: Altered basal lipid metabolism underlies the functional impairment of naive CD8+ T cells in elderly humans
date: 2020-09-25
journal: bioRxiv
DOI: 10.1101/2020.09.24.311704
sha: 19ded2debb33e040c6f3306cc849039667e28778
doc_id: 275825
cord_uid: e7dk3dhg

Aging is associated with functional deficits in the naive T cell compartment, which compromise the generation of de novo immune responses against previously unencountered antigens. The mechanisms that underlie this phenomenon have nonetheless remained unclear. We identified an age-related link between altered basal lipid metabolism and impaired antigen responsiveness in the naive CD8+ T cell compartment. These abnormalities were associated with an enhanced susceptibility to activation-induced apoptosis and could be recapitulated in vitro by exposure to the homeostatic cytokine interleukin (IL)-7. Importantly, reversal of the bioenergetic anomalies with lipid-altering drugs, such as rosiglitazone, almost completely restored the functional capabilities of naive CD8+ T cells. Interventions that favor lipid catabolism may therefore find utility as adjunctive therapies in the elderly to promote vaccine-induced immunity against emerging pathogens, such as seasonal influenza viruses and SARS-CoV-2.

Life expectancy has increased considerably over the last century as a consequence of advances in 54 medicine and improved public health systems. However, old age is associated with a high 55 prevalence of chronic diseases and an increased susceptibility to cancer and emerging pathogens, 56 such as SARS-CoV-2 (F. Nicoli et al., 2020) . Age-related deficits in the immune system are 57 thought to play a key role in the development of many pathological conditions (Dorshkind & Systemic metabolic disturbances are common in elderly individuals, and increased levels of 70 adipokines and proinflammatory lipid species in particular have been implicated as critical 71 mediators of inflammaging, which is thought to exacerbate many age-related diseases (Baylis, 72 Bartlett, Patel, & Roberts, 2013) . In this study, we investigated the bioenergetic features of naive 73 CD8 + T cells in middle-aged and elderly humans, aiming to establish a link between metabolic 74 disturbances and immunosenescence. Naive CD8 + T cells displayed a constellation of functional 75 and metabolic abnormalities in elderly people, including enhanced lipid influx and storage 76 accompanied by a marked susceptibility to apoptosis and impaired proliferation in response to activation via the TCR. Importantly, these deficits were rectified in the presence of lipid-altering 78 drugs, opening potential therapeutic avenues to slow the process of immunosenescence.

Naive CD8 + T cells in the elderly are susceptible to apoptosis and exhibit impaired 82 proliferation after stimulation via the TCR 83

In preliminary experiments, we compared the activation profiles of naive CD8 + T cells in middle-84 aged and elderly individuals, mimicking antigen-driven signals with plate-bound anti-CD3. No 85 age-related differences in activation per se were detected 24 hr after stimulation, as determined by 86 measuring the upregulation of CD69 and CD134 ( Figure 1A ). Signals transduced via the TCR 87 elicit an mTOR-driven metabolic switch that supports the function and the viability of activated 88 naive CD8 + T cells (F. Nicoli, L. Papagno, et al., 2018). We therefore assessed mTOR activity in 89 parallel by quantifying phospho-S6 (pS6). In line with the comparable activation profiles, naive 90 CD8 + T cells in middle-aged and elderly individuals upregulated mTOR activity to a similar 91 extent after stimulation with plate-bound anti-CD3 ( Figure 1B) . 92

To extend these findings, we measured the expression of metabolism-related genes, comparing 93 unstimulated and activated naive CD8 + T cells ( Figure 1C ). Genes encoding various enzymes 94 involved in glycolysis were upregulated similarly in flow-sorted naive CD8 + T cells from middle-95 aged and elderly individuals after stimulation with plate-bound anti-CD3. In contrast, genes 96 associated with lipid metabolism or signaling pathways involved in metabolic regulation were not 97 generally upregulated in response to stimulation, with the exception of MYC, which was 98 overexpressed in activated naive CD8 + T cells, irrespective of age. Genes that play a critical role 99 in the metabolic switch were also overexpressed in activated naive CD8 + T cells, irrespective of 100 age, with the exception of HIF1 and RPS6KB1, which were upregulated to a greater extent in 101 activated naive CD8 + T cells from middle-aged versus elderly individuals. 102 PBMCs from middle-aged (Mid) and elderly individuals (Old) were incubated in the absence or 106 presence of plate-bound anti-CD3. Surface expression of the activation markers CD69 and CD134 107 was measured after 24 hr (A), and intracellular expression of mTOR activity marker pS6 was 108 measured after 3 hr (B Despite these phenotypic and transcriptional similarities, naive CD8 + T cells from middle-aged 116 individuals proliferated to a greater extent than naive CD8 + T cells from elderly individuals in 117 response to stimulation with plate-bound anti-CD3 (Figure 2A ), consistent with a previous report 118 (Briceno et al., 2016) . Early apoptosis was also less commonly observed among activated naive 119 CD8 + T cells from middle-aged versus elderly individuals, as determined by measuring the expression of active caspase-3 ( Figure 2B ). In addition, we noted a strong inverse correlation 121 between the frequency of naive CD8 + T cells that proliferated after stimulation and the frequency 122 of naive CD8 + T cells that expressed active caspase-3 after stimulation, irrespective of age (Figure 123 2C). 124

Collectively, these data revealed that enhanced susceptibility to apoptosis was associated with an 125 age-related deficit in the proliferative capabilities of naive CD8 + T cells, despite a largely 126 unaltered response to activation via the TCR. 127 frequency of naive CD8 + T cells that proliferated after stimulation and the frequency of naive 135 CD8 + T cells that expressed active caspase-3 after stimulation. Each dot represents one donor. 136

Significance was determined using Spearman's rank correlation. 137 138 139

Unstimulated naive CD8 + T cells from middle-aged individuals expressed CD134 less commonly 141 than unstimulated naive CD8 + T cells from elderly individuals ( Figure 1A ). To consolidate this 142 observation, which suggested increased levels of basal activation in the elderly, we measured the 143 expression of T-bet, which is classically upregulated in response to activation via the TCR. The 144 basal expression frequencies of T-bet mirrored the basal expression frequencies of CD134 ( Figure 3A ). Equivalent results were obtained using a more stringent definition of naive CD8 + T cells 146 (Figures S1A and S1B), which excluded phenotypically similar memory CD8 + T cells (Pulko et 147 al., 2016) . The basal expression frequency of T-bet also correlated directly with the activation-148 induced expression frequency of active caspase-3 among naive CD8 + T cells ( Figure 3B ). 149

Glycolysis is the main metabolic pathway that supports the activation of naive CD8 + T cells (F. Whitney U test. 171

Collectively, these data revealed an age-related increase in the basal activation status of 172 unstimulated naive CD8 + T cells, which appeared to be independent of the glycolysis pathway or 173 triggering via the TCR. 174 175 176 Naive CD8 + T cells in the elderly are metabolically distinct 177

To explore these age-related homeostatic changes in more detail, we investigated the potential 178 roles of other metabolic pathways. Interestingly, fatty acid (FA) uptake was decreased among 179 unstimulated naive CD8 + T cells from middle-aged versus elderly individuals ( Figure 4A ), but 180 this difference was not associated with significant changes in the expression levels of genes 181 encoding various enzymes involved in FA oxidation (FAO) or FA synthesis (FAS). However, we 182 noted that DGAT1, which encodes diacylglycerol O-acyltransferase 1, a key enzyme involved in 183 the storage of FAs as triacylglycerol (TAG), was expressed at lower levels in unstimulated naive 184 CD8 + T cells from middle-aged versus elderly individuals ( Figure S2B ). In line with these results, 185

we also found that unstimulated naive CD8 + T cells from middle-aged individuals stored lower 186 amounts of neutral lipids (NLs) than unstimulated naive CD8 + T cells from elderly individuals 187 ( Figure 4B ). mitochondrial mass was largely unaffected by age ( Figure 4E ). We also noted a direct correlation 217 between ΔΨM and the frequency of unstimulated naive CD8 + T cells that expressed T-bet, 218 suggesting a link with the loss of quiescence ( Figure S3 ). 219 220 Collectively, these data revealed an age-related shift in the basal metabolic properties of naive 221 CD8 + T cells, typified by high levels of FA uptake and NL storage and a supranormal ΔΨM. Figure 5C ). In contrast, no inhibition was observed if IL-7 was 240 added to the cultures on day 4 or day 7, after the initial encounter with cognate antigen (Fig.  241   S4A) . Moreover, the addition of IL-7 on day 10 enhanced the recovery of EV10-specific CD8 + T 242 cells on day 30, consistent with a survival effect in the memory compartment ( Figure S4B) . 243 244 Collectively, these data revealed an association between altered basal lipid metabolism and 245 impaired antigen responsiveness as a function of age in the naive CD8 + T cell compartment, both 246 of which could be recapitulated with exogenous IL-7. 247 T cells that expressed active caspase-3 correlated directly with basal levels of FA uptake (Figure 265 6A) and NL content ( Figure 6B ). To determine the biological relevance of these associations, we 266 treated naive CD8 + T cells with rosiglitazone, a drug known to foster lipid catabolism by 267 activating triglyceride lipase (Kershaw et al., 2007) and preventing the conversion of FAs into 268

NLs (Askari et al., 2007) . Pretreatment with rosiglitazone inhibited activation-induced apoptosis 269 among naive CD8 + T cells, irrespective of age ( Figure 6C ). Similar results were obtained using 270 fenofibrate, which induces lipid catabolism by enhancing FAO (Figure S5 7A). We also found that proliferation under otherwise identical conditions was inhibited in the 287 presence of hexadecyltrimethylammonium bromide (CTAB), which disrupts FA uptake (Figure 288 7B). This result was expected in light of the fact that FAs are essential components of cell membranes. However, preincubation with palmitic acid, a common saturated FA, similarly 290 inhibited the proliferation of naive CD8 + T cells in response to stimulation with plate-bound anti-291 CD3 ( Figure 7C ). To explore these opposing effects in more detail, we tested our system using 292 two interventions, namely serum starvation, which inhibits FA uptake, and exposure to 293 rosiglitazone, which boosts NL catabolism ( Figure 7D ). Naive CD8 + T cells from elderly 294 individuals proliferated to a greater extent after serum starvation, and the addition of rosiglitazone 295 further enhanced these activation-induced proliferative responses to levels approaching those 296 observed in direct comparisons with untreated naive CD8 + T cells from middle-aged individuals 297 ( Figure 7E ). Of note, these interventions suppressed the activation-induced proliferation of naive 298 CD8 + T cells from middle-aged individuals, highlighting the importance of basal activation and 299 lipid metabolism as determinants of therapeutic efficacy. 300

To assess the potential relevance of these findings in the context of antigen-driven immune 302 responses, we used our in vitro model to prime naive CD8 + T cells specific for EV10 (Briceno et 

Bystander hyperactivation of preimmune CD8+ T cells in chronic HCV patients

Metabolic pathways in T cell 551 activation and lineage differentiation

Fatty acid metabolic reprogramming via mTOR-mediated inductions of 555 PPARgamma directs early activation of T cells

Grubeck-558

T cell receptor-mediated activation is a potent inducer of 559 macroautophagy in human CD8(+)CD28(+) T cells but not in CD8(+)CD28(-) T cells

Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to 563 diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-564 gamma-independent mechanism in human arterial smooth muscle cells and 565 macrophages

Understanding how we age: 567 insights into inflammaging

Metabolic syndrome, aging and involvement of 569 oxidative stress

Reduced naive CD8(+) T-cell priming efficacy in elderly adults

Posttranscriptional control of T cell effector function by aerobic glycolysis

Combination therapy strategies for improving 577 PD-1 blockade efficacy: a new era in cancer immunotherapy

PPAR-Induced Fatty Acid Oxidation 580 in T Cells Increases the Number of Tumor-Reactive CD8(+) T Cells and Facilitates Anti

Dramatic increase in naive T cell turnover is linked to loss of naive T cells from old 584 primates

IL-7-586 Induced Glycerol Transport and TAG Synthesis Promotes Memory CD8+ T Cell Longevity

Fatty acids, lipid 589 mediators, and T-cell function

Age-associated declines in immune system development and 591 function: causes, consequences, and reversal

Elderly human hematopoietic progenitor cells express cellular senescence 595 markers and are more susceptible to pyroptosis

Mechanisms involved in Jurkat cell death induced by oleic and linoleic acids

The many faces of IL-7: from lymphopoiesis to peripheral T cell 601 maintenance

As Two Sides of the Same Coin: Friends or Foes? 604 Front Immunol

Obesity altered T cell metabolism and the response to 606 infection

Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic 609 switch

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The STING ligand cGAMP potentiates the efficacy of vaccine-induced CD8+ T cells

ID2 616 (inhibitor of DNA binding 2) is a rhythmically expressed transcriptional repressor required 617 for circadian clock output in mouse liver

Foxp3 drives oxidative phosphorylation and protection from lipotoxicity

Early Inhibition of Fatty Acid Synthesis Reduces Generation of 624 Memory Precursor Effector T Cells in Chronic Infection

PPARgamma regulates adipose triglyceride lipase in adipocytes in vitro and in vivo

Aging is associated with a shift of fatty 630 metabolism toward lipogenesis

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Computationally Modeling Lipid Metabolism and 640 Aging: A Mini-review

PGC-1alpha overexpression results in increased hepatic fatty acid oxidation 644 with reduced triacylglycerol accumulation and secretion

Angry, Hungry T-Cells: How Are T-Cell Responses Induced in Low Nutrient 647 Conditions? Immunometabolism

The HIV-1 Tat protein affects human CD4+ T-cell programing and activation, and favors 650 the differentiation of naive CD4+ T cells

Naive CD8(+) T-Cells Engage a Versatile Metabolic Program Upon Activation in Humans 654 and Differ Energetically From Memory CD8(+) T-Cells. Front Immunol

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A guide to immunometabolism for 666 immunologists

Memory CD8(+) T cells use cell-intrinsic lipolysis to support the 669 metabolic programming necessary for development

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Figure S1. T-bet expression in naive CD8 + T cells. (A) Flow cytometric gating strategy used to 745 exclude memory CD8 + T cells with a naive phenotype (MNP). (B) T-bet expression in naive

Each dot represents one donor. Horizontal lines indicate 748 median values

Expression of metabolism-related genes in naive CD8 + T cells. (A & B) Expression 751 levels of genes related to glucose (A) and fatty acid metabolism (B) were measured in 752 unstimulated naive CD8 + T cells flow-sorted from middle-aged (n = 5; Mid) and elderly 753 individuals (n = 5; Old). Bars indicate mean ± SEM

Effect of IL-7 on primed epitope-specific CD8 + T cells. (A & B) EV10-specific

CD8 + T cells were primed and expanded for 10 days in the presence of Flt3 ligand and TLR8L

Cells were left unmanipulated or treated with IL-7 on day 4 or day 7 and tetramer-stained on 767 day 10

Each dot represents one donor. * p < 0.05 769 (Wilcoxon signed rank test). NT: not treated

Active caspase-3 expression was measured after 24 hr. Data are 777 shown for naive CD8 + T cells. Left panel: representative flow cytometry profiles. Right panel: 778 data summary. Bars indicate mean ± SEM

All peptides were synthesized at >95% purity (BioSynthesis Inc.). The EV20 peptide 482 (YTAAEELAGIGILTVILGVL, Melan-A 21-40/A27L ) was used for in vitro priming studies. 483Fluorochrome-labeled tetrameric complexes of HLA-A*02:01-EV10 (ELAGIGILTV, Melan-484 A 26-35/A27L ) were generated in-house as described previously (Price et al., 2005) .