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. 2020 Dec 15;9:e57950. doi: 10.7554/eLife.57950

Autophagy in T cells from aged donors is maintained by spermidine and correlates with function and vaccine responses

Ghada Alsaleh 1,†,✉, Isabel Panse 1,†, Leo Swadling 2, Hanlin Zhang 1, Felix Clemens Richter 1, Alain Meyer 3, Janet Lord 4, Eleanor Barnes 5,6,7, Paul Klenerman 5,6,7, Christopher Green 8, Anna Katharina Simon 1,✉
Editors: David Furman9, Jessica K Tyler10
PMCID: PMC7744099  PMID: 33317695

Abstract

Vaccines are powerful tools to develop immune memory to infectious diseases and prevent excess mortality. In older adults, however vaccines are generally less efficacious and the molecular mechanisms that underpin this remain largely unknown. Autophagy, a process known to prevent aging, is critical for the maintenance of immune memory in mice. Here, we show that autophagy is specifically induced in vaccine-induced antigen-specific CD8+ T cells in healthy human volunteers. In addition, reduced IFNγ secretion by RSV-induced T cells in older vaccinees correlates with low autophagy levels. We demonstrate that levels of the endogenous autophagy-inducing metabolite spermidine fall in human T cells with age. Spermidine supplementation in T cells from old donors recovers their autophagy level and function, similar to young donors’ cells, in which spermidine biosynthesis has been inhibited. Finally, our data show that endogenous spermidine maintains autophagy via the translation factor eIF5A and transcription factor TFEB. In summary, we have provided evidence for the importance of autophagy in vaccine immunogenicity in older humans and uncovered two novel drug targets that may increase vaccination efficiency in the aging context.

Research organism: Human

Introduction

The outbreak of coronavirus disease 2019 (COVID-19) caused a great threat to global public health in 2020 with the majority of deaths occurring in older adults. The development of effective treatments and vaccines against COVID-19 is now more than ever becoming a pressing and urgent challenge to overcome (Zhang et al., 2020; Lurie et al., 2020). However, the successful vaccination of the elderly against pathogens is considered one of the big challenges in our society (Weinberger, 2018; Chen et al., 2009). Immunosenescence, which is characterized by poor induction and recall of B and T memory responses upon exposure to new antigens, can lead to reduced immune responses following immunization of older adults. While most vaccines are less immunogenic and effective in the older population (Weinberger, 2018), little is known about the molecular mechanisms that underpin immune senescence. Autophagy is thought to be one of the few cellular processes that underlie many facets of cellular aging including immune senescence (Zhang et al., 2016). By delivering unwanted cytoplasmic material to the lysosomes for degradation, autophagy limits mitochondrial dysfunction and accumulation of reactive oxygen species (ROS) (Rubinsztein et al., 2011). Autophagy degrades protein aggregates that accumulate with age and its age-related decline could contribute to ‘inflamm-aging’ (Salminen et al., 2012), the age-related increase in inflammatory cytokines in the blood and tissue. Loss of autophagy strongly promotes production of the inflammatory cytokines TNFα, IL-6, and IL1-β (Saitoh et al., 2008; Stranks et al., 2015). We previously found autophagy levels decline with age in human peripheral CD8+ T cells (Phadwal et al., 2012). Deletion of key autophagy genes leads to a prematurely aged immune phenotype, with loss of function in mouse memory CD8+ T cells (Xu et al., 2014; Puleston et al., 2014), hematopoietic stem cells (Mortensen et al., 2011), and macrophages (Stranks et al., 2015) with a myeloid bias (Mortensen et al., 2011). In addition, we find in autophagy-deficient immune cells the same cellular phenotype that cells display in older organisms; they accumulate ROS and damaged mitochondria (Stranks et al., 2015; Puleston et al., 2014).

Importantly, we can improve CD8+ T memory responses from aged mice with spermidine (Puleston et al., 2014), an endogenous metabolite synthesized from arginine. It was shown in yeast and other model organisms that spermidine extends life-span via increased autophagy (Madeo et al., 2018). Several downstream mechanisms of spermidine-induced autophagy have been described in mice, including the inhibition of histone de-acetylases (Madeo et al., 2018). Recently, we uncovered a novel pathway in which spermidine donates a residue for the hypusination of the translation factor eIF5a, which is necessary for the translation of a three proline motif present in the master transcription factor of autophagy and lysosomal biogenesis, called TFEB (Zhang et al., 2019). We demonstrated that this pathway operates in activated B cells, which upon activation have an unusually high-protein synthesis rate, owing to the high production of immunoglobulins. It is likely that B cells may be particularly reliant on the unfolded protein response, the proteasome, and autophagy, to cope with this high rate of protein synthesis. B cells may have evolved special coping strategies including the translational signaling for autophagy via eIF5A and TFEB. We therefore sought to extend our findings to another immune cell type, CD8+ T cells, to investigate whether this pathway may be conserved in a related adaptive immune subset and possibly broadly applicable.

Here, we show for the first time that autophagy is indeed highly active in human CD8+ T cells after the in vivo encounter of antigens in donors from two different experimental vaccination trials. Our data show that polyamine levels fall with age in peripheral mononuclear cells. When supplemented with spermidine, the dysfunctional autophagic flux can be rejuvenated in CD8+ T cells from old donors, and levels of the important effector molecules IFNγ and perforin are enhanced as a consequence. Moreover, autophagy and effector function are maintained by spermidine in T cells from young donors. Lastly, in human CD8+ T cells we show that spermidine signals via eIF5A and TFEB to maintain autophagy levels. This study demonstrates that the function of human CD8+ T cells can be improved with spermidine. Taken together with our previous work on B cells, this leads us to the hypothesis that both T and B cell responses to infections and vaccinations are exquisitely reliant on sufficient autophagy levels, which are maintained by intracellular spermidine. This work highlights the potential of spermidine as a vaccine adjuvant in the older adults.

Results

First, we optimized a flow cytometry-based assay to reliably and reproducibly measure autophagy, before applying it to measure autophagy after in vivo antigen-stimulated T cells post-vaccination. To inhibit the autophagic flux and thereby degradation of LC3-II, the lysosomal inhibitor bafilomycin A was added to the culture for 2 hr before washing out non-membrane-bound LC3-I and staining. In Jurkat cells, a human lymphocyte line, we confirmed that treatment with bafilomycin A1 increases LC3-II by both flow cytometry and western blot (Figure 1—figure supplement 1a and b). Similar results were obtained in PBMCs stimulated in vitro (Figure 1—figure supplement 1c and d). Next, we used in vitro stimulated control PBMCs obtained from five young donors (22–50 years old), bled on three different occasions, 2 weeks apart, were stimulated with either IFNγ/LPS, anti-CD40 /-IgM or anti-CD3/CD28 to induce autophagy in monocytes (Figure 1—figure supplement 2a), B cells (Figure 1—figure supplement 2b), CD4+ T (Figure 1—figure supplement 2c), or CD8+ T (Figure 1—figure supplement 2d) cells, respectively.

Taken together, the data show: (a) autophagic flux varies little within one individual between blood draws; (b) there is limited variation between individuals; (c) bafilomycin A treatment leads to accumulation of LC3-II in all cell types; (d) the respective stimulations via TCR/BCR/TLR or IFNγ-R weakly induce autophagic flux which is consistently further increased in the presence of bafilomycin A.

Published human studies so far have limited their analysis of TCR-induced autophagy to non-specific stimulation in vitro such as anti-CD3/CD28. Here, we took advantage of an existing cohort of healthy human donors that were vaccinated with a candidate HCV vaccine encoding HCV non-structural proteins (NSmut). Healthy volunteers received an adenoviral vector prime vaccination (ChAd3-NSmut or Ad6-NSmut) followed by a heterologous adenoviral boost vaccination (ChAd3-NSmut or Ad6-NSmut) or an MVA-NSmut boost vaccination (Figure 1a and Figure 1—figure supplement 3; Swadling et al., 2016; Barnes et al., 2012).

Figure 1. Autophagy is induced by vaccination in antigen-specific T cells and correlates with donor age.

PBMCs were isolated from blood samples of vaccinated healthy donors. LC3-II was measured in CD8+ cells using flow cytometry after 2 hr treatment with 10 nM bafilomycin A1 (BafA1) or vehicle. Autophagic flux was calculated as LC3-II mean fluorescence intensity (BafA1-Vehicle)/Vehicle. (a) Vaccine regimen for HCV and RSV trials. (b) Representative plots showing BafA1 in light blue and vehicle pink. (c) Quantification in HCV non-specific CD8+ T cells and HCV-specific CD8+ T cells detected by HCV pentamers from 10 vaccinees (includling duplicates) using different HCV vaccine regimens, priming with ChimAd and boosting with MVA or AD6 vectors. Autophagy was measured at the peak of the T cell response post prime vaccination, peak of the T cell response post boost vaccination and at the end of the study. (d) Autophagic flux was measured in CD8+ cells from young donors (N = 12,<65 years) and old donors (N = 21,>65 years) vaccinated with respiratory syncytial virus (RSV) 7 days after last boost, quantification calculated as mentioned above. Data represented as mean ± SEM. (e, f) Correlation of autophagic flux with total response to peptide pools specific T cell IFNγ response to RSV exposure measured by ELISpot in CD8+ cells from old donors (e) and young donors (f), donors as in (d). Linear regression with 95% confidence intervals from old and young donors. The goodness of fit was assessed by R2. The p value of the slope is calculated by F test.

Figure 1—source data 1. Autophagy is induced by vaccination in antigen-specific T cells and correlates with donor age.

Figure 1.

Figure 1—figure supplement 1. Autophagy levels by flow cytometry-based assay and conventional LC3 western blot in Jurkat cell line and PBMCs.

Figure 1—figure supplement 1.

(a–b) Human Jurkat T cell line was cultured for 24 hr and treated with or without Bafilomycin A1 for the last 2 hr. Cells were split into two aliquots, representative flow cytometry-based assay (a), representative western blot for LC3-II and GAPDH for the same sample (b). (c–d) PBMCs from young human donors were cultured with anti-CD3/CD28 for 3 days in the absence/presence of Bafilomycin A1 for the last 2 hr. Cells were split into two aliquots, representative flow cytometry-based assay (c), representative western blot for LC3-II and GAPDH for the same sample (d).

Figure 1—figure supplement 2. LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws.

Figure 1—figure supplement 2.

PBMCs were generated from blood taken at 3 weeks intervals (samples 1, 2, 3) from young human donors and were cultured for 24 hr, in the abence/presence of Bafilomycin A1 for the last 2 hr. Here basal autophagic flux was calculated as LC3-II mean fluorescence intensity (treatment-basal)/basal. Monocytes gated on CD14+ treated with IFNγ or LPS (a), B cells gated on CD19+ treated with anti-CD40L and anti-IgM (b), CD4+ T cells gated on CD3+CD4+ treated with anti-CD3/CD28 (c), CD8+ T cells gated on CD3+ CD8+ treated with anti-CD3/CD28 (d).
Figure 1—figure supplement 2—source data 1. LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws.

Figure 1—figure supplement 3. Regimen of immunizations and blood sampling for HCV trail.

Figure 1—figure supplement 3.

HCV = Hepatitis C virus, ChAd = Chimpanzee Adenoviral Vector, MVA = Modified Ankara Virus vector.

Figure 1—figure supplement 4. Regimen of immunizations and blood sampling for RSV trail.

Figure 1—figure supplement 4.

RSV = respiratory syncitial virus, ChAd = Chimpanzee Adenoviral Vector, MVA = Modified Ankara Virus vector, Unlike for HCV, the adults in the RSV study will have prior immune responses that have been boosted by natural exposure throughout life. In the context of RSV, we still use the term ‘prime’ to mean the first dose of vaccine. Similarly, the term ‘boost’ means the second dose of vaccine and not exposure.

Figure 1—figure supplement 5. Correlation of age with total and peptide-pool specific T cell IFNγ response to RSV exposure measured by ELISpot in CD8+ cells, donors as in Figure 1e.

Figure 1—figure supplement 5.

Linear regression with 95% confidence intervals from old and young donors. The goodness of fit was assessed by R2. The p value of the slope is calculated by F test.
Figure 1—figure supplement 5—source data 1. Correlation of age with total and peptide-pool specific T cell IFNγ response to RSV.
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To identify HCV-specific CD8+ T cells, PBMCs were co-stained with an MHC class I pentamer (HLA-A*02:01, HCV peptide KLSGLGINAV) and anti-LC3-II (Figure 1b). When measured at the peak magnitude of the T cell response to vaccination (2–4 weeks post Ad, 1 week post MVA) HCV-specific CD8+ T cells show a significant increase in autophagic flux that is not observed in HCV non-specific CD8+ T cells (Figure 1c). In antigen-specific T cells, autophagic flux is highest shortly after vaccination but had declined to levels equivalent to antigen-non-specific T cells cells by the end of the study (week 36 or 52). Together, these data show that antigen exposure induces autophagic flux in the antigen-reactive CD8+ T cell subset in humans in vivo. In previous work, we found that autophagic flux was reduced in CD8+ T cells from 24-month-old mice 12. To test whether this is true in human CD8+ T cells from human individuals, and whether this correlates with vaccine immunogenicity, we measured autophagic flux in vaccinees of various ages that were given an experimental RSV vaccine (Figure 1a and Figure 1—figure supplement 4).

As older adults are particularly susceptible to severe disease from RSV infection, the vaccine was given to two age groups (18–50 years and 60–75 years of age). As expected, the naturally derived population of RSV-specific IFNγ-producing CD8+ T cells in peripheral circulation in response to the infection declines with age (Figure 1—figure supplement 5).

No MHC class I pentamers were available for RSV to identify antigen-specific T cells; however, bulk T cells of the >60 year vaccinees showed significantly lower basal autophagic flux (Figure 1d). We correlated autophagy levels in T cells with IFNγ ELISpot responses in individual vaccinees and found a strong inverse correlation in the aged group (Figure 1e) between autophagy and IFNγ responses, but not in the young group (Figure 1f). Taken together these data suggest that reduced T cell autophagy in aged T cells may underpin reduced T cell responses to vaccination. To gain better insight into the role of autophagy in T cells function, we sorted and activated splenocytes from control and autophagy-deficient mice, in which the essential autophagy gene Atg7 was deleted with Cre driven by the CD4 promotor (Atg7Δcd4). Both intracellular IFNγ expression and secretion was significantly decreased in Atg7-deficient T cells (Figure 2a and b). Similarly, intracellular perforin expression was reduced in Atg7-deficient T cells (Figure 2c). Next, we sought to confirm our data in human T cells using pharmacological autophagic flux inhibitors, namely hydroxychloroquine (HcQ) that inhibits autophagosomal-lysosomal fusion (Mauthe et al., 2018) and the AMPK1 and Ulk1 inhibitor SBI-0206965 (SbI) (Dite et al., 2018; Egan et al., 2015). Using these drugs, we could recapitulate the data observed in the genetic mouse model of autophagy deletion showing an inhibition of IFNγ (Figure 2d and e) and perforin production (Figure 2f) in CD8+ T cells. Conversely, activation of autophagy by Resveratrol (Res) (Rubinsztein et al., 2011; Morselli et al., 2010) in human CD8+ T cell increased the production of IFNγ and perforin (Figure 2g–i). In summary, these data suggest that autophagy is required for CD8+ T cell function by controlling IFNγ and perforin production.

Figure 2. Autophagy is required for CD8+ T cell function.

Figure 2.

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(a–c) Splenocytes from control mice (Ctrl: CD4-cre;Atg7+/+) and Atg7 knockout mice (Atg7Δcd4: CD4-cre;Atg7-/-) were cultured with anti-CD3/CD28 for 4 days and IFNγ assessed by ELISA in tissue culture supernatant (a), intracellular IFNγ by flow cytometry (b), intracellular perforin by flow cytometry (c), all gated on CD8+ T cells. (d–i) PBMCs from human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM Hydroxychloroquine (HcQ), 10 µM BSI-0206965 (SbI), 10 µM Resveratrol (Res) and IFNγ assessed by ELISA in tissue culture supernatant (d, g), intracellular IFNγ by flow cytometry (e, h), intracellular perforin by flow cytometry (f, i), all gated on CD8+ cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. Statistics by paired t-test for (d–i).

Figure 2—source data 1. Autophagy is required for CD8+ T cell function.

We have recently shown that treating old but not young mice with the metabolite spermidine improves autophagy levels in B lymphocytes due to an age-related decline of endogenous spermidine (Zhang et al., 2019). Here, we sought to confirm this in human lymphocytes. Thus, we determined spermidine, spermine and putrescine levels in PBMCs by gas chromatography-mass spectrometry (GC-MS), and found an inverse correlation between age and spermidine but not with spermine or putrescine (Figure 3a). We hypothesized that low levels of spermidine are responsible for low levels of autophagy and poor T cell function in PBMCs from old donors. We therefore tested whether supplementation with spermidine recovers T cell autophagy and function. As activation of PBMCs with anti-CD3/CD28 optimally induces autophagy levels on day 4 (Watanabe et al., 2014), we activated PBMCs from old donors in the presence of spermidine for 4 days and tested their autophagic flux and function by flow cytometry. Both autophagic flux and the secretion of IFNγ measured by ELISA was improved significantly in T cells from older vaccinees (Figure 3b and c). Similarly, increased IFNγ can be detected after spermidine treatment by intracellular staining for flow cytometry (Figure 3d). Interestingly, spermidine supplementation also increases the expression of perforin (Figure 3e) but not of Granzyme B (Figure 3f). Moreover, to investigate whether spermidine acts through autophagy to improve CD8+ T cell function, we added spermidine together with pharmacological autophagy inhibitors. Indeed, HcQ or Sbl ablates the rejuvenating effects of spermidine supplementation in old donor cells (Figure 3c–f). In contrast, spermine, another polyamine from the same pathway, which is not significantly reduced with age (Figure 3a) and does not serve as a direct substrate for eIF5A hypusination (Lee and Park, 2000), does not rescue CD8+ T cell function from old donors (Figure 3—figure supplement 1a–c). These data underline the importance of cellular spermidine levels in aged CD8+ T cells.

Figure 3. Spermidine declines with age and supplementing spermidine improves autophagy and CD8+ T cell function in old donors.

(a) Spermidine (Spd), spermine (Spm), and putrescine (Put) content of PBMCs collected from healthy donors were measured by GC-MS. Linear regression with 95% confidence intervals. The goodness of fit was assessed by R (Lurie et al., 2020). The p value of the slope is calculated by F test. (b–f) PBMCs from old human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM spermidine alone or with 10 µM Hydroxychloroquine (HcQ), 10 µM SBI-0206965 (SbI), and autophagic flux measured by flow cytometry (b), IFNγ assessed by ELISA in tissue culture supernatant (c), intracellular IFNγ by flow cytometry (d), intracellular perforin by flow cytometry (e), intracellular granzyme B (f), all gated on CD8+ cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. Statistics by paired t-test for (b–f).

Figure 3—source data 1. Spermidine declines with age and supplementing spermidine improves autophagy and CD8+ T cell function in old donors.

Figure 3.

Figure 3—figure supplement 1. Spermine does not improve function of CD8+ T cell from old donors.

Figure 3—figure supplement 1.

PBMCs from old human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM spermidine or 10 µM spermine and intracellular IFNγ assessed by flow cytometry (a), intracellular perforin by flow cytometry (b), intracellular granzyme B (d), all gated on CD8+ cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. Statistics by paired t-test for (a–c).
Figure 3—figure supplement 1—source data 1. Spermine does not improve function of CD8+ T cell from old donors.

Figure 3—figure supplement 2. Spermidine reduces mitochondrial mass in CD8+ T cell from old donors.

Figure 3—figure supplement 2.

PBMCs from old human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM spermidine and quantified for mitochondrial mass by flow cytometry using MitoTracker Green (MTG) (a) or nonylacridine orange (NAO) (b). Mitochondrial membrane potential was assessed by flow cytometry using TMRM dye (c) and mitochondrial ROS (mtROS) by mitoSOX staining (d). All gated on CD8+ cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. MFI normalized to Old untreated group. Statistics by paired t-test for (a–d).
Figure 3—figure supplement 2—source data 1. Spermidine reduces mitochondrial mass in CD8+ T cell from old donors.
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Polyamine biosynthesis has previously been shown to increase mitochondrial protein synthesis (Madeo et al., 2018; Puleston et al., 2019; Wang et al., 2020). Moreover, autophagy can ensure mitochondrial quality by selective degradation of damaged or dysfunctional mitochondria, a process called mitophagy, which is critical for maintaining cellular function (Palikaras et al., 2018; Vara-Perez et al., 2019). Since autophagy levels decline with age and damaged mitochondria accumulate in aged T cells (Bektas et al., 2019; Callender et al., 2020), we hypothesized that spermidine-induced autophagy may improve mitochondrial function in aged T cells. To test this, we measured mitochondrial function in CD8+ T cells isolated from old donors after spermidine treatment. We indeed show that mitochondrial mass was reduced after spermidine treatment as assessed by MitoTracker Green (MTG) and nonylacridine orange (NAO) staining, two fluorescent probes which have been commonly used to assess mitochondrial mass (Doherty and Perl, 2017; Figure 3—figure supplement 2a and b). The reduction of mitochondrial mass during spermidine treatment could be related to improved clearance of damaged mitochondria by mitophagy or decreased biogenesis. Damaged or dysfunctional mitochondria often show altered membrane potential and increased ROS) production (Puleston et al., 2014); however, we did not observe any changes in these parameters after spermidine treatment (Figure 3—figure supplement 2c and d). Our data suggest that spermidine treatment lowers mitochondrial content, presumably by mitophagy, but does not improve mitochondrial health. Mitochondria are not the only type of autophagic cargo and other cargos need to be investigated to understand exactly how autophagy induction improves T cell function.

Next, we investigated whether endogenous polyamine maintains autophagy levels in activated CD8+ T cells from young donors. The drug DFMO (α-difluoromethylornithine) inhibits polyamine biosynthesis by irreversibly inhibiting ornithine decarboxylase (ODC), a key enzyme conversion of ornithine into putrescine. Addition of DFMO almost completely blocked autophagy in CD8+ T cells activated over 7 days;, however, when cells were supplemented with spermidine autophagic flux was recovered (Figure 4a). DFMO also partially blocks IFNγ and perforin expression in anti-CD3/CD28 activated CD8+ T cells, which can be rescued by spermidine (Figure 4b and c). As before, granzyme B expression was not affected by DFMO or spermidine supplementation (Figure 4d). T cells from young donors, with their high endogenous spermidine levels, do not respond to spermidine supplementation (Figure 4e–g).

Figure 4. Endogenous spermidine maintains levels of autophagy and T cell function.

Figure 4.

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(a–d) PBMCs cells from young human donors (<65 years) were activated with anti-CD3/CD28 for 7 days and treated with spermidine synthesis inhibitor 1 mM DFMO alone or together with 10 µM spermidine (Spd). Autophagic flux (a) was assessed each day and IFNγ (b), Perforin (c), Granzyme B (d) were measured by flow cytometry in CD8+ cells on day 4. (e–f) PBMCs cells from young human donors (<65 years) were cultured with anti-CD3/CD28 for 4 days and streated with 10 µM spermidine. (e) Intracellular IFNγ, (f) intracellular perforin, (g) and intracellular granzyme B were measured in CD8+ cells by flow cytometry. Data represented as mean ± SEM.

Figure 4—source data 1. Endogenous spermidine maintains levels of autophagy and T cell function.

We previously found that spermidine maintains autophagic flux via hypusination of eIF5A which regulates TFEB expression (Zhang et al., 2019), and we sought to test this pathway in human T cells. Spermidine provides a moiety to the translation factor eIF5A, which in addition to its role in initiation and termination also promotes the translation of polyproline-rich domains, which are difficult to translate (Gutierrez et al., 2013). One such triproline motif-containing protein is TFEB, with mouse TFEB containing one triproline motif while human TFEB contains two. TFEB is the key master transcription factor of autophagosomal and lysosomal gene expression (Napolitano and Ballabio, 2016; Lapierre et al., 2015; Settembre et al., 2011). Here, we addressed whether this pathway operates in human T cells and accounts for the loss of autophagy and T cell function. We first verified in the human lymphocyte line Jurkat that the inhibitor GC7 inhibits the hypusinated/activated form of eIF5A (Figure 5a) and also confirmed that it decreases LC3-II expression in a dose-dependent manner (Figure 5b). GC7 reduces TFEB and hypusinated eIF5A in CD8+ T cells activated for 4 days (Figure 5c). It also diminishes the autophagic flux in activated CD8+ T cells over a time course of 7 days (Figure 5d). We then tested whether spermidine maintains eIF5A hypusination and TFEB levels in CD8+ T cells from young donors by depleting endogenous spermidine with DFMO. While anti-CD3/CD28 increases expression levels of TFEB and eIF5A, a reduction of hypusinated eIF5A and TFEB levels in PBMCs treated with DFMO was observed, which is rescued with spermidine (Figure 5e). As expected, spermidine treatment of PBMCs from young donors demonstrated to have high levels of spermidine does not induce this pathway (Figure 5—figure supplement 1) nor T cell function (Figure 3e–g). Finally, we sought to address whether naturally low endogenous spermidine levels can be rescued in PBMCs from old donors activated with anti-CD3/CD28. Again, we observed that activation induces levels of protein expression of both hypusinated eIF5A and TFEB, and spermidine further improves both eIF5A and TFEB two-fold (Figure 5f).

Figure 5. Spermidine’s mode of action is via eIF5A and TFEB in human CD8+ T cells.

(a–d) Human T cell line Jurkat was cultured for 24 hr with 100 µM GC7, then eIF5A and hypusinated eIF5A were measured by WB (a). Jurkat cell line was stimulated with increasing concentrations of GC7 and cell lysates blotted for LC3B (b). (c–d) PBMCs from young human donors were cultured with anti-CD3/CD28 for 7 days and treated with GC7. The protein levels of TFEB and eIF5A hypusination were measured in CD8+ cells by Western blot on day 4 (c) and autophagic flux was determined as in Figure 1 (d). PBMCs from young human donors were cultured with anti-CD3/CD28 for 4 days and treated with spermidine synthesis inhibitor DFMO alone or together with 10 µM spermidine and the protein levels of TFEB and eIF5A hypusination were measured in CD8+ cells by wWestern blot (e), representative images (left) and quantified (right). PBMCs from old human donors (>65 years) were cultured with anti-CD3/CD28 for 4 days and where indicated treated with 10 µM spermidine and the protein levels of TFEB and eIF5A hypusination were measured in CD8+ cells by wWestern blot (f), representative images (left) and quantified (right). Target band intensity was normalized to eIF5A (for Hyp) or GAPDH (for TFEB). Data represented as mean ± SEM.

Figure 5—source data 1. Dpermidine’s mode of action is via eIF5A and TFEB in human CD8+ T cells.

Figure 5.

Figure 5—figure supplement 1. Spermidine does not improve eIF5A and TFEB in young donors.

Figure 5—figure supplement 1.

PBMCs from young human donors were cultured with anti-CD3/CD28 for 4 days and treated with 10 µM spermidine for 4 days. The protein levels of TFEB and eIF5A hypusination were measured in sorted CD8+ cells by wWestern blot, representative images (left) and quantified (right). Target band intensity was normalized to eIF5A (for Hyp) or GAPDH (for TFEB). Data represented as mean ± SEM.
Figure 5—figure supplement 1—source data 1. Spermidine does not improve eIF5A and TFEB in young donors.

Figure 5—figure supplement 2. TFEB is required for CD8+ T cell function.

Figure 5—figure supplement 2.

Splenic T cells from wildtype C57BL/6 mice (WT) or tamoxifen-inducible Tfeb knockout mice (KO: CAG-Cre;Esr1+;Tfeb-/-) were cultured with anti-CD3/CD28 and 4-Hydroxytamoxifen (4-OHT) for 4 days. The protein levels of TFEB and GAPDH were measured in sorted CD8+ cells by wWestern blot (a) representative images (left) and quantified (right). Target band intensity was normalized to GAPDH. Intracellular IFNγ by flow cytometry (b), intracellular perforin by flow cytometry (c), intracellular granzyme B (d). All cells were gated on CD8+ T cells. Data represented as mean ± SEM, MFI = mean fluorescence intensity. Statistics by unpaired t-test.
Figure 5—figure supplement 2—source data 1. TFEB is required for CD8+ T cell function.
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To investigate if TFEB is required for T cell function, we knocked out Tfeb in cultured mouse T cells ex vivo (Figure 5—figure supplement 2a). Interestingly, both intracellular IFNγ and perforin expression were significantly decreased in Tfeb KO CD8+ T cells upon stimulation (Figure 5—figure supplement 2b and c). Moreover, Tfeb KO CD8+ T cells failed to upregulate IFNγ and perforin in the presence of spermidine (Figure 5—figure supplement 2b and c). Analogous to previous observation, loss of TFEB had no impact on granzyme B expression (Figure 5—figure supplement 2d). Taken together, these data suggest that TFEB itself is essential for T cell function and that spermidine was unable to improve T cell function when TFEB was knocked out.

Discussion

Several studies have addressed the induction of autophagy by antigenic stimulation in murine T cells in vivo and in response to anti-CD3/CD28 in vitro stimulation of human T cells (Macian, 2019). In mouse studies Xu et al., 2014 measured a peak in autophagy induction shortly after the CD8+ effector phase with subsequent failure to mount memory responses. In line with this, we and Xu et al showed that deletion of autophagy mostly affects T cells in the memory phase of CD8+ T cell responses as opposed to the effector phase (Xu et al., 2014; Puleston et al., 2014). The question remains whether it is initiation or maintenance of memory responses that are affected. Interestingly, the work of Schlie et al., 2015 showed that the memory response could be rescued by N-acetyl cysteine, arguing that memory cells may be formed but not maintained without autophagy. While it has been shown in human T cells that autophagy peaks after 4 days of anti-CD3/CD28 stimulation, later time points are more difficult to mimic accurately in vitro. Here, we show for the first time that antigenic encounter in vivo induces autophagy after the effector phase, arguing that autophagy may be important in human CD8+ T cells during the memory phase. Interestingly, we could not observe any bystander effect on CD8+ T cells that are not specific for HCV, suggesting that it is a cell-intrinsic effect through specific stimulation via their TCR.

The role of autophagy in these long-term antigen-specific T cells remains still elusive; autophagy could either degrade molecules no longer needed comparable to the transcription factor PU.1 in Th9 cells (Rivera Vargas et al., 2017) or cell cycle regulator CDKN1 in the effector phase of CD8+ T cells (Jia et al., 2015) or organelles such as mitochondria or ER (Puleston et al., 2014; Jia et al., 2011). Alternatively, autophagy could provide building blocks including amino acids, free fatty acids and nucleotides that are important for the maintenance of essential cellular processes through provision of ATP (Shi et al., 2011; Pearce et al., 2009) or transcription and translation for T cell function or a combination of these mechanisms. Our data is in agreement with previous reports showing that spermidine increases the autophagy-dependent selective degradation of mitochondria (mitophagy) and thus contributes to mitochondrial health and functionality (Eisenberg et al., 2016; García-Prat et al., 2016; Fan et al., 2017; Qi et al., 2016). The positive impact on cellular mitochondrial content is in line with two recent studies showing that TFEB promotes autophagy and improves mitophagy turnover in neuronal and hepatocytes (Tan et al., 2019; Kim et al., 2018).

With increasing life expectancy, the number of people over 60 years of age is expected to double by 2050, reaching 2.1 billion worldwide. The severity of many infections is higher in the older population compared to younger adults as particularly notable during the SARS2-CoV pandemic. Moreover, the success of childhood vaccination is widely recognized but the importance of vaccination of the older population is frequently underestimated (Weinberger, 2018). Immune responses to vaccines are known to be particularly ineffective in the older population and yet some vaccines such as for influenza and SARS2-Cov are primarily needed for the older adults. The development of drugs that improve vaccination in this expanding population is therefore an urgent socio-economic need.

Targeting mTOR with Rapamycin was the first drug both in mice and in human clinical trials shown to have a beneficial effect on T cell responses in mice and older humans (Araki et al., 2009; Mannick et al., 2018). However, whether Rapamycin triggers autophagy at the administered dose has not been investigated. With this study we show for the first time that the autophagy-inducing drug spermidine has an immune boosting effect on the T cell compartment in humans in vitro and that low autophagy levels correlate with low responses to vaccination. The translation of TFEB is one of the limiting factors for sufficient autophagy levels required to mount an immune response. In addition, to be active, TFEB needs to be dephosphorylated for its translocation into the nucleus, which mTOR inhibition facilitates (Martina et al., 2012; Roczniak-Ferguson et al., 2012; Settembre et al., 2012). Therefore, spermidine and an mTOR inhibitor may have to be combined to optimally restore immune responses in the older adults.

We find that spermidine levels decline in PBMCs in the older humans. This confirms earlier study in plasma in which spermidine levels were found to be low in the >65 age group and rising again in centenarians (Pucciarelli et al., 2012). Studies of this kind usually indicate that the phenotype is maladaptive with age as centenarians do not display many of the aging features of the age group below. However, the reasons for the age-related decline in spermidine in blood cells are not clear, and currently under investigation. Overall it is evident that endogenous spermidine maintains autophagy in human T cells, a novel metabolic pathway that has not been investigated before.

Spermidine has recently been administered to humans in a small experimental trial with a beneficial effect on cognitive function without adverse effects (Schwarz et al., 2018; Wirth et al., 2019). It remains to be shown whether at such low doses it has an effect on autophagy. Our study provides a good starting point for a small experimental trial to assess whether spermidine can be used to improve vaccination efficiency in older adults. Furthermore, this trial could help to determine if eIF5A, TFEB and autophagy in PBMCs could be used as biomarkers for the antiaging effect of spermidine and related drugs during vaccination. Based on this information, the vaccination protocol in older adults could in the future be adjusted according to whether this pathway is functional in individual vaccinees.

In conclusion, we validated a novel anti-immune senescence pathway in humans with druggable targets and biomarkers, which could also be used for other broader anti-aging drug trials.

Materials and methods

Key resources table.

Reagent type
(species) or resource		 Designation Source or reference Identifiers Additional
information
Genetic reagent (M. musculus) Atg7flox PMID:15866887 MGI:3587769 Dr. Masaaki Komatsu (Juntendo University)
Genetic reagent (M. musculus) TFEBflox PMID:23604321 Prof. Andrea Ballabio (Telethon Institute of Genetics and Medicine (TIGEM)).
Cell line (H. sapiens) Jurkat E6.1 ATCC RRID:CVCL_0367
Antibody Rabbit anti-GAPDH Millipore MAB374, RRID:AB_2107445 WB (1:5,000)
Antibody Rabbit anti-LC3 Sigma L8918, RRID:AB_1079382 WB (1:1500)
Antibody Mouse anti-eIFA5 BD Biosciences 611976, RRID:AB_399397 WB (1:3000)
Antibody Rabbit anti-hypusine Millipore ABS1064, RRID:AB_2631138 WB (1:1500)
Antibody Rabbit anti-TFEB Bethyl A303-673A, RID:AB_11204751 WB (1:1500)
Antibody Mouse anti-Actin Cell Signaling 3700, RRID:AB_2242334 WB (1:10,000)
Antibody IRDye 800CW Donkey Anti-Rabbit IgG (H+L) LI-COR 926–32213, RRID:AB_621848 WB (1:10,000)
Antibody IRDye 680RD Donkey Anti-Mouse IgG (H+L) LI-COR 926–68022, RRID:AB_10715072 WB (1:10,000)
Antibody BV605 anti-human CD14 BioLegend 301834, Clone: M5E2 FACS (1:200)
Antibody PE anti-human Granzyme B eBioscience 12-8899-41, Clone: GB11 FACS (1:100)
Antibody PE/Cy7 anti-human CD8a BioLegend 344712, Clone: SK1 FACS (1:100)
Antibody PE/Cy5 anti-human CD19 BioLegend 302210, Clone:HIB19 FACS (1:100)
Antibody BV711anti-human CD19 BioLegend 302245, Clone:HIB19 FACS (1:200)
Antibody APC anti-human CD3 BioLegend 300312, Clone:HIT3a FACS (1:200)
Antibody APC anti-human Perforin BioLegend 353311, Clone:B-D48 FACS (1:100)
Antibody APC anti-human CD4 BioLegend 317416, Clone:OKT4 FACS (1:200)
Antibody Alexa Fluor 700 anti-human IFNγ BioLegend 506515, Clone:B27 FACS (1:100)
Antibody PE/Cy7 anti-mouse CD8a Antibody BioLegend 100722, RRID:AB_312761 FACS (1:200)
Antibody BV605 anti-mouse CD4 Antibody BioLegend 100451, Clone: GK1.5 FACS (1:200)
Antibody Alexa Fluor 700 anti-mouse IFNγ BioLegend 505823, Clone: XMG1.2 FACS (1:100)
Antibody APC anti-mouse Perforin BioLegend 154303, Clone: S16009A FACS (1:100)
Antibody PE anti-mouse Granzyme B BioLegend 372207, Clone: QA16A02 FACS (1:100)
Antibody APC anti-human CD19 BioLegend 302212, RRID:AB_314242 FACS (1:200)
Antibody Alexa Fluor 700 anti-mouse CD8a BioLegend 100730, RRID:AB_493703 FACS (1:100)
Antibody PE-labeled MHC class I pentamer (HLA-A*02:01, HCV peptide KLSGLGINAV) ProImmune (HLA-A*02:01, HCV peptide KLSGLGINAV) FACS (1:50)
Commercial assay or kit FlowCellect Autophagy LC3 Antibody-based Detection Kit Merck Millipore FCCH100171 FACS (1:20)
Commercial assay or kit LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit Invitrogen
ThermoFisher		 L10119 FACS (1:1000)
Commercial assay or kit LIVE/DEAD Fixable Aqua Dead Cell Stain Kit Invitrogen
ThermoFisher		 L34957 FACS (1:1000)
Commercial assay or kit CellTrace Violet Cell Proliferation Kit Invitrogen,ThermoFisher C34557 FACS (1:1000)
Commercial assay or kit MitoTracker Green Invitrogen,ThermoFisher M7514 FACS (150 nM)
Commercial assay or kit MitoSox Invitrogen,ThermoFisher M36008 FACS (5 uM)
Commercial assay or kit CD3/CD28 activation Dynabeads Thermo Fisher 11161D
Commercial assay or kit Human IFN-γ ELISpotPLUS kit (ALP) Mabtech 3420-4APT-2
Commercial assay or kit IFNγ ELISA Kit Life Technologies Ltd 88-7316-22
Commercial assay or kit EasySep Human CD8+ T Cell Isolation Kit Stemcell 17953
Commercial assay or kit BCA Assay Thermo Fisher 23227 100 μl/sample
Chemical compound, drug Spermidine Cayman Chemical 14918 10 μM
Chemical compound, drug Spermine Cayman Chemical 18041 10 μM
Chemical compound, drug Hydroxychloroquine Sulfate Stratech Scientific Ltd B4874-APE-10mM 10 μM
Chemical compound, drug Bafilomycin A1 Cayman Chemical 11038 10 μM
Chemical compound, drug Resveratrol Stratech Scientific Ltd A4182-APE 10 μM
Chemical compound, drug BSI-0206965 Stratech Scientific Ltd A8715-APE 10 μM
Chemical compound, drug AICR Stratech Scientific Ltd A8184-APE 10 μM
Chemical compound, drug NAO Thermo Fisher Scientific A1372 100 nM
Chemical compound, drug TRMR Thermo Fisher Scientific T668 FACS (100 nM)
Chemical compound, drug Difluoromethylornithine (DFMO) Enzo Life Sciences ALX-270–283 M010 1 mM
Chemical compound, drug GC7 Millipore 259545–10 MG 10 μM
Chemical compound, drug 4-Hydroxytamoxifen Sigma H7904 100 nM
Chemical compound, drug LPS Santa Cruz sc-3535 10 µg/ml
Chemical compound, drug Aanti-IgM Jackson Immuno Research 109-005-043 5 µg/ml
Chemical compound, drug Aanti-CD40L Enzo Life science ALX-522–015 C010 100 ng/ml,
Peptide, recombinant protein Human IFNγ Enzo Life science ENZ-PRT141-0100 20 ng/ml
Software, algorithm Image Studio Lite LI-COR
Software, algorithm Prism GraphPad
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Human samples

Human peripheral blood mononuclear cells (PBMCs) were obtained under the ethics reference NRES Berkshire 13/SC/0023, from phase I clinical trials of novel viral-vectored vaccines for hepatitis-C virus (HCV; NCT0100407 and NCT01296451) or respiratory syncytial virus (RSV), described in more detail elsewhere (Swadling et al., 2016; Barnes et al., 2012; Green et al., 2015a; Green et al., 2015b; Green et al., 2019). Volunteers were self-selected adults who provided written informed consent and who were carefully screened for being healthy before vaccination. The vaccine schedules are described in Diagrams (Figure 1—figure supplements 3 and 4). Blood samples were collected in heparinized tubes for assays that required PBMCs. PBMCs were isolated within 6 hr of sample collection. An aliquot of PBMCs was immediately used for fresh ELISpot assays and the remainder cryopreserved in RecoveryTM Cell Freezing Medium. Serum samples were obtained by centrifugation of whole blood collected in clotted tubes, and then cryopreserved.

Control PBMCs

Control PDBMCs were isolated from blood or blood cones of healthy donors using Ficoll-Paque density gradient separation. All volunteers provided written informed consent. The study was approved by the Local Ethics Committee Oxford and Birmingham. Freshly isolated PBMCs were cultured directly or were frozen in 90% FBS and 10% DMSO in liquid nitrogen. Fresh or thawed PBMCs were cultured with RPMI 1640, 10% FCS, 2 mM L-Glutamine, 100 U/mL Penicillin (Invitrogen).

ELISpot assay

Ex vivo IFNγ ELISpot assays were performed according to manufacturers’ instructions (Mabtech) on freshly isolated PBMCs plated in triplicates at 2 × 105 PBMCs per well. In brief, peptide pools consisted of mainly 15-mer sequences with 11 amino acid overlaps and covering the sequence of proteins F, N, and M2- 1. Peptides were dissolved in 100% DMSO and arranged in four pools. DMSO (the peptide diluent) and Concanavalin A (ConA) were used as negative and positive controls, respectively. The mean + 4 StDev of the DMSO response from all samples identified a cut off whereby individual samples with background DMSO values ≥ 50 spot forming cells per million PBMCs were excluded from analysis. Calculation of triplicate well variance was performed as described previously (Green et al., 2019).

Human T cell assays

The mean age of young donors was 40.7 ± 11.3 years and the mean age of old donors was 77.6 ± 6.6. PBMCs were activated with either soluble anti-CD3 (1 μg/mL, Jackson Immuno Research) and anti-CD28 (1 μg/mL, Jackson Immuno Research) with or without 10 μM spermidine, 10 μM spermine or 1 mM difluoromethylornithine (DFMO, Enzo Life Sciences) or 10 µM GC7 (or as indicated, Millipore), 10 µM hydroxychloroquine (Stratech Scientific Ltd), 10 µM SBI-0206965 (Stratech Scientific Ltd) 10 µM Resveratrol (Stratech Scientific Ltd) for 4 days. After MACS sorting of CD8+ T cell using a negative selection kit (CD8+ T Cell Isolation Kit II, human, Miltenyi Biotec), cells were lysed for western blotting or stained for Autophagy flux assay as described below. IFNγ release in culture supernatants was measured by heterologous two-site sandwich ELISA, according to the manufacturer’s protocol (Invitrogen).

Cell line

Human Jurkat T cell line was cultured with increasing concentrations of GC7 (10 µM, 30 µM, 100 µM, Millipore) for 24 hr and then treated with or without Bafilomycin A1 for the last 2 hr. The cell line was mycoplasma negative.

Mice T cell assays

Splenocytes from: CD4-cre;Atg7-/- (Atg7Δcd4) or CD4-cre;Atg7+/+ mice were cultured with anti-CD3/CD28 for 4 days. For 4-Hydroxytamoxifen (4-OHT) 4-OHT-inducible Tfeb knockout splenic T cells (CAG-Cre/Esr1+,Tfebf/f), cells were stimulated were anti-CD3/CD28 and 100 nM 4-Hydroxytamoxifen (4-OHT) for 4 days. Animal experiments were approved by the local ethical review committee and performed under UK project licenses PPL 30/3388.

Autophagy flux assay for flow cytometry

PBMCs from healthy donors, activate for T cells with CD3/CD28 beads (Dynabeads Thermo Fisher) (1:1), for B cells with anti-IgM (5 µg/mL, Jackson Immuno Research) and CD40L (100 ng/mL, Enzo Life science) and for monocytes activated with IFNγ (20 ng/mL, Enzo Life science) and/or LPS (10 µg/mL, Santa Cruz) (all 24 hr except for T cells which were stimulated overnight). Autophagy levels were measured after 2 hr treatment with bafilomycin A1 (10 nM BafA1) or vehicle. We adapted the FlowCellect Autophagy LC3 antibody-based assay kit (FCCH100171, Millipore) as follows: In brief, cells were stained with surface markers (as above) and washed with Assay Buffer in 96 well U bottom plates. 0.05% Saponin was added to each well and spun immediately, followed by anti-LC3 (FITC) at 1:20 in Assay Buffer, (30–50 µL/ well) at 4°C for 30 min, and washed once with 150 µL Assay Buffer. Stained cells were fixed with 2% PFA before FACS analysis. Autophagic flux was calculated as LC3-II mean fluorescence intensity of (BafA1-Vehicle)/Vehicle.

Surface staining for flow cytometry

For CellTrace staining, CellTrace Violet (C34557, Thermo Fisher) was used according to the manufacturer’s protocol. Cells were transferred to a round bottom 96 well plate and centrifuged (300xg, 5 min). The pellet was resuspended in PBS containing the viability dye Live/Dead (Life Technologies) or fixable Zombie Aqua Live/Dead (423102, Biolegend) for 10 min in the dark at room temperature (RT) to exclude dead cells during analysis. After washing with PBS/5% FCS, cells were resuspended in PBS/2% FCS/5 mM EDTA (FACS buffer) containing a cocktail of antibodies relevant to the desired cell surface markers. Fc block was typically added to the antibody mix to minimize non-specific staining. Surface antibody staining was performed at 4°C for 20 mins in the dark. A list of all surface antibodies utilized and their working concentrations are in Key resources table. Following incubation cells were washed with FACS buffer and immediately analyzed on a four-laser LSR Fortessa X-20 flow cytometer. Acquired data were analyzed using FlowJo 10.2.

Mitochondrial staining was performed after Live/Dead and surface marker staining by incubating cells at 37°C for 15 min with 5 µM MitoSOX red, 37°C for 25 min with 150 nM MitoTracker Green, 37°C for 25 min with 100 nM nonylacridine orange (NAO) or at 37°C for 15 min with 100 nM tetramethylrhodamine methyl ester (TMRM) (all from Thermo Fisher Scientific).

Intracellular staining for flow cytometry

For intracellular staining, PBMCs were stimulated in R10 with anti-CD3 (1 μg/ml, Jackson Immuno Research) and anti-CD28 (1 μg/mL, Jackson Immuno Research) with or without 10 μM spermidine for 4 days. On day 4, cells were re-stimulated with the same concentrations of anti-CD3/CD28 for 6 hr at 37°C in the presence of 1 μg/mL brefeldin-A (Sigma-Aldrich). As a control, cells were left unstimulated. Following surface marker staining as described above, cells were fixed with 100 μL Fixation buffer (eBioscience) for 20 min at RT in the dark. Next, cells were permeabilized with 100 μL of 1x Permeabilization buffer (eBioscience) for 15 min in the dark at RT. Then, cells were resuspended in the intracellular antibody mix (anti-IFN-γ, anti-Granzyme B, anti-Perforin) and incubated for 30 min in the dark at RT. After being washed twice with Permeabilization buffer the cells were resuspended in 200 μL of FACS buffer for analysis.

MHC class I pentamer staining to identify antigen-specific T cells

An HCV-specific HLA-A*02-restricted pentamer, peptide sequence KLSGLGINAV (Proimmune) was used to identify HCV-specific CD8+ T cells ex vivo. PBMCs were washed in PBS and were stained with pentamers at room temperature (20 min) in PBS, washed twice in PBS before further mAb staining as described above. During analysis, stringent gating criteria were applied with doublet and dead cell exclusion to minimize nonspecific binding contamination.

Western blot

Cells in suspension were washed with PBS and lysed using NP-40 lysis buffer containing proteinase inhibitors (Sigma) and phosphatase inhibitors (Sigma) on ice. After spinning down the debris, protein concentration in the supernatant was quantified by BCA Assay (23227, Thermo Fisher). Reducing Laemmli Sample Buffer was then added to the supernatant and heated at 100°C for 5 min. 5–20 µg protein was loaded for SDS-PAGE analysis. NuPAGE Novex 4–12% Bis-Tris gradient gel (Thermo Fisher) with MES running buffer (Thermo Fisher) was used. Proteins were transferred to a PVDF membrane (IPFL00010, Millipore) and blocked with 5% skimmed milk-TBST. Membranes were incubated with primary antibodies dissolved in 1% milk overnight and secondary antibodies dissolved in 1% milk with 0.01% SDS for imaging using the Odyssey CLx Imaging System. Data were analyzed using Image Studio Lite.

Spermidine measurement in cell lysates by GC-MS

This protocol was used as published previously (Yu et al., 2017). Briefly, cells were washed with PBS and the pellet resuspended in lysis buffer (80% methanol + 5% Trifluoroacetic acid) spiked with 2.5 µM 1,7-diaminoheptane (Sigma). The cell suspension, together with acid-washed glass beads (G8772, Sigma), was transferred to a bead beater tube and homogenized in a bead beater (Precellys 24, Bertin Technologies) for four cycles (6500 Hz, 45 s) with 1 min of ice incubation between each cycle. The homogenized samples were centrifuged at 13,000 g for 20 min at 4°C. The supernatant was collected and dried overnight. For chemical derivatization, 200 µL trifluoroacetic anhydride was added to the dried pellet and incubated at 60°C for 1 hr, shaking at 1200 rpm. The derivatization product was dried, re-suspended in 30 µL isopropanol and transferred to glass loading-vials. The samples were analyzed using a GCxGC-MS system as described (Yu et al., 2017). The following parameters were used for quantification of the 1D-GC-qMS data: Slope: 1000/min, width: 0.04 s, drift 0/min and T. DBL: 1000 min without any smoothing methods used.

Statistical analyses

Prism software (GraphPad) was used for statistical analyses. Data are represented as mean ± SEM. All comparative statistics were post-hoc analyses. Paired or unpaired two-tailed Student’s t-test was used for comparisons between two normally distributed data sets with equal variances. Linear regression with a 95% confidence interval was used to assess the relationships between age and the expression of target proteins or spermidine levels, in which R (Lurie et al., 2020) was used to assess the goodness of fit and the p value calculated from F test was used to assess if the slope was significantly non-zero. p Values were used to quantify the statistical significance of the null hypothesis testing. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns, not significant.

Acknowledgements

We acknowledge Reithera/Okairos for RSV trial, Zhanru Yu for Spermidine and Putrescine measurements, Kirsty McGee and Christos Ermogenous for ethical approval and provision of samples from Birmingham healthy older donors. We thank all donors, volunteers and phlebotomists. K.S. is a Wellcome Investigator, which funded this study. Wellcome funding (WT109665MA) and NIHR SF was also awarded to P.K. We acknowledge funding from the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Tfebflox mice were obtained from A Ballabio.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Ghada Alsaleh, Email: ghada.alsaleh@kennedy.ox.ac.uk.

Anna Katharina Simon, Email: katja.simon@kennedy.ox.ac.uk.

David Furman, Buck Institute For Research On Aging.

Jessica K Tyler, Weill Cornell Medicine, United States.

Funding Information

This paper was supported by the following grants:

  • Wellcome WT109665MA to Anna Katharina Simon.

  • National Institute for Health Research to Paul Klenerman.

  • Oxford Biomedical Research Center to Paul Klenerman.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing.

Validation, Investigation, Methodology, set up the autophagy flux protocol in the primary cells and performed the experiments for the HCV vaccine samples and some of the RSV samples.

Visualization, Writing - original draft.

Investigation.

Methodology, Writing - review and editing.

Resources.

Resources, Writing - original draft.

Resources.

Resources, Writing - original draft.

Resources, Writing - original draft.

Conceptualization, Resources, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing - review and editing.

Ethics

Clinical trial registration NCT01070407 and NCT01296451.

Human subjects: Human vaccine samples: Human peripheral blood mononuclear cells (PBMC) were obtained under the ethics reference NRES Berkshire 13/SC/0023, from phase I clinical trials of novel viral-vectored vaccines for hepatitis-C virus (HCV; NCT01070407 and NCT01296451)or respiratory syncytial virus (RSV). Human healthy control: The study was approved by the Local Ethics Committee Oxford and Birmingham: The acquisition of normal control human tissue for medical research Kennedy Institute of Rheumatology. University of Oxford, Rec: 11/h0711/7 collection. University of Birmingham Research Ethics Committee, Reference ERN_12-1184R2, Investigations of the ageing immune system" Application for Ethical Review ERN_12-1184R2, UoB Ref: 17-1106.

Animal experimentation: Animal experiments were approved by the local ethical review committee and performed under UK project licenses PPL 30/3388.

Additional files

Transparent reporting form

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-5 and figure supplements.

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Decision letter

Editor: David Furman1

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This paper involving autophagy and immune function is of high importance for the scientific community and public in general, especially in these times where the immune system is in a central focus. The paper now provide evidence that spermidine, in addition to its known healthful effects similar to those observed during caloric restriction including autophagy and protein deacetylation, it has the potential to become a new intervention for improving immune system health.

Decision letter after peer review:

Thank you for submitting your article "Autophagy in T cells from aged donors is maintained by spermidine, and correlates with function and vaccine responses" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Your article provides robust evidence that spermidine administration reinvigorates T-Cell function and autophagy in cells from older subjects. We believe that the following suggested revisions will greatly improve the clarify of your manuscript and thus, we hope you can address these promptly before we can accept it for publication. Some of the major conclusions require a modest amount of additional data as pointed by reviewer #2 and an extended Discussion as suggested by reviewer #3. Especially the proposed link between mitochondrial function and the improvement in autophagy is not supported by data. Please incorporate data on mitochondrial function or discuss prior published work. In addition, kindly provide evidence to demonstrate that spermidine effects on INF and T-cell function are blunted when autophagy is blocked, as suggested by reviewer #3.

Please find below the detailed suggestions from the reviewers.

Reviewer #2:

Simon and colleagues show that spermidine administration reinstalls autophagy and T-Cell function taken ex vivo from aged donors. They also point to hypusination and TFEB translation as an underlying mechanism. This is an intriguing and technically well done work and I have only a few suggestions for improvement.

The data showing that in young donors Autophagy is blocked by DFMO and can be rescued by spermidine administration are impressive and should be mentioned in the Abstract.

The sentence: “Here, we show induction of autophagy is specifically induced in human vaccine-induced antigen-specific T cells in vivo" is unclear and even flawed.

The authors correctly point out that mitochondria/mitophagy/improved mitochondrial function could play a role in the phenotypes observed. They should either show markers of mitochondrial capacity/quality in their assays (e.g. respiration, mitophagy) or at least discuss this in the light of published works.

Reviewer #3:

The manuscript entitled "Autophagy in T cells from aged donors is maintained by spermidine and correlates with function and vaccine responses" shows that antigen exposure induces autophagic flux in CD8+ T cells in humans in vivo, and this induction declines with age. Moreover, it evidences that the autophagy-inducing metabolite spermidine, falls with age and when supplemented increases autophagy and T cell function in old T cells.

The manuscript is overall well written and the message is potentially important. However, some conclusion are forced, and additional data are needed to support main author's conclusions.

– The correlation between INFɣ response and autophagic flux is positive in old, and negative in young individuals. The authors concluded that "reduced autophagy in aged T cells may cause low T cell response to vaccination". This is an interesting hypothesis that should be corroborated by direct evidences. For example, the authors should investigate INFɣ response in T cells in which autophagy was stimulated/inhibited using different compounds that modulate autophagy through different mechanism of action.

– The use of metabolite Spermidine has been previously shown to induce autophagy. However, the authors should include additional controls (i.e. other amino acid metabolites) to prove that the effects are indeed specific for spermidine.

– Spermidine's mode of action via EiF5A and TFEB in human CD8+ T cells requires additional experiments, i.e. real time of TFEB genes targets upon spermidine administration. Can the authors inhibit TFEB in spermidine treated cells to formally prove the involvement of TFEB in spermidine activity? Indeed, spermidine can activate autophagy through different mechanisms.

– There is no formal demonstration that Spermidine improve T-cell function through TFEB-mediated autophagy. The authors should use pharmacological inhibitors of autophagy to demonstrate that spermidine effects on INF and T-cell function are blunted when autophagy is blocked.

eLife. 2020 Dec 15;9:e57950. doi: 10.7554/eLife.57950.sa2

Author response

Reviewer #2:

Simon and colleagues show that spermidine administration reinstalls autophagy and T-Cell function taken ex vivo from aged donors. They also point to hypusination and TFEB translation as an underlying mechanism. This is an intriguing and technically well done work and I have only a few suggestions for improvement.

The data showing that in young donors Autophagy is blocked by DFMO and can be rescued by spermidine administration are impressive and should be mentioned in the Abstract.

We thank the reviewer for his/her recommendation and added a sentence describing this observation in the Abstract.

“We demonstrate in human donors that levels of the endogenous autophagy-inducing metabolite spermidine fall in T cells with age. Spermidine supplementation of T cells from old donors recovers their autophagy level and function, similar to young donor cells, in which spermidine biosynthesis has been inhibited.”

The sentence: “Here, we show induction of autophagy is specifically induced in human vaccine-induced antigen-specific T cells in vivo" is unclear and even flawed.We apologize for the unclear sentence and we are happy to clarify it. We replaced the sentence with the following in the Abstract:“Here, we show that autophagy is specifically induced in vaccine-induced antigen-specific CD8+ T cells in healthy human volunteers.”.

The authors correctly point out that mitochondria/mitophagy/improved mitochondrial function could play a role in the phenotypes observed. They should either show markers of mitochondrial capacity/quality in their assays (e.g. respiration, mitophagy) or at least discuss this in the light of published works.

We appreciate the reviewer’s comment regarding the mitochondria. We addressed this point and added it to the Results, new Figure 3—figure supplement 2, Materials and methods and Discussion.

“Polyamine biosynthesis has previously been shown to increase mitochondrial protein synthesis (Madeo et al., 2018; Puleston et al., 2019; Wang et al., 2020). […] Mitochondria are not the only type of autophagic cargo and other cargos need to be investigated to understand exactly how autophagy induction improves T cell function.”

“Our data is in agreement with previous reports showing that spermidine increases the autophagy-dependent selective degradation of mitochondria (mitophagy) and thus contributes to mitochondrial health and functionality (Eisenberg et al., 2016; Garcia-Prat et al., 2016; Fan et al., 2017; Qi et al., 2016). The positive impact on cellular mitochondrial content is in line with two recent studies showing that TFEB promotes autophagy and improves mitophagy turnover in neuronal and hepatocytes (Tan et al., 2019; Kim et al., 2018).”.

“Mitochondrial staining was performed after Live/Dead and surface marker staining by incubating cells at 37 °C for 15 min with 5 µM MitoSOX red, 37 °C for 25 min with 150 nM MitoTracker Green, 37 °C for 25 min with 100 nM nonylacridine orange (NAO) or at 37 °C for 15 min with 100 nM tetramethylrhodamine methyl ester (TMRM) (all from Thermo Fisher Scientific).”

Reviewer #3:

The manuscript entitled "Autophagy in T cells from aged donors is maintained by spermidine and correlates with function and vaccine responses" shows that antigen exposure induces autophagic flux in CD8+ T cells in humans in vivo, and this induction declines with age. Moreover, it evidences that the autophagy-inducing metabolite spermidine, falls with age and when supplemented increases autophagy and T cell function in old T cells.

The manuscript is overall well written and the message is potentially important. However, some conclusion are forced, and additional data are needed to support main author's conclusions.

– The correlation between INFɣ response and autophagic flux is positive in old, and negative in young individuals. The authors concluded that "reduced autophagy in aged T cells may cause low T cell response to vaccination". This is an interesting hypothesis that should be corroborated by direct evidences. For example, the authors should investigate INFɣ response in T cells in which autophagy was stimulated/inhibited using different compounds that modulate autophagy through different mechanism of action.

We appreciate the reviewer’s comment and we hope the newly added data provides additional information on the effects of decreased autophagy on T cell function.

To address this, we first used splenocytes from mice which were deleted for the essential autophagy gene Atg7 using the CD4Cre promotor. After stimulation, we found that autophagy loss leads to reduced IFNɣ, Perforin and Granzyme B expression in comparison to autophagy-sufficient cells (new Figure 2A-C). In addition, we treated old and young blood cells with either autophagy inhibitors (Hydroxychloroquine (HCQ)) and BSI-0206965 or with autophagy activators (Resveratrol) to assess their functional response. We performed intracellular cytokine staining to quantify IFNɣ, Perforin and Granzyme B response and ELISA for soluble IFNɣ in the supernatant (new Figure 2D-I). Our data shows that autophagy is important for T cell function. This was added to the Results, Materials and methods and new Figure 2.

“To gain better insight into the role of autophagy in T cells function, we sorted and activated splenocytes from control and autophagy-deficient mice, in which the essential autophagy gene Atg7 was deleted with Cre driven by the CD4 promotor (Atg7Δcd4). […] In summary, these data suggest that autophagy is required for CD8+ T cell function by controlling IFNγ and perforin production.”

“Mice T cell assays

Splenocytes from CD4-cre;Atg7+/+ and CD4-cre;Atg7-/- mice were cultured with anti-CD3/CD28 for 4 days. For 4-Hydroxytamoxifen (4-OHT) 4-OHT- inducible Tfeb knockout splenic T cells (CAG-Cre/Esr1+, Tfebf/f), cells were stimulated were anti-CD3/CD28 and 100 nM 4-Hydroxytamoxifen (4-OHT) for 4 days.”

– The use of metabolite Spermidine has been previously shown to induce autophagy. However, the authors should include additional controls (i.e. other amino acid metabolites) to prove that the effects are indeed specific for spermidine.

Based on the reviewer’s suggestion, we treated the cells with spermine, another amino acid metabolite from the same pathway. Spermine was unable to rescue T cell function from old donors (new Figure 3—figure supplement 1). This was added to the Results, Materials and methods and new Figure 3—figure supplement 1 and is in line with our finding that spermine is not reduced with age (Figure 3A) and does not serve as substrate for eIF5A hypusination (Lee and Park, 2000).

“In contrast, spermine, another polyamine from the same pathway, which is not significantly reduced with age (Figure 3A) and does not serve as a direct substrate for eIF5A hypusination (Lee and Park, 2000), does not rescue CD8+ T cell function from old donors (Figure 3—figure supplement 1A-C). These data underline the importance of cellular spermidine levels in aged CD8+ T cells.”

– Spermidine's mode of action via EiF5A and TFEB in human CD8+ T cells requires additional experiments, i.e. real time of TFEB genes targets upon spermidine administration. Can the authors inhibit TFEB in spermidine treated cells to formally prove the involvement of TFEB in spermidine activity? Indeed, spermidine can activate autophagy through different mechanisms.

We appreciate the reviewer’s comment addressing the question of spermidine’s mode of action acting via TFEB. In an attempt to address this comment, we tested the Amaxa nucleofector kit optimised for human T cells. Unfortunately, transfection efficiency for plasmids expressing GFP was just under 16%, and no significant reduction of Tfeb mRNA was observed when primary human T cells were transfected with siTfeb for 3 days.

Instead, we obtained inducible Tfeb knockout splenic T cells from Andrea Ballabio’s lab. We tested several culture conditions and found an optimal protocol for inducing Tfeb knockout in these T cells ex vivo. Upon 100 nM 4-Hydroxytamoxifen (4-OHT) treatment for 4 days, T cells stimulated with anti-CD3/CD28 achieved a good inhibition (new Figure 5—figure supplement 2A).Functionally, IFNsecretionwassignificantlyreducedasmeasured in culture supernatants with or without spermidine (new Figure 5—figure supplement 2B). Taken together, these data suggest that TFEB is required for T cell function and that spermidine supplementation did not improve T cell function in this condition. This was added to the Results, Materials and methods and new Figure 5—figure supplement 2.

“To investigate if TFEB is required for T cell function, we knocked out Tfeb in cultured mouse T cells ex vivo (Figure 5—figure supplement 2A). […] Taken together, these data suggest that TFEB itself is essential for T cell function and that spermidine was unable to improve T cell function when TFEB was knocked out.”“Mice T cell assays

Splenocytes from: CD4-cre;Atg7-/- (Atg7Δcd4) or CD4-cre;Atg7+/+ mice were cultured with anti-CD3/CD28 for 4 days. For 4-Hydroxytamoxifen (4-OHT) 4-OHT- inducible Tfeb knockout splenic T cells (CAG-Cre/Esr1+,Tfebf/f), cells were stimulated were anti-CD3/CD28 and 100 nM 4-Hydroxytamoxifen (4-OHT) for 4 days.”

– There is no formal demonstration that Spermidine improve T-cell function through TFEB-mediated autophagy. The authors should use pharmacological inhibitors of autophagy to demonstrate that spermidine effects on INF and T-cell function are blunted when autophagy is blocked.

We want to thank the reviewer for this comment and have added the requested experiment to the manuscript in the new Figure 3 with text in the Results and Materials and methods. In this figure we show that inhibition of autophagy using either HcQ or Sbl blocks spermidine-induced production of IFNγ measured in the supernatant or in each cell by flow cytometry. Interestingly, we observe similar changes for Perforin, whereas Granzyme B remains unchanged, similar to what we observed for the other assays. This data suggests the significance of autophagy in T cell function and cytokine secretion.

“Moreover, to investigate whether spermidine acts through autophagy to improve CD8+ T cell function, we added spermidine together with pharmacological autophagy inhibitors. Indeed, HcQ or Sbl ablates the rejuvenating effects of spermidine supplementation in old donor cells (Figure 3C-F).”

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Figure 1—source data 1. Autophagy is induced by vaccination in antigen-specific T cells and correlates with donor age.
    elife-57950-fig1-data1.xlsx (12.8KB, xlsx)
    Figure 1—figure supplement 2—source data 1. LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws.
    elife-57950-fig1-figsupp2-data1.xlsx (13.5KB, xlsx)
    Figure 1—figure supplement 5—source data 1. Correlation of age with total and peptide-pool specific T cell IFNγ response to RSV.
    elife-57950-fig1-figsupp5-data1.xlsx (9.2KB, xlsx)
    Figure 2—source data 1. Autophagy is required for CD8+ T cell function.
    elife-57950-fig2-data1.xlsx (16.2KB, xlsx)
    Figure 3—source data 1. Spermidine declines with age and supplementing spermidine improves autophagy and CD8+ T cell function in old donors.
    elife-57950-fig3-data1.xlsx (15.2KB, xlsx)
    Figure 3—figure supplement 1—source data 1. Spermine does not improve function of CD8+ T cell from old donors.
    elife-57950-fig3-figsupp1-data1.xlsx (11.5KB, xlsx)
    Figure 3—figure supplement 2—source data 1. Spermidine reduces mitochondrial mass in CD8+ T cell from old donors.
    elife-57950-fig3-figsupp2-data1.xlsx (12.4KB, xlsx)
    Figure 4—source data 1. Endogenous spermidine maintains levels of autophagy and T cell function.
    elife-57950-fig4-data1.xlsx (15.7KB, xlsx)
    Figure 5—source data 1. Dpermidine’s mode of action is via eIF5A and TFEB in human CD8+ T cells.
    elife-57950-fig5-data1.xlsx (12.4KB, xlsx)
    Figure 5—figure supplement 1—source data 1. Spermidine does not improve eIF5A and TFEB in young donors.
    elife-57950-fig5-figsupp1-data1.xlsx (9.1KB, xlsx)
    Figure 5—figure supplement 2—source data 1. TFEB is required for CD8+ T cell function.
    elife-57950-fig5-figsupp2-data1.xlsx (11.8KB, xlsx)
    Transparent reporting form
    elife-57950-transrepform.docx (66.6KB, docx)

    Data Availability Statement

    All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-5 and figure supplements.

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