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.

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).

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

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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 (Atg7^Δcd4). 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.

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Autophagy is required for CD8⁺ T cell function.

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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.

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Spermidine declines with age and supplementing spermidine improves autophagy and CD8+ T cell function in 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).

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Endogenous spermidine maintains levels of autophagy and T cell function.

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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).

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

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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.

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.

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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Author details

1. Ghada Alsaleh

   The Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, United Kingdom

   Contribution

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

   Contributed equally with

   Isabel Panse

   For correspondence

   ghada.alsaleh@kennedy.ox.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4211-3420

2. Isabel Panse

   The Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, United Kingdom

   Contribution

   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

   Contributed equally with

   Ghada Alsaleh

   Competing interests

   No competing interests declared

3. Leo Swadling

   Division of Infection and Immunity, University College London, London, United Kingdom

   Contribution

   Visualization, Writing - original draft

   Competing interests

   No competing interests declared

4. Hanlin Zhang

   The Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Felix Clemens Richter

   The Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, United Kingdom

   Contribution

   Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

6. Alain Meyer

   Fédération de médecine translationnelle Université de Strasbourg, Strasbourg, France

   Contribution

   Resources

   Competing interests

   No competing interests declared

7. Janet Lord

   MRC-Versus Arthritis Centre for Musculoskeletal Ageing Research, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom

   Contribution

   Resources, Writing - original draft

   Competing interests

   No competing interests declared

8. Eleanor Barnes

   1. Peter Medawar Building for Pathogen Research,Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
   2. Translational Gastroenterology Unit, John Radcliffe Hospital, Oxford, United Kingdom
   3. NIHR Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom

   Contribution

   Resources

   Competing interests

   No competing interests declared

9. Paul Klenerman

   1. Peter Medawar Building for Pathogen Research,Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
   2. Translational Gastroenterology Unit, John Radcliffe Hospital, Oxford, United Kingdom
   3. NIHR Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom

   Contribution

   Resources, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4307-9161

10. Christopher Green

    Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Oxford, United Kingdom

    Contribution

    Resources, Writing - original draft

    Competing interests

    No competing interests declared

11. Anna Katharina Simon

    The Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, United Kingdom

    Contribution

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

    For correspondence

    katja.simon@kennedy.ox.ac.uk

    Competing interests

    No competing interests declared

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