MAIT cell activation during chronic viral infection in vivo

MAIT cells are found at high frequencies within the liver in both healthy and diseased states^3,5,13,24. Therefore, we analysed MAIT cell activation during chronic infection with HCV, like DENV, a member of the Flaviviridae family of positive-sense RNA viruses. We examined MAIT cell frequency and phenotype in the PBMC of patients with persistent and resolved HCV infection (spontaneously or after therapy). In all HCV patients, regardless of status, we observed a reduction in MAIT cell frequencies compared to healthy controls (Fig. 2a). However, we only observed upregulation of Granzyme B in patients with prolonged HCV infection (including those who had subsequently responded to antiviral therapy; Fig. 2b,c), and not in those patients with prior short-lived viremia at a distant time-point associated with acute resolving infection (thus, more akin to convalescent DENV infection). Our results indicate substantial activation of MAIT cells in vivo both during acute and chronic viral infections.

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Figure 2

MAIT cell activation during chronic viral infection in vivo.

PBMC's from healthy controls (n=20–23) or patients (n=12–25) with persistent (treatment naive, REL, NR) and resolved HCV infection (SVR, SC) were analysed by flow cytometry by gating on live CD3⁺CD8⁺CD161⁺⁺Vα7.2⁺ (MAIT) cells. (a) MAIT cell frequency as a proportion of the CD8⁺ T cells. (b,c) Granzyme B expression by MAIT cells. (b) Representative flow cytometry plots. Bars represent means±s.e.m. Statistical significance was determined with the Kruskal–Wallis test followed by the Dunns' test. ns>0.05, *P 0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. HC, healthy control; Gr B, Granzyme B; REL/NR, relapse/non-response; SC, spontaneous clearance; SVR, sustained virological response; tr., treatment.

Viral MAIT cell activation in vitro

Having observed MAIT cell activation in vivo during acute and chronic viral infections, we next established in vitro models for viral infections using PBMCs or human CD8⁺ T cells, co-incubated with infected or virus-treated dendritic cells (DCs) or macrophages as antigen presenting cells (APC).

MAIT cells were readily and specifically activated in response to DENV-treated APCs (multiplicity of infection (MOI)=1), as indicated by production of IFN-γ and Granzyme B (Fig. 3a,b), as well as, expression of CD38, with minimal production of TNF-α (Supplementary Fig. 1a–c). Activation was dependent on the presence of APCs as DENV alone did not activate MAIT cells (data not shown).

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Figure 3

Viral MAIT cell activation in vitro.

MAIT cells from healthy individuals were analysed by flow cytometry, gated on live CD3⁺CD161⁺⁺Vα7.2⁺ cells. (a,b) PBMC's (n=7) were co-cultured with autologous monocyte-derived DC's exposed to DENV (MOI=1) as described in ‘Methods'. (c–e) CD8⁺ T cells isolated from PBMC's (n=11–12) were co-cultured with IAV-exposed macrophages (MOI=1) as described in ‘Methods', unless indicated otherwise. (f) CD8⁺ T cells isolated from PBMC's (n=11–12) were co-cultured with macrophages exposed to the clinical H3N2 influenza A strain (A/England/691/2010 (n=7)) or influenza B (B/Florida/04/2006 (n=8)) (MOI=1) as described in ‘Methods'. (g,h) CD8⁺ T cells isolated from PBMC's (n=7–12) co-cultured with macrophages exposed to HCV (MOI=1) as described in ‘Methods'. Proportion of MAIT cells producing IFN-γ (a,c,d,f,g), TNF-α (c,d), CD69 (c,d) or Granzyme B (b,e,h). (d) Representative flow cytometry plots. All data are representative from at least two independent experiments. Bars represent means±s.e.m. Statistical significance was determined with the Kruskal–Wallis test followed by the Dunns' test (f) or the Mann–Whitney test (a,b,e,g,h). ns>0.05, *P 0.05, ***P≤0.001, ****P≤0.0001. Eng, England; Flor, Florida; HC, healthy control; Gr B, Granzyme B; MΦ, macrophage; REL/NR, relapse/non-response; SC, spontaneous clearance; SVR, sustained virological response; tr., treatment.

We next examined activation of MAIT cells in vitro in response to influenza virus. APCs incubated with influenza virus (MOI=1) promoted strong MAIT cell activation, inducing upregulation of CD69 and IFN-γ (with minimal TNF-α secretion), in a dose-dependent manner (Fig. 3c,d). Also, Granzyme B expression was induced (Fig. 3e). This activation was not restricted to laboratory-adapted influenza virus strains, but also was seen in response to recently-circulating strains of influenza A and B (Fig. 3f). Interestingly, MAIT cell activation was also readily induced using PBMCs instead of purified APCs (Supplementary Fig. 1d). Moreover, influenza virus exposure of purified monocyte, macrophage and DC populations all led to MAIT cell activation (Supplementary Fig. 1e).

Finally, we tested the impact of HCV on MAIT cells in vitro. MAIT cells expressed IFN-γ and Granzyme B (Fig. 3g,h), as well as CD69 in response APC's treated with HCV (MOI=1), in a dose-dependent and specific manner (Supplementary Fig. 1f–i).

Taken together, we demonstrate that MAIT cells are activated in vitro in response to DENV, influenza virus and HCV-exposed APCs.

Viral MAIT cell activation is dependent on cytokines

Next, we investigated the mechanisms of MAIT cells activation in response to viruses. The ligand presented by MR1 is derived from the riboflavin synthesis pathway, which is not found in host cells or viruses⁷. Consistent with this, using the models described above, we confirmed by antibody blocking of MR1 that MAIT cell activation was TCR-independent for all the viruses tested (Fig. 4a–c).

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Figure 4

Viral MAIT cell activation is dependent on cytokines.

(a–d,f,h) Isotype control, anti-MR1, anti-IL-12, anti-IL-15 and/or anti-IL-18 antibodies were added to a co-culture exposed to DENV (a,d, n=3–6), IAV (b,f, n=5–29) or HCV (c,h, n=7–16). IFN-γ expression by MAIT cells (gated on live CD3⁺CD8⁺CD161⁺⁺Vα7.2⁺ cells) was analysed by flow cytometry and is shown relative to the isotype control. (e,g,i) IL-12p70, IL-15 and IL-18 levels secreted by virus-exposed APC's. (e) DENV-exposed DC's (n=4) at 42h. (g) IAV-exposed macrophages (n=7) at 48h. (i) HCV-exposed macrophages (n=7) at 48h. Data are representative from at least two independent experiments. Bars represent means±s.e.m. Statistical significance was determined with the Mann–Whitney test (a–c,e,g,i) or the Kruskal–Wallis test followed by the Dunns' test or the Mann–Whitney test (d,f,h). ns>0.05, *P 0.05, ***P≤0.001, ****P≤0.0001. ND, not detected.

As activation was TCR-independent, we explored triggering of MAIT cells by cytokines. Previously, we have shown that TLR8 is capable of inducing IFN-γ expression in MAIT cells via IL-12 and IL-18 (ref. ²⁴). In addition, IL-15 can specifically activate distinct functions of MAIT cells in synergy with IL-12 and/or IL-18, in a dose-dependent manner (Supplementary Figs 2 and 3)²⁵. We extended this finding by exploring responses to a range of TLR ligands in PBMCs and found endosomal TLR3 was also a potent activator (Supplementary Fig. 4). As with TLR8, TLR3 induced MAIT cell activation via IL-18 and IL-12 and not MR1^12,13. TLR sensing by APC's can occur in the absence of viral replication^26,27. To assess the requirement of viral replication for MAIT cell activation, we used ultraviolet-irradiation of the viruses, which prevents transcription or replication. Ultraviolet-irradiated DENV was no longer able to activate MAIT cells (Supplementary Fig. 5a). In contrast, ultraviolet-irradiated HCV and influenza virus were still able to activate MAIT cells, although less efficiently compared to untreated virus (Supplementary Fig. 5b,c). Accordingly, DENV productively infects APCs, whereas productive influenza virus and HCV infection is limited in both primary and stem cell-derived human APCs (Supplementary Fig. 5d–f)^28,29,30. Furthermore, the level of DENV infection correlated with MAIT cell IFN-γ expression (Supplementary Fig. 5e).

We next assessed blockade of IL-12, IL-18 and IL-15 in the in vitro virus models. Figure 4d reveals that IL-12 and IL-18 had the most significant impact on DENV-dependent MAIT cell activation using DC's, with anti-IL-18 blockade alone leading to complete suppression of IFN-γ expression. IL-18 was the most abundant cytokine induced in DC's in response to DENV (Fig. 4e).

Anti-IL-18 also inhibited activation of MAIT cells induced by influenza virus, a result observed using purified APCs (Fig. 4f) or PBMC's (Supplementary Fig. 6a). Blockade of IL-12 and IL-15 alone or in combination had a limited impact in influenza virus-induced MAIT cell activation using macrophages (Fig. 4f). Accordingly, IL-18, but not IL-12 or IL-15 was secreted by macrophages after incubation with influenza virus (Fig. 4g).

Similar to DENV and influenza virus, IL-18 had a dominant role in HCV-induced MAIT cell activation (Fig. 4h). Addition of further blockade with anti-IL-15 was required to achieve maximal suppression. We assessed expression of IL-12, IL-15 and IL-18 on macrophages after incubation with HCV (Fig. 4i). No IL-15 protein was detected, yet IL-15R (responsible for IL-15 trans-presentation³¹) was upregulated by macrophages in response to HCV, as measured by quantitative PCR (Supplementary Fig. 6b).

Overall, these experiments indicate a mechanism by which viruses can activate MAIT cells, dependent on viral sensing and cytokines, with a key role for IL-18, and independent of MR1 and TCR.

Virally-triggered IL-18 correlates with MAIT cell activation

Having identified IL-18 as an important cytokine for viral MAIT cell activation in vitro, we further explored its relevance in vivo. In DENV infection, IL-18 levels in plasma were markedly increased during the acute phase compared to the convalescent phase of infection and healthy controls (Fig. 5a). In addition, patients suffering from the severe form of dengue (DHF) had significantly higher levels of IL-18 over the course of acute infection compared to patient with DF (Fig. 5b). Interestingly, expression of the IL-18 receptor (IL-18R) on MAIT cells was upregulated during the acute phase of infection and resolved to healthy control levels in the convalescent samples (Fig. 5c). As observed for IL-18, patients with DHF revealed higher expression of IL-18R on MAIT cells compared to patient with DF (Fig. 5d,e). Thus, levels of IL-18 in plasma and expression of IL-18R on MAIT cells in vivo are upregulated during the acute phase of infection, especially in those patients suffering from DHF. These findings show an association with increased activation of MAIT cells and disease severity in dengue patients (Figs 1f and ​and5f).5f). These results are not restricted to DENV infection since IL-18 protein (Fig. 5g) and mRNA (Fig. 5h) were found readily in liver tissues from patients with HCV infection.

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Figure 5

Virally triggered IL-18 correlates with MAIT cell activation.

(a–d) PBMC's from healthy controls (n=10) or patients suffering from severe dengue (DHF, n=2–12) or dengue (DF, n=4–12) were analysed. (a,b) Plasma levels of IL-18. (c,d) IL-18Ra expression on MAIT cells. (a,c) Comparison between acute (day 0) and convalescent (conv) phase (day >10) of infection. (b,d) Comparison between DHF and DF patients. Correlation of plasma levels of IL-18 (e) or MAIT cell CD38 expression (f) against MAIT cell IL-18Ra expression by the Spearman-rank correlation test. (g) Paraffin-embedded human liver tissues sections from HCV or control patients were stained for IL-18 (brown-coloured). Scale bar, 10mm. (h) IL-18 mRNA expression in liver biopsies from HCV patients (n=55) relative to uninfected control liver samples is shown (n=6). Bars represent means±s.e.m. Statistical significance was determined with the Mann–Whitney test (b,d,h) or the Wilcoxon matched-paired test (a,c). ns>0.05, *P 0.05, ***P≤0.001, ****P≤0.0001. conv, convalescent; HC, healthy control.

Overall, these data indicate that IL-18 is not only a dominant factor during in vitro viral MAIT cell activation, but also has an important role in vivo.

MAIT cells respond to type I IFNs in vitro and in vivo

Type I IFNs are also important cytokines known for their strong antiviral activity. Previous reports have determined a crucial role of IFN-α/β during DENV³², influenza³³ and HCV³⁴ infections in limiting viral replication and disease severity. We therefore addressed whether IFN-α/β can also influence MAIT cell activation in vitro and in vivo.

First, we tested whether IFN-α/β could trigger MAIT cells alone or in combination. We found IFN-γ secretion by MAIT cells in response to IFN-α/β in combination with IL-12 and IL-18, but not IL-15 (Fig. 6a), in a dose-dependent manner (Supplementary Fig. 7). Next, we confirmed that these TCR-independent responses to cytokines are shared with other CD161⁺⁺ T-cell subsets, but not CD161⁻ T-cell populations. CD161⁺⁺CD8⁺ T cells that lack expression of the Vα7.2Jα33 TCR (Supplementary Fig. 8a), CD161⁺⁺ Vα7.2Jα33⁺ CD4 T cells (Supplementary Fig. 8b) and CD4⁻CD8⁻ T cells (Supplementary Fig. 8c) could all be triggered in response to cytokines, unlike CD161⁻ T-cell populations (Supplementary Fig. 8d). This is in agreement with our previous findings which suggest that CD161-expressing lymphocytes display a shared innate response to cytokines¹⁶.

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Figure 6

MAIT cells respond to type I interferons in vitro and in vivo.

(a) PBMC's from healthy individuals were directly stimulated for 24h with IFN-α, IFN-β, IL-12, IL-15, IL-18 or indicated combinations thereof. IFN-γ expression by MAIT cells (gated on live CD3⁺CD8⁺CD161⁺⁺Vα7.2⁺ cells) was analysed by flow cytometry. (b–d) B18R (IFN-α/β neutralizing protein) or PBS control were added to the co-culture (n=6–11). IFN-γ (b), CD69 (c) and Granzyme B (d) expression is shown relative to the control. Data are representative from at least two independent experiments. (e) PBMC's from healthy controls or HCV patients at baseline, during or end of treatment with either SOF+RBV or SOF+RBV/PEG-IFN were analysed by flow cytometry. CD69 expression on MAIT cells (gated on live CD3⁺CD8⁺CD161⁺⁺Vα7.2⁺ cells) was measured. Bars represent means±s.e.m. Statistical significance was determined with the Kruskal–Wallis test followed by the Dunns' test (a) or the Mann–Whitney test (b–e). ns>0.05, *P 0.05, ***P≤0.001, ****P≤0.0001. HC, healthy controls.

To further explore the role of type I IFNs during viral MAIT cell triggering, we used a vaccinia virus-encoded, soluble type I IFN receptor (B18R)³⁵. Macrophages treated with HCV in vitro were incubated in the presence of B18R and T cells overnight. Blockade of type I IFNs by B18R inhibited MAIT cell IFN-γ secretion (Fig. 6b), CD69 expression (Fig. 6c) and Granzyme B upregulation (Fig. 6d).

IFN-α has been an important component of HCV treatment. This gave us an opportunity to explore whether type I IFNs could contribute to MAIT cell activation in vivo. We measured the activation marker CD69 on MAIT cells from HCV patients taking part in a clinical trial in which they were treated with either sofosbuvir (SOF)+ribavirin (RBV)+pegylated IFN (PEG-IFN), or only SOF+RBV, without PEG-IFN. Interestingly, CD69 expression on MAIT cells was upregulated only in the treatment group receiving PEG-IFN (Fig. 6e), which corresponded with a significantly higher sustained virologic response rate³⁶. High CD69 upregulation was specific to CD161⁺⁺MAIT cells and not observed on CD161⁻CD8⁺ or CD4⁺ T cells (Supplementary Fig. 9). These data demonstrate that type I IFNs can contribute to MAIT cell activation in vitro and in vivo.

Tissue staining

Paraffin-embedded human liver tissues sections were dewaxed using Clearene and IMS. Endogenous peroxidase was blocked by using 5% hydrogen peroxide followed by antigen retrieval using 10% EDTA buffer. Slides were blocked using casein buffer. Primary Rabbit IL-18 antibody LS-B2809, 1:50 dilution was applied for an hour followed by Vector Impress anti-rabbit secondary for 30min in a staining chamber with rocking. Slides were then washed with PBS+Tween and Impress DAB substrate was applied for 2½ mins. Slides were then counterstained in Mayer's haematoxylin and mounted in DPX.

Cell culture

Human PBMCs were isolated from leukocyte cones from healthy donors supplied by the NHS blood service. CD8+ T-cells were isolated from PBMCs using positive selection with MACS CD8+ microbeads (Miltenyi). Monocytes were enriched from PBMCs using MACS CD14+ microbeads (Miltenyi). Monocytes were differentiated into macrophages (macrophages or GM-MΦ), by incubating the CD14+ cells for 7 days in X-VIVO15 (Lonza), Pen/strep (Sigma-Aldrich), L-glutamine (Sigma-Aldrich) with 50ngml⁻¹ GM-CSF (Miltenyi). Immature dendritic cells (imDC's or DC's) were obtained by culturing CD14⁺ cells for 4–5 days in the presence of 20–100ngml⁻¹ GM-CSF (Miltenyi/First Link) and 25ngml⁻¹ IL-4 (Miltenyi/eBioscience). Where stated, MAIT cells were sorted using a Beckman Coulter MoFlo XDP.

Viruses

DENV serotype 2, strain 16681, was grown in C6/36 cells in Leibovitz's L-15 medium with L-glutamine and supplemented with 2% fetal calf serum. Culture medium was centrifuged and stored at −80°C. The titres of virus were determined by a focus-forming assay on Vero cells and expressed as focus-forming units per ml. Briefly, virus was serially diluted and incubated with Vero cells for 2h at 37°C. The monolayers were then overlaid with 1.5% carboxymethylcellulose and incubated at 37°C for 3 days. Virus foci were stained with anti-DENV E antibody (4G2) followed by peroxidase-conjugated anti-mouse Ig and visualized by the addition of DAB substrate. Infection rates of DENV infected DCs were assessed using an antibody detecting the non-structural DENV protein, NS3, by flow cytometry. The mouse monoclonal anti-DENV NS3 (E1D8) and anti-DENV E (4G2) were gifts from E. Harris and AFRIMS.

A total of 10ng RNA transcribed from genotype 2a HCV strain J6CF-JFH1 (obtained from Prof. Bartenschlager⁶²) was electroporated into Huh-7.5 cells (obtained from Apath) and were cultured for up to 3 weeks. Cell culture supernatants were collected for up to 20 days post electroporation centrifuged and concentrated using an Amicon Ultra-15 (Millipore). This inoculum was used to infect huh-7.5 cells. The HCV titre was determined by immunofluorescence. Huh-7.5 cells were fixed with methanolacetone, blocked with BSA/phosphate-buffered saline (PBS) solution, washed with PBS, stained with anti-HCV core primary antibody (Cambridge Biosciences), washed with PBS and Alexa Fluor 488 donkey polyclonal secondary antibody to Mouse IgG (ab150105, Abcam). To measure HCV replication a HCV luciferase reporter Jc1FLAGp7-nsGluc2A²⁹ virus was used. Jc1FLAG(p7-nsGluc2A) was grown in naïve Huh-7.5 cells⁶³ and the infectivity titre was determined by limiting dilution titration on naive Huh-7.5 cells as median tissue culture infective dose (TCID50). To measure HCV replication, Huh-7.5 cells were plated in a 96-well plate and infected at an MOI of 0.1 for 6h. After washing the Huh-7.5 cells, supernatants from (un)stimulated MAIT cells were added in the presence or absence of a neutralizing IFN-γ (clone MD-1, 14-7317-85 eBioscience). Supernatants were collected 4 days post infection and luciferase expression determined using the Renilla Luciferase Assay System (Promega) and a Berthold TriStar2 multimode reader LB 942.

Influenza A/WSN/33 (H1N1) virus (WSN) was grown in Madin Darby Bovine Kidney (MDBK, obtained from the European Collection of Cell Cultures) cells in Minimum Essential Medium Eagle (Sigma) with 2mM L-glutamine and 0.5% fetal calf serum. To produce virus stocks, six 175cm² tissue culture flasks containing sub-confluent MDBK cells were infected at low-multiplicity. After 48h of culture in a 37°C humidified incubator, supernatants were collected and clarified by low-speed centrifugation (30min, 2,000g and 30min, 18,000g, 4°C). Next, WSN was concentrated through a 30% sucrose cushion by ultracentrifugation (90min, 112,000g, 4°C). Finally, WSN was purified on a 30–60% sucrose gradient by ultracentrifugation (150min, 209,000g, 4°C). The visible band of virus was drawn off with a needle. (Hutchinson, E. and Fodor, E., Nature ProtocolExchange, Purification of influenza virions by haemadsorption and ultracentrifugation, ‘ http://www.nature.com/protocolexchange/protocols/3315')^64,65. Plaque assays were performed on MDBK cells using standard techniques. Influenza A (H3N2) virus A/England/691/2010 is a clinical strain isolated by Public Health England (PHE), as part of the MOSAIC project. Influenza B virus, B/Florida/04/2006 was derived from reverse genetics systems by de novo synthesis (GeneArt).

In vitro virus experiments

Unless specified differently, for co-culture experiments using viruses, APCs were treated with virus at a MOI of 1, as determined based on the viral titre and number of cells plated, for 90 to 120min at 37°C. Virus was washed off and isolated CD8+ T-cells were added for an additional 10–24h, unless specified differently. In the case of the DENV experiments, PBMC's were added instead of isolated CD8+ T-cells. In the case of influenza virus, WSN virus was used, unless specified differently.

In vitro cytokine stimulations

For cytokine stimulation experiments, isolated CD8+ T-cells or PBMC's were stimulated for 24h with 50ngml⁻¹ IL-12 (Miltenyi Biotech), 50ngml⁻¹ IL-18 (R&D Systems Europe), 50ngml⁻¹ IL-15 (Miltenyi Biotech) or 2,000Uml⁻¹ IFN-α (Sigma-Aldrich) or 50ngml⁻¹ IFN-β (Miltenyi Biotech) or combinations thereof. For blocking experiments, neutralizing agents were added into the culture together with the CD8⁺ T-cells or PBMC's. Blocking antibodies against IL-12p40/70 (508804, eBioscience), IL-12p70 (MAB219-100, R&D), IL-15 (MAB2471, R&D) or IL-18 (D044-3, MBL International, USA), IFN-γ (14-7317-85, eBioscience) or MR1 (361102, Biolegend) were used at 5–10μgml⁻¹. Type I IFN was blocked using 1μgml⁻¹ B18R (34-8185-81, eBioscience).

Flow cytometry

Antibodies/dyes and dilutions used were: viability dye live/dead fixable-violet (L34955, Invitrogen, 1:1250), CD3-eFluor450 (48-0038, eBioscience, 1:100), CD3-PECy7 (25-0038, eBioscience, 1:100), CD4-VioGreen (130-096-900, Miltenyi Biotech, 1:50), CD8-VioGreen (130-098-062, Miltenyi Biotech, 1:50), CD8-V450 (560347, BD, 1:50), CD8-PerCP.Cy5.5/PerCP (301032, Biolegend, 1:100), CD38-APC (555462, BD, 1:50), CD69-FITC (11-0699, eBioscience, 1:40), CD161-APC (130-098-908, Miltenyi Biotech, 1:100), CD161-PE (130-099-193, Miltenyi Biotech, 1:100), IFN-γ-FITC (130-091-641, Miltenyi Biotech, 1:50), IFN-γ-APCCy7 (502529 Biolegend, 1:50), Vα7.2-PE/PeCy7/APC/FITC (351705/351711/351707/351703, Biolegend, 1:50). Granzyme B-APC (MHGB05, Invitrogen), IL-18Ra-APC (17-7183-41, eBioscience, 1:50), TNF-α-PeCy7 (502929, Biolegend, 1:100). All data was acquired on a MACSQuant (Miltenyi Biotech) or a BD FACSVerse (BD) and analyzed on FlowJo (Tree Star Inc.). Gating strategy is shown in Supplementary Fig. 12.

Cytokine measurements

Cytokines levels in this study were measured from cell culture supernatants or heparin plasma samples. Cell culture supernatants from macrophages treated with mock, HCV or influenza A were collected at 48h. Cell culture supernatants from DCs treated with mock or DENV were collected at 42h. Heparin plasma samples were collected from DENV infected patients at different phases of illness or from healthy controls. Cell culture supernatants and heparin plasma samples were frozen at −80̊C until further use. Cytokine levels were analysed using Bio-Plex human cytokine kits (BioRad) and acquired on a Luminex 100 (Luminex) or a Bio-Plex 200 reader (BioRad) according to the manufacturer's instructions.

This research was supported by the NIHR Biomedical Research Program (Oxford), the Wellcome Trust (WT091663MA) and National Institutes for Health Research Biomedical Research Centre (Imperial College), the NIH (U19AI082630) and the Oxford Martin School. I.S. contribution was supported by the Wellcome Trust programme Grant PS2186_WMII. The STOP-HCV consortium is funded by a grant from the Medical Research Council. E.C.H. contribution was supported by MRC programme Grant MR/K000241/1 awarded to Ervin Fodor. L.H. contribution was supported by MRC Project Grant G0600371. Y.H.O. contribution was supported by MRC intermediate fellowship programme. The authors wish to acknowledge the role of the HCV Research UK Biobank in collecting and making available the samples/data used in the generation of this publication. We thank Wendy Barclay for providing Influenza A (H3N2) virus A/England/691/2010 and Influenza B virus, B/Florida/04/2006. We thank Mark Ainsworth for overseeing the HCV patients at the JR hospital. We thank Amin Moghaddam, Jacob Hurst and Leo Swadling for their scientific advice. We also want to acknowledge Nattaya Tangthawornchaikul and Thaneeya Duangchinda.