Influenza vaccine-specific T cell responses are impaired in older adults

Influenza vaccine-specific T cell responses are impaired in older adults | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Influenza vaccine-specific T cell responses are impaired in older adults Pauline Saint-Charles, Magdalena Hagen, Gaelle Autaa, Elske Bijvank, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7818000/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Apr, 2026 Read the published version in npj Vaccines → Version 1 posted 9 You are reading this latest preprint version Abstract Immune defences decline with age, rendering older adults vulnerable to infectious diseases such as influenza. Vaccination remains the most effective means of preventing severe illness and death caused by influenza, although its efficacy is diminished in older adults. Vaccine-specific antibody responses and effectiveness are generally lowest against the influenza A(H3N2) strain, and particularly in individuals over 65 years of age. The mechanisms underlying this age-related decline in vaccine responsiveness remains unclear, prompting the present investigation into the quantity and quality of influenza specific T-cell responses upon vaccination. Frequencies of T cells specific to two influenza A strains H1N1 and H3N2, and two influenza B strains Victoria and Yamagata were measured in adults before and after quadrivalent inactivated influenza vaccination, stratified by age, under (n = 100) or over (n = 120) 65 years. Polyfunctionality of vaccine induced CD4 + and CD8 + T cells and immune ageing markers were also assessed in a selection of responders (n = 34). Older individuals showed significantly reduced H3N2-specific CD4⁺ T cell frequencies ( P = 0.003) and polyfunctionality ( P = 0.04) which correlated with lower H3N2 hemagglutination inhibition antibody titers ( r = 0.42, P = 0.008). Cytomegalovirus seropositivity was associated with diminished influenza specific CD8⁺ T cell responses in the older age group ( P = 0.01). These findings highlight both quantitative and qualitative deficiencies of influenza-specific memory T cells in older vaccinees, which could explain suboptimal humoral responses with advanced age, notably against H3N2. This underscores the need for vaccines designed to boost cellular immunity in this vulnerable population, potentially through improved H3N2 antigen design or alternative vaccine platforms promoting stronger T cell induction. EU Clinical Trials Registration 2019–000836–24 Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology Influenza vaccination immunogenicity T cells older adults antigen design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Influenza or flu is a contagious respiratory illness caused by influenza viruses, which primarily affect the upper respiratory tract and lungs, and can lead to hospitalization, as well as severe complications, such as pneumonia, myocarditis, diabetes and kidney failure. Influenza viruses infect 5–15% of the population worldwide annually, causing 3–5 million cases of severe illness and possibly up to half a million deaths each year 1 , 2 . Therefore, influenza remains a major public health issue. Due to age-related weakening of their immune defenses, people over 65 years of age are at high risk for developing severe illness and complications 3 . Vaccination remains the most effective protective measure, reducing severe influenza cases by inducing antibodies and T cells targeting the virus 4 , 5 . Annual influenza vaccination is thus strongly recommended for the most vulnerable populations, including older adults. As a consequence of the constant genetic changes of influenza viruses and their variability from one season to another, the composition of the influenza vaccine is adapted every year to incorporate the most recent circulating influenza A (H1N1 and H3N2) and influenza B strains 4 . Vaccine efficacy depends on the match between the vaccine and circulating strains, of which the selection may now gain from artificial intelligence-based predicting assistance 6 . Vaccine efficacy is also impacted by the age of the vaccinees. While influenza vaccines offer some protection in older adults, the level of protection is generally lower and varies among influenza strains. Meta-analyses of studies published between 2004–2016 on influenza vaccine effectiveness (IVE) against several virus strains reveal a pooled seasonal IVE of 51% in adults aged 18–64, but only 37% in those above 65 7,8 . Furthermore, IVE against H3N2 most notably lower at 33% in older adults compared to 50% in younger adults 9 . This reduced efficacy is reflected in lower seroprotection and antibody titers following vaccination, particularly against H3N2, limiting the ability to neutralize the virus 10 , 11 . These effects may be attributable to age-associated immune alterations as well as repeated vaccination 12 , which could result in weakened and exhausted B cell responses and decreased number of vaccine-specific plasmablasts in older adults 13 , 14 . However, factors underlying lower humoral responses and IVE in older adults are not fully understood. Gaining further insights is important to improve vaccine design and efficacy, especially for vulnerable populations. Given the significant age-related alterations observed in the T lymphocyte compartment 15 , 16 , studying T cell responses to vaccination in older adults is therefore particularly relevant in this context. Defects of CD4 + T cell responses, such as reduced activation of T follicular helper (Tfh), which provide help to B cells, may have a direct impact on humoral immunity and antibody titers in older adults 17 , 18 . Moreover, CD8 + T cell responses are important for providing protection from severe influenza illness 19 . Their impaired induction may contribute to reduced IVE in older adults. However, studies of influenza vaccine cellular responses in relation to age and virus strains are scarce, relative to the number of studies on humoral responses. Earlier studies of soluble factors released by T cells, like IFNγ, IL-10 or Granzyme B, upon activation with influenza viruses suggested defective vaccine-specific cellular responses with advanced age 20 – 23 . Here, we studied the T lymphocyte compartment in young (< 65 years) and older (≥ 65 years) adults vaccinated with a standard quadrivalent inactivated subunit vaccine containing viral antigens from two influenza A and two influenza B virus strains 24 , 25 . We examined both quantitative and qualitative aspects of T cell responses in vaccinees. Frequencies of total vaccine-specific T cells were measured using IFNγ Elispot in older adults, and their functional potential was assessed by intracellular staining of cytokines upon vaccine antigen mediated stimulation. Markers of immune ageing were also analysed and included the naive-memory phenotype, the activity of senescence-associated-β-galactosidase (SA-β-gal) and the proliferation potential of total T cell populations, as well as the seropositivity for cytomegalovirus. The latter is known to alter the T cell subset homeostasis, and to play an important role in changes of the immunological landscape associated with ageing 26 – 28 . Our findings underscore reduced magnitude and functional impairment of influenza vaccine-induced T cell responses in older adults. This may participate to suboptimal antibody titers upon vaccination and IVE with old age, particularly against H3N2. Results Older adults have reduced influenza-specific T cell responses To characterize T cell responsiveness to influenza vaccination, we analyzed 220 of the 326 participants of the Vaccines and InfecTious diseases in the Aging popuLation (VITAL) cohort 24 , 25 , who were immunized with one dose of the quadrivalent inactivated influenza vaccine (QIV) in autumn 2019 ( Figure S1 ). The 2019 QIV contained haemagglutinin (HA) and neuraminidase (NA) antigens from the influenza A virus strains H1N1 (A/H1N1 Brisbane/02/2018) and H3N2 (A/H3N2/Kansas/14/2017), and influenza B virus strains Victoria (B/Vic/Maryland/15/2016) and Yamagata (B/Yamagata/16/88 lineage). Participants were selected for being vaccinated against influenza the year before to minimize the influence of disparate influenza vaccination history on T cell responses and were separated in two age groups (< 65y n = 100 and ≥ 65y n = 120). Frequencies of influenza vaccine-specific T cells were measured at days 0, 7, and 28 as well as at month 6 post-vaccination, by IFNγ Elispot upon stimulation with HA and NA antigens contained in the vaccine. Levels of flu-specific T cells were globally similar for all virus strains at a given time point and age group, except H3N2, for which T cell frequencies were lowest in both age groups at all time points ( P < 0.0001 at day 0, P = 0.0015 at day 7 and P = 0.0008 at day 28 compared to H1N1) ( Figure S2 ). This highlights the disparate immunity against different influenza viruses. At peak response (i.e., day 7), T cell frequencies specific for 3 out of the 4 virus strains were lower ( P = 0.003 for H3N2, P = 0.02 for H1N1, and P = 0.008 for Victoria) in the older group compared to the younger group (Fig. 1 A). The difference between age groups was confirmed for H3N2 ( P = 0.003) and Victoria ( P = 0.03) with regards to vaccine responsiveness, i.e. assessing the frequency of responders (defined as vaccinees with an increase in T cell frequencies from day 0 to day 7 of at least 5 spots) for each strain (Fig. 1 B). For instance, only 38% of participants above 65 years were considered responders to H3N2, in contrast to 68% of participants below 65 years. Overall, H3N2-specific T cell responses appeared therefore lower than responses to other strains, and were particularly reduced in older versus younger adults. We next evaluated the capacity of vaccinees from the two age groups to respond to several strains of the 4 influenza A and B viruses simultaneously (corresponding to the frequency of participants responding from none to the four strains). Fewer participants of the older age group were able to respond to multiple strains ( P = 0.02), with 18% of them showing responses to all four vaccine strains, compared to 36% in the younger age group (Fig. 1 C). Moreover, we calculated an influenza specific T cell score for each participant, to account for their overall vaccine responsiveness at day 7, considering both its intensity (i.e. T cell frequency) and its breadth (i.e. response to the four strains). To this end, we assigned a value (1, 2, 3 or 4) based on the quartiles of the response for each influenza strain and calculated the mean value for the four strains. This score was lower ( P = 0.01) in older versus younger subjects (Fig. 1 D). These results highlight a globally reduced T cell responsiveness, in terms of frequency at peak response and reactivity to multiple virus strains, to recall vaccination against influenza in older adults. Influenza vaccine-specific CD4 T cells have limited helper potential in older adults In order to distinguish between CD4 + and CD8 + T lymphocytes among influenza vaccine-specific cells, we performed flow cytometry-based assays on a selection of participants (< 65y n = 17 and ≥ 65y n = 17), arbitrarily chosen among strong responders in Elispot assays. Using this approach, we assessed the capacity of the responding cells to upregulate CD40L + and secrete a variety of cytokines (namely IFNγ, TNF, IL-2, and IL-21), which reflect functional properties of T cells, in particular the helper potential of CD4 + T lymphocytes (Fig. 2 A and Figure S3 ). Responsiveness was tested for two strains, H3N2 and Yamagata (as the most and least affected responses by age in terms of frequency, respectively), using the same antigens as for the Elispot assay. In line with the Elispot data, cytokine-secreting cells were, in general, fewer to respond to H3N2 compared to Yamagata (Fig. 2 B). IL21 + cells were infrequent and often undetectable. We observed clear trends towards lower frequencies of CD40L + CD4 + T cells secreting each individual cytokine upon stimulation with H3N2 or Yamagata antigens in older versus younger responders (Fig. 2 B). Of note, vaccine antigen-specific CD8 + T cell responses showed high inter-individual diversity in younger and older groups, and did not enable us to draw any association with age ( Figure S4 ). This may be partly related to the poor efficacy of inactivated subunit vaccines, such as QIV, to induce CD8 + T cell responses. We next consider the polyfunctional profile of influenza vaccine-specific CD4 + T cells, i.e., their capacity to secrete multiple helper cytokines simultaneously. Older vaccine responders displayed lower frequencies of polyfunctional CD4 + T cells (i.e. CD40L + cells secreting IFNγ, TNF and IL-2, or TNF and IL-2) specific for either H3N2 ( P = 0.04) or Yamagata ( P = 0.01) (Fig. 2 C), and these cells had lower polyfunctionality indexes (Fig. 2 D), compared to younger vaccinees. We found a correlation between polyfunctionality indexes of H3N2-specific CD4 + T cells and the hemagglutination inhibition (HI) antibody titers against H3N2 measured in the same subjects ( r = 0.42, P = 0.008) (Fig. 2 E). In contrast, IFNγ-production alone, as measured by Elispot or intracellular cytokine staining does not correlate with antibody titers ( r = 0.07, P = 0.28 or r = -0.03, P = 0.84, respectively) (data not shown). This highlights the importance of polyfunctional T cells as a measure for efficient T cell help. We next evaluated the magnitude of total and vaccine-specific circulating CD4 + T follicular helper cells (cTfh), and their relationship to age and antibody responses in the same vaccinees. For this purpose, we used CXCR5 as a marker of cTfh on total CD4 + T cells or activated CD40L + CD4 + T cells upon stimulation with vaccine antigens (Fig. 3 A). We found a lower frequency of total cTfh in people above versus below 65 years of age ( P = 0.002) (Fig. 3 B), which aligns with previous reports 29 . CXCR5 + CD40L + CD4 + T cells were usually scarce, particularly those specific for H3N2. There was a trend towards lower frequencies of Yamagata reactive cTfh in older vaccinees (Fig. 3 C ) . Of note, the frequency of total cTfh correlated with HI antibody titers against H3N2 ( r = 0.37, P = 0.027) (Fig. 3 D ) . The correlation was particularly evident for the younger group ( r = 0.59, P = 0.012), which displays the highest levels of cTfh. Altogether, these observations support a qualitative skewing of influenza vaccine-specific CD4 + T cells, and their limited help to B cells and the production of HI inducing antibodies in older vaccinees. Influenza vaccine-specific CD8 T cells are low in CMV seropositive older vaccinees Lastly, we aimed to determine if suboptimal T cell responsiveness to the influenza vaccine in older adults could be related to certain hallmarks of immune ageing in CD4 + and CD8 + T lymphocytes, namely the alteration of the naive versus memory T cell homeostasis, markers of cellular senescence, as well as the influence of CMV infection. T cell differentiation subset homeostasis was assessed by looking at the frequencies of naive (CD45RA + CCR7 + ) versus memory (non CD45RA + CCR7 + ) CD4 + and CD8 + T cells (Fig. 4 A). Cellular senescence was informed by assessing the activity of SA-β-gal by flow cytometry (Fig. 4 B) in naive and memory CD4 + and CD8 + T lymphocytes, and the proliferation capacity upon TCR-mediated stimulation (Fig. 4 C) of the same T cell subsets. Although alterations of the CD8 + T cell subset homeostasis, together with increased levels of SA-β-gal and lower proliferation capacity in naive CD8 + T cells were observed in older subjects (Fig. 4 ), this was not the case for CD4 + T cells. Naive and memory CD4 + T cell subsets did not present obvious change with regards to frequency or cellular senescence markers comparing younger and older age groups. Suboptimal CD4 + T cell responsiveness to influenza vaccine could therefore not be attributed to intrinsic alterations of this lineage. Initial analyses did not reveal apparent effects of CMV seropositivity on total H3N2 or Yamagata-specific total T cell responses measured by Elispot, nor on H3N2 HI titers in younger or older vaccinees (Fig. 5 A and Figure S5) . However, CD8 + T cell responses specific for both H3N2 and Yamagata, measured by intracellular cytokine staining, were lower in the CMV-seropositive vs CMV-seronegative subjects of the older ( P = 0.03), but not of the younger group (Fig. 5 B). In contrast, CMV-serostatus did not have an influence on the influenza vaccine-specific CD4 + T cells (Fig. 5 C). In line with these observations, the fraction of memory CD8 + , but not CD4 + , T cells was altered ( P = 0.01) in CMV-seropositive older individuals (Fig. 5 D). This suggests that CMV-related alteration of the CD8 + T cell compartment homeostasis has an influence on the memory CD8 + T cell responses to influenza vaccines with ageing. Discussion Factors underlying the limited seroprotection against influenza viruses, specially H3N2, in older vaccinees remain poorly understood. Low antibody titers and quality upon influenza vaccination in older adults may be in part attributed to less functional B cells, weaker germinal centers, and impaired affinity maturation associated with ageing 30 . Our findings underline a quantitative and qualitative impairment of CD4 + T cell responses to recall influenza antigens in older adults. Previous studies in aged mice also indicate that impaired cytokine production by CD4 + T cells following antigenic stimulation affects B cell help functions, causing reduced humoral immunity to influenza vaccine 17 , 23 , 31 , 32 . In young adults, cTfh cell frequencies was associated with increased antibody titers after vaccination 18 . In older vaccinees, the waning functional capacity of CD4 + T cells, in particular Tfh, to confer help to B cells likely participates to the reduced capacity to stimulate strong antibody responses and therefore reduced effectiveness of influenza vaccines. Of note, we found that H3N2-specific CD4 + T cell responses were lowest in older adults, compared to CD4 + T cell responses specific for other influenza A or B strains. Although the underlying reasons are unclear, it may explain the particularly reduced seroprotection against H3N2 reported in vaccinated older adults 7 , 9 . This indicates that efforts should be undertaken to enhance H3N2-specific T cell responses using vaccines. This may be achieved through specific optimization of H3N2 antigens to make them more immunogenic. Although the use of whole protein antigens in our ex vivo assays primarily promotes the activation and detection of CD4⁺ T cells, we were nonetheless able to detect influenza-specific CD8⁺ T cells in vaccinees. Notably, these cells were found in lower frequencies among older individuals who were CMV-seropositive. CMV seropositivity has also been reported to negatively affect B cell and antibody responses to the influenza vaccine 33 . Reduced CD4 + T cell responses to CMV-unrelated antigens in older CMV-infected individuals have been previously documented in the context of primary vaccination (i.e., de novo T cell responses from the naive T cell pool) 34 . Here, our data suggest that CMV infection can also affect memory CD8⁺ T cell responses upon recall vaccination. Although precise mechanisms remain to be elucidated, one possible explanation is that overcrowding of the “immunological space” by T cells specific for persistent viruses like CMV, by disrupting T cell homeostasis, may hinder the activation of heterologous memory T cell responses 35 . Consistent with this, influenza virus-specific T cell frequencies were actually reported to be lower in CMV-seropositive older individuals compared to CMV-seronagative older individuals 36 , 37 . Robust CD8 + T cell responses are important not only for mitigating disease severity but also for providing cross-reactive protection 19 . The potential for CD8⁺ T cell cross-reactivity across influenza A, B, and C viruses further supports the rationale for developing universal T cell-based influenza vaccines 38 . While current inactivated vaccines are not known to elicit strong CD8 + T cell responses, the use of recombinant vaccines such as mRNA vaccines may offer significant opportunities to promote cellular immune responses against influenza 5 . However, it will be important to consider the impact of CMV seropositivity in these contexts in particular for older adults. Overall, our work highlights that it is imperative to promote stronger and more functional T cell responses upon immunization against influenza in older adults. To address weaker induction of immune responses, vaccines should be designed specifically for adults aged 65 and older. Ongoing research aims to develop vaccines that better address the unique immunological challenges of ageing. High-dose seasonal influenza vaccines (e.g. Fluzone High-Dose), which can contain four times the antigen amount can help stimulate stronger responses in older adults 39 . Moreover, adjuvants are likely to be very important in this context. For instance, oil-in-water adjuvanted influenza vaccines (e.g. Flurarix® and Fluad®, which contain the adjuvants AS03 and MF59 respectively) can enhance immune responses in older adults 40 , 41 . Recent studies show that adjuvanted and recombinant vaccines can even enhance the breadth of protection against H3N2 viruses in older adults 42 , 43 . These approaches are likely to be more potent than standard vaccines at inducing cellular immune responses to support the production of high antibody titers against H3N2 in older adults. Together with the monitoring of antibody titers and potential secondary effects associated with these new approaches, the induced T cell responses and their benefit in terms of seroconversion and seroprotection need to be precisely examined to evaluate their true efficacy. Materials and Methods Cohort description, study subjects and sampling Individuals (n = 326) were recruited in The Vaccines and InfecTious diseases in the Aging popuLation (VITAL) cohort started in 2019 in the Netherlands (September 2019–October 2022) 25 . All participants were adult and categorized for the present analyses into two age groups: young (< 65 years) and older (≥ 65 years) adults. Younger-aged adults were staffs of the Dutch National Institute for Public Health and the Environment (Rijksinstituut voor Volksgezondheid en Milieu, RIVM) (Bilthoven), University Medical Center Utrecht (Utrecht), and Spaarne Hospital (Hoofddorp). Older adults were recruited from a previous study on influenza-like-illness in older adults from the Spaarne Hospital region (Hoofddorp) 44 . Only individuals who were vaccinated with the previous year's seasonal influenza vaccine (2018–2019) were included in the study, to normalize recent influenza vaccination history across the participants. Individuals under immune-modulatory drug treatments, including corticosteroids or chemotherapy in the last 3 years, or with evidence of compromised immunity, including recipient of an organ- or bone marrow transplant, were excluded from the study. In addition, individuals with known allergic reaction to vaccine components and factors that may interfere with blood collection, like anemia or coagulation disorder, or immunological analyses were excluded 24 , 25 . Participants were immunized with the seasonal quadrivalent inactivated subunit influenza vaccine (QIV) (2019–2020), containing neuraminidase and hemagglutinin from the following viral strains: A/Brisbane/02/2018, IVR-190(H1N1); A/Kansas/ 14/2017, NYMC X-327 (H3N2); B/Maryland/15/2016, NYMC BX-69A (B/ Victoria/2/87 lineage); and B/Phuket/3073, wildtype (B/Yamagata/16/ 88 lineage) (Abbott Biologicals B.V. The Netherlands). Blood samples were collected for immunomonitoring pre-vaccination and at Day 0, 7, 28 and Month 6 after vaccination. PBMCs were isolated from heparine pre-coated tubes and cryopreserved at -150°C. CMV seropositivity was determined by quantifying IgG levels against CMV in serum collected prior to vaccination 45 . Seropositivity thresholds was defined a concentration of < 4 relative units (RU) ml − 1 was categorized as seronegative, ≥ 4 and < 7.5 RU ml − 1 as borderline, and ≥ 7.5 RU ml − 1 as seropositive 46 . Standard assessment of vaccine immunogenicity Influenza vaccine specific T cell frequencies were measured using IFN-γ Elispot assays. PVDF membranes of MultiScreen Immobilon-P Filtration Plates (Merck Millipore, Germany) were activated and coated with5 µg/ml α-IFN-γ antibody (Mabtech, Sweden). Membranes were blocked with RPMI-1640 (Sigma; USA) with 10% foetal calf serum (FCS) (Sigma, USA). Cryopreserved PBMCs were thawed at 37°C, treated with DNase, rested for 18h and seeded at 4 x 10 5 cells/well. Stimulation was performed for 20h using hole hemagglutinin proteins from the influenza A virus strains H1N1 and H3N2 and influenza B virus strains Victoria and Yamagata directly issued from the vaccine manufacturing process (kindly provided by our Vital consortium partner Sanofi) at a final concentration of 1 µg/mL. IFN-γ was detected with biotinylated α-IFN-γ (Mabtech, Sweden), Streptavidin-Alkaline phosphatase (ALP) (Mabtech, Sweden), and NBT-BCIP substrate (Moss Inc., USA). IFN-γ spot forming units (SFU)/4x10 5 PBMCs were counted with a CTL Analyzer (ImmunoSpot, USA). Unspecific SFC counts (unstimulated control) were subtracted, and samples with > 25 SFC in the negative control were excluded. Vaccine responders were defined as individuals with a ≥ 5 spot increase between Day 0 and Day 7. We generated a Flu-specific T cell score for each participant by assigning a value (1, 2, 3 or 4) based on the quartiles of the response for each influenza strain (i.e. an Elispot value in the lowest quartile equals 1, and an Elispot value in the highest quartile equals 4), and calculating the mean value of responses to the four strains. Hemagglutination inhibition (HI) titers of antibodies against the H3N2 (A/Kansas/14/2017) strain were measured by Viroclinics (Rotterdam, the Netherlands) 25 . Ex vivo characterization of influenza-specific CD4 and CD8 T cells PBMCs were thawed and rested for 2 h in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (all reagents from Thermo Fisher Scientific). Cells were then incubated in the absence or presence of whole protein directly issued from the vaccine manufacturing process (kindly provided by our Vital consortium partner Sanofi), each at a final concentration of 1 µg/mL. After 1 h, protein transport was blocked using GolgiPlug (1 µL/mL, BD Biosciences) and GolgiStop (0.7 µL/mL, BD Biosciences), and cells were cultured for a further 12h. Cells were washed and stained with directly conjugated antibodies specific for CCR7 (clone G043H7, BioLegend), CD3 (clone REA613, Miltenyi Biotec), CD4 (clone REA623, Miltenyi Biotec), CD8 (clone REA734, Miltenyi Biotec), CD45RA (clone REA1047, Miltenyi Biotec), and CXCR5 (clone RF8B2, BD Biosciences). Nonviable events were excluded using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Cells were then fixed/permeabilized using an Inside Stain Kit (Miltenyi Biotec) and stained with directly conjugated antibodies specific for CD40L/CD154 (clone REA238, Miltenyi Biotec), IFN-γ (clone REA600, Miltenyi Biotec), TNF (clone MAb11, BioLegend), IL-2 (clone REA689, Miltenyi Biotec), and IL-21 (clone 3A3-N2, Miltenyi Biotec). Nonviable events were excluded using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Data were acquired using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo version 10.8.1 (FlowJo LLC). Combinatorial analysis of influenza-specific CD4 + T cell functionality were performed with SPICE version 6 ( https://niaid.github.io/spice/ ) 47 and polyfunctionality indexes calculated using the Funky cells web software ( https://funkycells.com/main/index.php ) 48 . Analysis of immune ageing markers in naive and memory CD4 + and CD8 + T cells T cells were phenotyped ex vivo via flow cytometry using directly conjugated antibodies specific for CCR7 (clone 3D12, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone SK1, BD Biosciences), CD27 (clone O323, BioLegend), and CD45RA (clone HI100, eBioscience). Nonviable events were excluded using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Data were acquired using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo version 10.8.1 (FlowJo LLC). For the measurement of senescence-associated β-galactosidase activity, PBMCs were thawed and cultured for 1 h in RPMI 1640 medium containing 1 mM sodium pyruvate, 1 mM nonessential amino acids, 1 mM L-glutamine, 1% penicillin/streptomycin, 25 mM HEPES, and 0.1 µM bafilomycin (all reagents from Thermo Fisher Scientific). Cells were then incubated for a further 2 h in the presence of SA-β-Gal Fluorescent Substrate (33 µM, Cell Signaling Technology). For the measurement proliferation using Cell Proliferation Dye (CPD), PBMCs were labeled with CPD eFluor450 (Thermo Fisher Scientific) and stimulated for 4 days with plate-bound anti-CD3 (clone OKT3; Thermo Fisher Scientific). Proliferation was measured using flow cytometry to quantify the dilution of CPD in CD4 + and CD8 + T cell subsets based on CCR7 and CD45RA expression. Data were acquired using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo version 10.8.1 (FlowJo LLC). Ethics The VITAL study was approved by the Medical Research Ethics Committee Utrecht (EU Clinical Trials Registration 2019–000836–24) ( https://www.clinicaltrialsregister.eu/ctr-search/trial/2019-000836-24/NL ). This study was performed according to Good Clinical Practice, the Declaration of Helsinki, and written informed consent was obtained from all participants. Statistics Simple group comparisons were performed using the Mann–Whitney U test or the chi-square test, and correlations were assessed using Spearman’s rank test. All basic statistical analyses were performed using Prism software version 9 (GraphPad). Declarations Conflicts of interest: The authors declare that they have no competing financial interests Funding: This work was supported by the University of Bordeaux (Senior IdEx Chair) and and the European Union (VITAL study). The VITAL project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No. 806776 and the Dutch Ministry of Health, Welfare and Sport. The JU receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA- members. PSC is a laureate of the University of Bordeaux doctorate school program. Author Contribution PSC, JvB, DvB, BW and VA designed the study and experiments. JvB and DvB wrote the medical ethical application, and performed the clinical trial. PSC, MH and GA performed experiments. PSC, MH, GA, LB, BW, and VA analyzed data. EB was responsible for the data management. PSC, BW and VA wrote the paper. All authors critically revised the manuscript before publication. Acknowledgement We thank the VITAL EFFPIA partners, namely Jim Janimak, Wivine Burny and Ellen Oe from GSK, and Daniel Larocque from Sanofi for productive discussions and for kindly providing influenza vaccine antigens. We are indebted to the VITAL PBMC preparation team. We are grateful to Martin Larsen for assistance with polyfunctionality analyses, to Atika Zouine, Jean-Michel Griffon and Vincent Pitard for technical assistance at the Flow cytometry facility, CNRS UMS 3427, INSERM US 005, Univ. Bordeaux, F-33000 Bordeaux, France, and all the participants of the VITAL cohort. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Petrova, V. N. & Russell, C. A. The evolution of seasonal influenza viruses. Nat Rev Microbiol 16, 47–60, doi: 10.1038/nrmicro.2017.118 (2018). Rolfes, M. A. et al. Effects of Influenza Vaccination in the United States During the 2017–2018 Influenza Season. Clin Infect Dis 69, 1845–1853, doi: 10.1093/cid/ciz075 (2019). Iuliano, A. D. et al. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet 391, 1285–1300, doi: 10.1016/S0140-6736(17)33293-2 (2018). Grohskopf, L. A. et al. Prevention and Control of Seasonal Influenza with Vaccines: Recommendations of the Advisory Committee on Immunization Practices - United States, 2020-21 Influenza Season. MMWR Recomm Rep 69, 1–24, doi: 10.15585/mmwr.rr6908a1 (2020). Mosmann, T. R., McMichael, A. J., LeVert, A., McCauley, J. W. & Almond, J. W. Opportunities and challenges for T cell-based influenza vaccines. Nat Rev Immunol 24, 736–752, doi: 10.1038/s41577-024-01030-8 (2024). Shi, W., Wohlwend, J., Wu, M. & Barzilay, R. Influenza vaccine strain selection with an AI-based evolutionary and antigenicity model. Nat Med , doi: 10.1038/s41591-025-03917-y (2025). Belongia, E. A. et al. Variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies. Lancet Infect Dis 16, 942–951, doi: 10.1016/S1473-3099(16)00129-8 (2016). Rondy, M. et al. Effectiveness of influenza vaccines in preventing severe influenza illness among adults: A systematic review and meta-analysis of test-negative design case-control studies. J Infect 75, 381–394, doi: 10.1016/j.jinf.2017.09.010 (2017). Allen, J. D. & Ross, T. M. H3N2 influenza viruses in humans: Viral mechanisms, evolution, and evaluation. Hum Vaccin Immunother 14, 1840–1847, doi: 10.1080/21645515.2018.1462639 (2018). Sasaki, S. et al. Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies. J Clin Invest 121, 3109–3119, doi: 10.1172/JCI57834 (2011). Khurana, S., Frasca, D., Blomberg, B. & Golding, H. AID activity in B cells strongly correlates with polyclonal antibody affinity maturation in-vivo following pandemic 2009-H1N1 vaccination in humans. PLoS Pathog 8, e1002920, doi: 10.1371/journal.ppat.1002920 (2012). Mosterin Hopping, A. et al. The confounded effects of age and exposure history in response to influenza vaccination. Vaccine 34, 540–546, doi: 10.1016/j.vaccine.2015.11.058 (2016). Frasca, D. et al. Intrinsic defects in B cell response to seasonal influenza vaccination in elderly humans. Vaccine 28, 8077–8084, doi: 10.1016/j.vaccine.2010.10.023 (2010). Henry, C. et al. Influenza Virus Vaccination Elicits Poorly Adapted B Cell Responses in Elderly Individuals. Cell Host Microbe 25, 357–366 e356, doi: 10.1016/j.chom.2019.01.002 (2019). Akbar, A. N., Henson, S. M. & Lanna, A. Senescence of T Lymphocytes: Implications for Enhancing Human Immunity. Trends Immunol 37, 866–876, doi: 10.1016/j.it.2016.09.002 (2016). Mittelbrunn, M. & Kroemer, G. Hallmarks of T cell aging. Nat Immunol 22, 687–698, doi: 10.1038/s41590-021-00927-z (2021). Lefebvre, J. S., Masters, A. R., Hopkins, J. W. & Haynes, L. Age-related impairment of humoral response to influenza is associated with changes in antigen specific T follicular helper cell responses. Sci Rep 6, 25051, doi: 10.1038/srep25051 (2016). Hill, D. L. et al. Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans. Elife 10, doi: 10.7554/eLife.70554 (2021). Grant, E. J., Quinones-Parra, S. M., Clemens, E. B. & Kedzierska, K. Human influenza viruses and CD8(+) T cell responses. Curr Opin Virol 16, 132–142, doi: 10.1016/j.coviro.2016.01.016 (2016). McElhaney, J. E. et al. T cell responses are better correlates of vaccine protection in the elderly. J Immunol 176, 6333–6339, doi: 10.4049/jimmunol.176.10.6333 (2006). McElhaney, J. E. et al. Granzyme B: Correlates with protection and enhanced CTL response to influenza vaccination in older adults. Vaccine 27, 2418–2425, doi: 10.1016/j.vaccine.2009.01.136 (2009). McElhaney, J. E., Kuchel, G. A., Zhou, X., Swain, S. L. & Haynes, L. T-Cell Immunity to Influenza in Older Adults: A Pathophysiological Framework for Development of More Effective Vaccines. Front Immunol 7, 41, doi: 10.3389/fimmu.2016.00041 (2016). Lorenzo, E. C., Bartley, J. M. & Haynes, L. The impact of aging on CD4(+) T cell responses to influenza infection. Biogerontology 19, 437–446, doi: 10.1007/s10522-018-9754-8 (2018). Van Baarle, D. et al. Preventing infectious diseases for healthy ageing: The VITAL public-private partnership project. Vaccine 38, 5896–5904, doi: 10.1016/j.vaccine.2020.07.005 (2020). van der Heiden, M. et al. Multiple vaccine comparison in the same adults reveals vaccine-specific and age-related humoral response patterns: an open phase IV trial. Nat Commun 15, 6603, doi: 10.1038/s41467-024-50760-9 (2024). Wertheimer, A. M. et al. Aging and cytomegalovirus infection differentially and jointly affect distinct circulating T cell subsets in humans. J Immunol 192, 2143–2155, doi: 10.4049/jimmunol.1301721 (2014). van den Berg, S. P. H. et al. Quantification of T-cell dynamics during latent cytomegalovirus infection in humans. PLoS Pathog 17, e1010152, doi: 10.1371/journal.ppat.1010152 (2021). Breznik, J. A. et al. Cytomegalovirus Seropositivity in Older Adults Changes the T Cell Repertoire but Does Not Prevent Antibody or Cellular Responses to SARS-CoV-2 Vaccination. J Immunol 209, 1892–1905, doi: 10.4049/jimmunol.2200369 (2022). Herati, R. S. et al. Circulating CXCR5 + PD-1 + response predicts influenza vaccine antibody responses in young adults but not elderly adults. J Immunol 193, 3528–3537, doi: 10.4049/jimmunol.1302503 (2014). Frasca, D., Blomberg, B. B., Garcia, D., Keilich, S. R. & Haynes, L. Age-related factors that affect B cell responses to vaccination in mice and humans. Immunol Rev 296, 142–154, doi: 10.1111/imr.12864 (2020). Lefebvre, J. S. et al. Vaccine efficacy and T helper cell differentiation change with aging. Oncotarget 7, 33581–33594, doi: 10.18632/oncotarget.9254 (2016). Linton, P. J., Haynes, L., Klinman, N. R. & Swain, S. L. Antigen-independent changes in naive CD4 T cells with aging. J Exp Med 184, 1891–1900, doi: 10.1084/jem.184.5.1891 (1996). Frasca, D., Diaz, A., Romero, M., Landin, A. M. & Blomberg, B. B. Cytomegalovirus (CMV) seropositivity decreases B cell responses to the influenza vaccine. Vaccine 33, 1433–1439, doi: 10.1016/j.vaccine.2015.01.071 (2015). Nicoli, F. et al. Primary immune responses are negatively impacted by persistent herpesvirus infections in older people: results from an observational study on healthy subjects and a vaccination trial on subjects aged more than 70 years old. EBioMedicine 76, 103852, doi: 10.1016/j.ebiom.2022.103852 (2022). Nicoli, F., Karrer, U. & Appay, V. Impact of CMV infection on heterologous vaccine responsiveness. Phil. Trans. R. Soc. B 380, In Press, doi: https://doi.org/10.1098/rstb.2024.0407 (2025). Derhovanessian, E. et al. Latent infection with cytomegalovirus is associated with poor memory CD4 responses to influenza A core proteins in the elderly. J Immunol 193, 3624–3631, doi: 10.4049/jimmunol.1303361 (2014). van den Berg, S. P. H. et al. Latent CMV Infection Is Associated With Lower Influenza Virus-Specific Memory T-Cell Frequencies, but Not With an Impaired T-Cell Response to Acute Influenza Virus Infection. Front Immunol 12, 663664, doi: 10.3389/fimmu.2021.663664 (2021). Koutsakos, M. et al. Human CD8(+) T cell cross-reactivity across influenza A, B and C viruses. Nat Immunol 20, 613–625, doi: 10.1038/s41590-019-0320-6 (2019). DiazGranados, C. A. et al. Efficacy of high-dose versus standard-dose influenza vaccine in older adults. N Engl J Med 371, 635–645, doi: 10.1056/NEJMoa1315727 (2014). Couch, R. B. et al. Superior antigen-specific CD4 + T-cell response with AS03-adjuvantation of a trivalent influenza vaccine in a randomised trial of adults aged 65 and older. BMC Infect Dis 14, 425, doi: 10.1186/1471-2334-14-425 (2014). Domnich, A. et al. Effectiveness of MF59-adjuvanted seasonal influenza vaccine in the elderly: A systematic review and meta-analysis. Vaccine 35, 513–520, doi: 10.1016/j.vaccine.2016.12.011 (2017). Fox, A. et al. Enhanced Influenza Vaccines Extend A(H3N2) Antibody Reactivity in Older Adults but Prior Vaccination Effects Persist. Clin Infect Dis , doi: 10.1093/cid/ciaf060 (2025). Essink, B. J. et al. A randomised phase 2 immunogenicity and safety study of a MF59-adjuvanted quadrivalent subunit inactivated cell-derived influenza vaccine (aQIVc) in adults aged 50 years and older. Vaccine 51, 126791, doi: 10.1016/j.vaccine.2025.126791 (2025). van Beek, J. et al. Influenza-like Illness Incidence Is Not Reduced by Influenza Vaccination in a Cohort of Older Adults, Despite Effectively Reducing Laboratory-Confirmed Influenza Virus Infections. J Infect Dis 216, 415–424, doi: 10.1093/infdis/jix268 (2017). Korndewal, M. J. et al. Cytomegalovirus infection in the Netherlands: seroprevalence, risk factors, and implications. J Clin Virol 63, 53–58, doi: 10.1016/j.jcv.2014.11.033 (2015). Samson, L. D. et al. In-depth immune cellular profiling reveals sex-specific associations with frailty. Immun Ageing 17, 20, doi: 10.1186/s12979-020-00191-z (2020). Roederer, M., Nozzi, J. L. & Nason, M. C. SPICE: exploration and analysis of post-cytometric complex multivariate datasets. Cytometry A 79, 167–174, doi: 10.1002/cyto.a.21015 (2011). Larsen, M. et al. Evaluating cellular polyfunctionality with a novel polyfunctionality index. PLoS One 7, e42403 (2012). Additional Declarations No competing interests reported. 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07:29:49","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140314,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7818000/v1/8ed8d0245265f049a4c7e6d9.html"},{"id":96141360,"identity":"f4ff49c2-3d51-46b0-9344-f94f082413e1","added_by":"auto","created_at":"2025-11-18 05:31:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT cell responsiveness to quadrivalent influenza vaccination in younger and older adults. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Frequencies of H3N2, H1N1, Victoria or Yamagata\u003cstrong\u003e \u003c/strong\u003einfluenza-specific T cells measured using IFN-γ Elispot on day 0, 7, 28 and month 6 post-vaccination among vaccinees below (n=100) or above (n=120) 65 years of age. Violon plots are shown with mean and lower and higher quartile values. Bars indicate median values. Significance was assessed using the Mann-Whitney U test. (\u003cstrong\u003eB\u003c/strong\u003e) Pie charts indicating the percentages of weak vs good responders (defined as individuals with a ≥ 5 spot increase using IFN-γ Elispot between day 0 and day 7) against H3N2, H1N1, Victoria or Yamagata\u003cstrong\u003e \u003c/strong\u003einfluenza strains among vaccinees below (n=100) or above (n=120) 65 years of age. Significance was assessed using the chi-square test. (\u003cstrong\u003eC\u003c/strong\u003e) Pie charts indicating the percentages of weak vs good responders (defined as individuals with a ≥ 5 spot increase using IFN-γElispot between day 0 and day 7) against 0, 1, 2, 3, 4\u003cstrong\u003e \u003c/strong\u003einfluenza strains simultaneously among vaccinees below (n=100) or above (n=120) 65 years of age. Significance was assessed using the chi-square test. (\u003cstrong\u003eD\u003c/strong\u003e) Influenza specific T cell score (calculated based on the quartiles of the response for all influenza strains) for each participant among vaccinees below (n=100) or above (n=120) 65 years of age. Each dot represents one donor. Significance was assessed using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7818000/v1/494ce0c04abae7a6b3e2c50f.png"},{"id":96141361,"identity":"f609cf91-3f2d-431e-91ef-11170e8ec94c","added_by":"auto","created_at":"2025-11-18 05:31:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional characterization of CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells specific for H3N2 or Yamagata influenza strains in younger and older vaccinees.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative flow cytometry plots showing the identification of influenza-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells without or with stimulation of PBMCs with vaccine antigens on day 7 post-vaccination. Plots are gated on CD4\u003csup\u003e+\u003c/sup\u003e T cells. Numbers indicate the frequencies of specific T cells. (\u003cstrong\u003eB\u003c/strong\u003e) Frequencies of H3N2 or Yamagata\u003cstrong\u003e \u003c/strong\u003einfluenza-specific CD4\u003csup\u003e+\u003c/sup\u003e (CD40L\u003csup\u003e+\u003c/sup\u003e and IFN-γ\u003csup\u003e+\u003c/sup\u003e, TNF\u003csup\u003e+\u003c/sup\u003e, IL-2\u003csup\u003e+\u003c/sup\u003e or IL-21\u003csup\u003e+\u003c/sup\u003e) T cells on day 7 post-vaccination among vaccinees below (n=17) or above (n=17) 65 years of age. Each dot represents one donor. Bars indicate median values. Significance was assessed using the Mann-Whitney U test. (\u003cstrong\u003eC\u003c/strong\u003e) Polyfunctional profiles of influenza-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells among younger (\u0026lt; 65y) and older (≥ 65y) vaccinees. Combinatorial analysis of influenza-specific CD4\u003csup\u003e+\u003c/sup\u003e T cell functionality. Bars indicate median values. Significance was assessed using the Mann-Whitney U test. (\u003cstrong\u003eD\u003c/strong\u003e) Polyfunctionality indexes of influenza-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells among younger (\u0026lt; 65y) and older (≥ 65y) vaccinees. Bars indicate median values. Significance was assessed using the Mann-Whitney U test. (\u003cstrong\u003eE\u003c/strong\u003e) Correlation between polyfunctionality indexes of H3N2-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells and H3N2 HI titers among younger (\u0026lt; 65y) and older (≥ 65y) vaccinees. Each dot represents one donor. Significance was assessed using Spearman’s rank test.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7818000/v1/2cce1bc46571ca7da3c19f7d.png"},{"id":96249841,"identity":"20967335-2808-45e8-9873-1d8c9e49cff5","added_by":"auto","created_at":"2025-11-19 07:36:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFrequencies of total or influenza-specific cTfh CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells in younger and older vaccinees.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative flow cytometry plots showing the identification of circulating T follicular helper (cTfh) CXCR5\u003csup\u003e+\u003c/sup\u003e cells in total CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes (left) or in influenza-specific\u003cstrong\u003e \u003c/strong\u003eCD40L\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes (right) upon stimulation of PBMCs with vaccine antigens on day 7 post-vaccination. Plots are gated on CD4\u003csup\u003e+\u003c/sup\u003e T cells. Numbers indicate the frequencies of specific T cells. (\u003cstrong\u003eB-C\u003c/strong\u003e) Frequencies of total (B) or influenza-specific\u003cstrong\u003e \u003c/strong\u003e(C) cTfh CD4\u003csup\u003e+\u003c/sup\u003e T cells on day 7 post-vaccination among vaccinees below (n=17) or above (n=17) 65 years of age. Each dot represents one donor. Bars indicate median values. Significance was assessed using the Mann-Whitney U test. (\u003cstrong\u003eD\u003c/strong\u003e) Correlation between total cTfh CD4\u003csup\u003e+\u003c/sup\u003e T cells and H3N2 HI titers among younger (\u0026lt; 65y) and older (≥ 65y) vaccinees. Each dot represents one donor. Significance was assessed using Spearman’s rank test.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7818000/v1/0bde1f32577423c8ad3bf253.png"},{"id":96249122,"identity":"1575c2fd-ef98-44fe-b425-e8a99de4bc9f","added_by":"auto","created_at":"2025-11-19 07:30:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of naive and memory CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells in younger and older vaccinees.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative flow cytometry plots (left) and summary data (right) showing the frequencies of naive and memory CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells from younger (\u0026lt; 65y, n=17) and older (≥ 65y, n=17) vaccinees. (\u003cstrong\u003eB\u003c/strong\u003e) Representative flow cytometry plots (left) and summary data (right) showing senescence-associated b-galactosidase (SA-b-gal) activity among naive and memory CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells from younger (\u0026lt; 65y, n=17) and older (≥ 65y, n=17) vaccinees. (\u003cstrong\u003eC\u003c/strong\u003e) Representative flow cytometry plots (left) and summary data (right) showing proliferative capacity (CPD low) among naive and memory CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells from younger (\u0026lt; 65y, n=17) and older (≥ 65y, n=17) vaccinees upon stimulation with anti-CD3 antibodies. Each dot represents one donor. Bars indicate median values.\u003cstrong\u003e \u003c/strong\u003eSignificance was assessed using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7818000/v1/523b5c83dced606ae5aee175.png"},{"id":96141362,"identity":"fc93f17c-127c-44ea-848f-8965d773be6e","added_by":"auto","created_at":"2025-11-18 05:31:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":44178,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT cell responsiveness to quadrivalent influenza vaccination according to CMV seropositivity. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Frequencies of H3N2 or Yamagata\u003cstrong\u003e \u003c/strong\u003einfluenza-specific T cells (IFN-γ Elispot data) and H3N2 HI titers measured on day 7 post-vaccination among younger (\u0026lt; 65y, n=17) and older (≥ 65y, n=17) vaccinees according to CMV seropositivity. (\u003cstrong\u003eB-C\u003c/strong\u003e) Frequencies of H3N2 or Yamagata\u003cstrong\u003e \u003c/strong\u003einfluenza-specific CD8\u003csup\u003e+\u003c/sup\u003e (B) or CD4\u003csup\u003e+\u003c/sup\u003e (C) (IFN-γ\u003csup\u003e+\u003c/sup\u003e or TNF\u003csup\u003e+\u003c/sup\u003e) T cells on day 7 post-vaccination among younger (\u0026lt; 65y, n=17) and older (≥ 65y, n=17) vaccinees according to CMV seropositivity. (\u003cstrong\u003eD\u003c/strong\u003e) Frequencies of naive and memory CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells among vaccinees below (n=17) or above (n=17) 65 years of age according to CMV seropositivity. Each dot represents one donor. Bars indicate median values. Significance was assessed using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7818000/v1/1c0c3b85e3ce03e2afc8aeed.png"},{"id":106809038,"identity":"c34260cd-d1cf-4d12-9f46-9dfa47907275","added_by":"auto","created_at":"2026-04-13 16:05:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1178228,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7818000/v1/9f7e10bc-97c4-4cde-8ad9-86108a3d154e.pdf"},{"id":96249864,"identity":"66b39f00-33c0-4ef9-8db5-70dc31f1e812","added_by":"auto","created_at":"2025-11-19 07:36:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1996111,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTALMATERIALS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7818000/v1/0fb5e708a303b707333b5018.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influenza vaccine-specific T cell responses are impaired in older adults","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfluenza or flu is a contagious respiratory illness caused by influenza viruses, which primarily affect the upper respiratory tract and lungs, and can lead to hospitalization, as well as severe complications, such as pneumonia, myocarditis, diabetes and kidney failure. Influenza viruses infect 5\u0026ndash;15% of the population worldwide annually, causing 3\u0026ndash;5\u0026nbsp;million cases of severe illness and possibly up to half a million deaths each year \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Therefore, influenza remains a major public health issue. Due to age-related weakening of their immune defenses, people over 65 years of age are at high risk for developing severe illness and complications \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Vaccination remains the most effective protective measure, reducing severe influenza cases by inducing antibodies and T cells targeting the virus \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Annual influenza vaccination is thus strongly recommended for the most vulnerable populations, including older adults. As a consequence of the constant genetic changes of influenza viruses and their variability from one season to another, the composition of the influenza vaccine is adapted every year to incorporate the most recent circulating influenza A (H1N1 and H3N2) and influenza B strains \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Vaccine efficacy depends on the match between the vaccine and circulating strains, of which the selection may now gain from artificial intelligence-based predicting assistance \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eVaccine efficacy is also impacted by the age of the vaccinees. While influenza vaccines offer some protection in older adults, the level of protection is generally lower and varies among influenza strains. Meta-analyses of studies published between 2004\u0026ndash;2016 on influenza vaccine effectiveness (IVE) against several virus strains reveal a pooled seasonal IVE of 51% in adults aged 18\u0026ndash;64, but only 37% in those above 65 \u003csup\u003e7,8\u003c/sup\u003e. Furthermore, IVE against H3N2 most notably lower at 33% in older adults compared to 50% in younger adults \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This reduced efficacy is reflected in lower seroprotection and antibody titers following vaccination, particularly against H3N2, limiting the ability to neutralize the virus \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These effects may be attributable to age-associated immune alterations as well as repeated vaccination \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, which could result in weakened and exhausted B cell responses and decreased number of vaccine-specific plasmablasts in older adults \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, factors underlying lower humoral responses and IVE in older adults are not fully understood. Gaining further insights is important to improve vaccine design and efficacy, especially for vulnerable populations.\u003c/p\u003e\u003cp\u003eGiven the significant age-related alterations observed in the T lymphocyte compartment \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, studying T cell responses to vaccination in older adults is therefore particularly relevant in this context. Defects of CD4\u003csup\u003e+\u003c/sup\u003e T cell responses, such as reduced activation of T follicular helper (Tfh), which provide help to B cells, may have a direct impact on humoral immunity and antibody titers in older adults \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Moreover, CD8\u003csup\u003e+\u003c/sup\u003e T cell responses are important for providing protection from severe influenza illness \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Their impaired induction may contribute to reduced IVE in older adults. However, studies of influenza vaccine cellular responses in relation to age and virus strains are scarce, relative to the number of studies on humoral responses. Earlier studies of soluble factors released by T cells, like IFNγ, IL-10 or Granzyme B, upon activation with influenza viruses suggested defective vaccine-specific cellular responses with advanced age \u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Here, we studied the T lymphocyte compartment in young (\u0026lt;\u0026thinsp;65 years) and older (\u0026ge;\u0026thinsp;65 years) adults vaccinated with a standard quadrivalent inactivated subunit vaccine containing viral antigens from two influenza A and two influenza B virus strains \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We examined both quantitative and qualitative aspects of T cell responses in vaccinees. Frequencies of total vaccine-specific T cells were measured using IFNγ Elispot in older adults, and their functional potential was assessed by intracellular staining of cytokines upon vaccine antigen mediated stimulation. Markers of immune ageing were also analysed and included the naive-memory phenotype, the activity of senescence-associated-β-galactosidase (SA-β-gal) and the proliferation potential of total T cell populations, as well as the seropositivity for cytomegalovirus. The latter is known to alter the T cell subset homeostasis, and to play an important role in changes of the immunological landscape associated with ageing \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Our findings underscore reduced magnitude and functional impairment of influenza vaccine-induced T cell responses in older adults. This may participate to suboptimal antibody titers upon vaccination and IVE with old age, particularly against H3N2.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eOlder adults have reduced influenza-specific T cell responses\u003c/h2\u003e\u003cp\u003eTo characterize T cell responsiveness to influenza vaccination, we analyzed 220 of the 326 participants of the Vaccines and InfecTious diseases in the Aging popuLation (VITAL) cohort \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, who were immunized with one dose of the quadrivalent inactivated influenza vaccine (QIV) in autumn 2019 (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The 2019 QIV contained haemagglutinin (HA) and neuraminidase (NA) antigens from the influenza A virus strains H1N1 (A/H1N1 Brisbane/02/2018) and H3N2 (A/H3N2/Kansas/14/2017), and influenza B virus strains Victoria (B/Vic/Maryland/15/2016) and Yamagata (B/Yamagata/16/88 lineage). Participants were selected for being vaccinated against influenza the year before to minimize the influence of disparate influenza vaccination history on T cell responses and were separated in two age groups (\u0026lt;\u0026thinsp;65y n\u0026thinsp;=\u0026thinsp;100 and \u0026ge;\u0026thinsp;65y n\u0026thinsp;=\u0026thinsp;120). Frequencies of influenza vaccine-specific T cells were measured at days 0, 7, and 28 as well as at month 6 post-vaccination, by IFNγ Elispot upon stimulation with HA and NA antigens contained in the vaccine.\u003c/p\u003e\u003cp\u003eLevels of flu-specific T cells were globally similar for all virus strains at a given time point and age group, except H3N2, for which T cell frequencies were lowest in both age groups at all time points (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 at day 0, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0015 at day 7 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0008 at day 28 compared to H1N1) (\u003cb\u003eFigure S2\u003c/b\u003e). This highlights the disparate immunity against different influenza viruses. At peak response (i.e., day 7), T cell frequencies specific for 3 out of the 4 virus strains were lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003 for H3N2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02 for H1N1, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008 for Victoria) in the older group compared to the younger group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The difference between age groups was confirmed for H3N2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) and Victoria (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) with regards to vaccine responsiveness, i.e. assessing the frequency of responders (defined as vaccinees with an increase in T cell frequencies from day 0 to day 7 of at least 5 spots) for each strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). For instance, only 38% of participants above 65 years were considered responders to H3N2, in contrast to 68% of participants below 65 years. Overall, H3N2-specific T cell responses appeared therefore lower than responses to other strains, and were particularly reduced in older versus younger adults.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next evaluated the capacity of vaccinees from the two age groups to respond to several strains of the 4 influenza A and B viruses simultaneously (corresponding to the frequency of participants responding from none to the four strains). Fewer participants of the older age group were able to respond to multiple strains (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), with 18% of them showing responses to all four vaccine strains, compared to 36% in the younger age group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, we calculated an influenza specific T cell score for each participant, to account for their overall vaccine responsiveness at day 7, considering both its intensity (i.e. T cell frequency) and its breadth (i.e. response to the four strains). To this end, we assigned a value (1, 2, 3 or 4) based on the quartiles of the response for each influenza strain and calculated the mean value for the four strains. This score was lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) in older versus younger subjects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These results highlight a globally reduced T cell responsiveness, in terms of frequency at peak response and reactivity to multiple virus strains, to recall vaccination against influenza in older adults.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInfluenza vaccine-specific CD4 T cells have limited helper potential in older adults\u003c/h3\u003e\n\u003cp\u003eIn order to distinguish between CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes among influenza vaccine-specific cells, we performed flow cytometry-based assays on a selection of participants (\u0026lt;\u0026thinsp;65y n\u0026thinsp;=\u0026thinsp;17 and \u0026ge;\u0026thinsp;65y n\u0026thinsp;=\u0026thinsp;17), arbitrarily chosen among strong responders in Elispot assays. Using this approach, we assessed the capacity of the responding cells to upregulate CD40L\u003csup\u003e+\u003c/sup\u003e and secrete a variety of cytokines (namely IFNγ, TNF, IL-2, and IL-21), which reflect functional properties of T cells, in particular the helper potential of CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u003cb\u003eand Figure S3\u003c/b\u003e). Responsiveness was tested for two strains, H3N2 and Yamagata (as the most and least affected responses by age in terms of frequency, respectively), using the same antigens as for the Elispot assay. In line with the Elispot data, cytokine-secreting cells were, in general, fewer to respond to H3N2 compared to Yamagata (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). IL21\u003csup\u003e+\u003c/sup\u003e cells were infrequent and often undetectable. We observed clear trends towards lower frequencies of CD40L\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells secreting each individual cytokine upon stimulation with H3N2 or Yamagata antigens in older versus younger responders (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Of note, vaccine antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cell responses showed high inter-individual diversity in younger and older groups, and did not enable us to draw any association with age (\u003cb\u003eFigure S4\u003c/b\u003e). This may be partly related to the poor efficacy of inactivated subunit vaccines, such as QIV, to induce CD8\u003csup\u003e+\u003c/sup\u003e T cell responses. We next consider the polyfunctional profile of influenza vaccine-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells, i.e., their capacity to secrete multiple helper cytokines simultaneously. Older vaccine responders displayed lower frequencies of polyfunctional CD4\u003csup\u003e+\u003c/sup\u003e T cells (i.e. CD40L\u003csup\u003e+\u003c/sup\u003e cells secreting IFNγ, TNF and IL-2, or TNF and IL-2) specific for either H3N2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04) or Yamagata (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and these cells had lower polyfunctionality indexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), compared to younger vaccinees. We found a correlation between polyfunctionality indexes of H3N2-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells and the hemagglutination inhibition (HI) antibody titers against H3N2 measured in the same subjects (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.42, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In contrast, IFNγ-production alone, as measured by Elispot or intracellular cytokine staining does not correlate with antibody titers (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.07, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.28 or \u003cem\u003er\u003c/em\u003e = -0.03, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.84, respectively) (data not shown). This highlights the importance of polyfunctional T cells as a measure for efficient T cell help.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next evaluated the magnitude of total and vaccine-specific circulating CD4\u003csup\u003e+\u003c/sup\u003e T follicular helper cells (cTfh), and their relationship to age and antibody responses in the same vaccinees. For this purpose, we used CXCR5 as a marker of cTfh on total CD4\u003csup\u003e+\u003c/sup\u003e T cells or activated CD40L\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells upon stimulation with vaccine antigens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We found a lower frequency of total cTfh in people above versus below 65 years of age (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which aligns with previous reports \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. CXCR5\u003csup\u003e+\u003c/sup\u003e CD40L\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells were usually scarce, particularly those specific for H3N2. There was a trend towards lower frequencies of Yamagata reactive cTfh in older vaccinees (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Of note, the frequency of total cTfh correlated with HI antibody titers against H3N2 (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.37, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. The correlation was particularly evident for the younger group (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.59, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012), which displays the highest levels of cTfh. Altogether, these observations support a qualitative skewing of influenza vaccine-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells, and their limited help to B cells and the production of HI inducing antibodies in older vaccinees.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eInfluenza vaccine-specific CD8 T cells are low in CMV seropositive older vaccinees\u003c/h3\u003e\n\u003cp\u003eLastly, we aimed to determine if suboptimal T cell responsiveness to the influenza vaccine in older adults could be related to certain hallmarks of immune ageing in CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes, namely the alteration of the naive versus memory T cell homeostasis, markers of cellular senescence, as well as the influence of CMV infection. T cell differentiation subset homeostasis was assessed by looking at the frequencies of naive (CD45RA\u003csup\u003e+\u003c/sup\u003eCCR7\u003csup\u003e+\u003c/sup\u003e) versus memory (non CD45RA\u003csup\u003e+\u003c/sup\u003eCCR7\u003csup\u003e+\u003c/sup\u003e) CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Cellular senescence was informed by assessing the activity of SA-β-gal by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) in naive and memory CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes, and the proliferation capacity upon TCR-mediated stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) of the same T cell subsets. Although alterations of the CD8\u003csup\u003e+\u003c/sup\u003e T cell subset homeostasis, together with increased levels of SA-β-gal and lower proliferation capacity in naive CD8\u003csup\u003e+\u003c/sup\u003e T cells were observed in older subjects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), this was not the case for CD4\u003csup\u003e+\u003c/sup\u003e T cells. Naive and memory CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets did not present obvious change with regards to frequency or cellular senescence markers comparing younger and older age groups. Suboptimal CD4\u003csup\u003e+\u003c/sup\u003e T cell responsiveness to influenza vaccine could therefore not be attributed to intrinsic alterations of this lineage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInitial analyses did not reveal apparent effects of CMV seropositivity on total H3N2 or Yamagata-specific total T cell responses measured by Elispot, nor on H3N2 HI titers in younger or older vaccinees (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003eand Figure S5)\u003c/b\u003e. However, CD8\u003csup\u003e+\u003c/sup\u003e T cell responses specific for both H3N2 and Yamagata, measured by intracellular cytokine staining, were lower in the CMV-seropositive vs CMV-seronegative subjects of the older (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03), but not of the younger group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In contrast, CMV-serostatus did not have an influence on the influenza vaccine-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In line with these observations, the fraction of memory CD8\u003csup\u003e+\u003c/sup\u003e, but not CD4\u003csup\u003e+\u003c/sup\u003e, T cells was altered (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) in CMV-seropositive older individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). This suggests that CMV-related alteration of the CD8\u003csup\u003e+\u003c/sup\u003e T cell compartment homeostasis has an influence on the memory CD8\u003csup\u003e+\u003c/sup\u003e T cell responses to influenza vaccines with ageing.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFactors underlying the limited seroprotection against influenza viruses, specially H3N2, in older vaccinees remain poorly understood. Low antibody titers and quality upon influenza vaccination in older adults may be in part attributed to less functional B cells, weaker germinal centers, and impaired affinity maturation associated with ageing \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Our findings underline a quantitative and qualitative impairment of CD4\u003csup\u003e+\u003c/sup\u003e T cell responses to recall influenza antigens in older adults. Previous studies in aged mice also indicate that impaired cytokine production by CD4\u003csup\u003e+\u003c/sup\u003e T cells following antigenic stimulation affects B cell help functions, causing reduced humoral immunity to influenza vaccine \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In young adults, cTfh cell frequencies was associated with increased antibody titers after vaccination \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In older vaccinees, the waning functional capacity of CD4\u003csup\u003e+\u003c/sup\u003e T cells, in particular Tfh, to confer help to B cells likely participates to the reduced capacity to stimulate strong antibody responses and therefore reduced effectiveness of influenza vaccines. Of note, we found that H3N2-specific CD4\u003csup\u003e+\u003c/sup\u003e T cell responses were lowest in older adults, compared to CD4\u003csup\u003e+\u003c/sup\u003e T cell responses specific for other influenza A or B strains. Although the underlying reasons are unclear, it may explain the particularly reduced seroprotection against H3N2 reported in vaccinated older adults \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This indicates that efforts should be undertaken to enhance H3N2-specific T cell responses using vaccines. This may be achieved through specific optimization of H3N2 antigens to make them more immunogenic.\u003c/p\u003e\u003cp\u003eAlthough the use of whole protein antigens in our \u003cem\u003eex vivo\u003c/em\u003e assays primarily promotes the activation and detection of CD4⁺ T cells, we were nonetheless able to detect influenza-specific CD8⁺ T cells in vaccinees. Notably, these cells were found in lower frequencies among older individuals who were CMV-seropositive. CMV seropositivity has also been reported to negatively affect B cell and antibody responses to the influenza vaccine \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Reduced CD4\u003csup\u003e+\u003c/sup\u003e T cell responses to CMV-unrelated antigens in older CMV-infected individuals have been previously documented in the context of primary vaccination (i.e., \u003cem\u003ede novo\u003c/em\u003e T cell responses from the naive T cell pool) \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Here, our data suggest that CMV infection can also affect memory CD8⁺ T cell responses upon recall vaccination. Although precise mechanisms remain to be elucidated, one possible explanation is that overcrowding of the \u0026ldquo;immunological space\u0026rdquo; by T cells specific for persistent viruses like CMV, by disrupting T cell homeostasis, may hinder the activation of heterologous memory T cell responses \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Consistent with this, influenza virus-specific T cell frequencies were actually reported to be lower in CMV-seropositive older individuals compared to CMV-seronagative older individuals \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Robust CD8\u003csup\u003e+\u003c/sup\u003e T cell responses are important not only for mitigating disease severity but also for providing cross-reactive protection \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The potential for CD8⁺ T cell cross-reactivity across influenza A, B, and C viruses further supports the rationale for developing universal T cell-based influenza vaccines \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. While current inactivated vaccines are not known to elicit strong CD8\u003csup\u003e+\u003c/sup\u003e T cell responses, the use of recombinant vaccines such as mRNA vaccines may offer significant opportunities to promote cellular immune responses against influenza \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, it will be important to consider the impact of CMV seropositivity in these contexts in particular for older adults.\u003c/p\u003e\u003cp\u003eOverall, our work highlights that it is imperative to promote stronger and more functional T cell responses upon immunization against influenza in older adults. To address weaker induction of immune responses, vaccines should be designed specifically for adults aged 65 and older. Ongoing research aims to develop vaccines that better address the unique immunological challenges of ageing. High-dose seasonal influenza vaccines (e.g. Fluzone High-Dose), which can contain four times the antigen amount can help stimulate stronger responses in older adults \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Moreover, adjuvants are likely to be very important in this context. For instance, oil-in-water adjuvanted influenza vaccines (e.g. Flurarix\u0026reg; and Fluad\u0026reg;, which contain the adjuvants AS03 and MF59 respectively) can enhance immune responses in older adults \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Recent studies show that adjuvanted and recombinant vaccines can even enhance the breadth of protection against H3N2 viruses in older adults \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. These approaches are likely to be more potent than standard vaccines at inducing cellular immune responses to support the production of high antibody titers against H3N2 in older adults. Together with the monitoring of antibody titers and potential secondary effects associated with these new approaches, the induced T cell responses and their benefit in terms of seroconversion and seroprotection need to be precisely examined to evaluate their true efficacy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCohort description, study subjects and sampling\u003c/h2\u003e\u003cp\u003eIndividuals (n\u0026thinsp;=\u0026thinsp;326) were recruited in The Vaccines and InfecTious diseases in the Aging popuLation (VITAL) cohort started in 2019 in the Netherlands (September 2019\u0026ndash;October 2022) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. All participants were adult and categorized for the present analyses into two age groups: young (\u0026lt;\u0026thinsp;65 years) and older (\u0026ge;\u0026thinsp;65 years) adults. Younger-aged adults were staffs of the Dutch National Institute for Public Health and the Environment (Rijksinstituut voor Volksgezondheid en Milieu, RIVM) (Bilthoven), University Medical Center Utrecht (Utrecht), and Spaarne Hospital (Hoofddorp). Older adults were recruited from a previous study on influenza-like-illness in older adults from the Spaarne Hospital region (Hoofddorp) \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Only individuals who were vaccinated with the previous year's seasonal influenza vaccine (2018\u0026ndash;2019) were included in the study, to normalize recent influenza vaccination history across the participants. Individuals under immune-modulatory drug treatments, including corticosteroids or chemotherapy in the last 3 years, or with evidence of compromised immunity, including recipient of an organ- or bone marrow transplant, were excluded from the study. In addition, individuals with known allergic reaction to vaccine components and factors that may interfere with blood collection, like anemia or coagulation disorder, or immunological analyses were excluded \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Participants were immunized with the seasonal quadrivalent inactivated subunit influenza vaccine (QIV) (2019\u0026ndash;2020), containing neuraminidase and hemagglutinin from the following viral strains: A/Brisbane/02/2018, IVR-190(H1N1); A/Kansas/ 14/2017, NYMC X-327 (H3N2); B/Maryland/15/2016, NYMC BX-69A (B/ Victoria/2/87 lineage); and B/Phuket/3073, wildtype (B/Yamagata/16/ 88 lineage) (Abbott Biologicals B.V. The Netherlands). Blood samples were collected for immunomonitoring pre-vaccination and at Day 0, 7, 28 and Month 6 after vaccination. PBMCs were isolated from heparine pre-coated tubes and cryopreserved at -150\u0026deg;C. CMV seropositivity was determined by quantifying IgG levels against CMV in serum collected prior to vaccination \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Seropositivity thresholds was defined a concentration of \u0026lt;\u0026thinsp;4 relative units (RU) ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was categorized as seronegative, \u0026ge;\u0026thinsp;4 and \u0026lt;\u0026thinsp;7.5 RU ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as borderline, and \u0026ge;\u0026thinsp;7.5 RU ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as seropositive \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStandard assessment of vaccine immunogenicity\u003c/h3\u003e\n\u003cp\u003eInfluenza vaccine specific T cell frequencies were measured using IFN-γ Elispot assays. PVDF membranes of MultiScreen Immobilon-P Filtration Plates (Merck Millipore, Germany) were activated and coated with5 \u0026micro;g/ml α-IFN-γ antibody (Mabtech, Sweden). Membranes were blocked with RPMI-1640 (Sigma; USA) with 10% foetal calf serum (FCS) (Sigma, USA). Cryopreserved PBMCs were thawed at 37\u0026deg;C, treated with DNase, rested for 18h and seeded at 4 x 10\u003csup\u003e5\u003c/sup\u003e cells/well. Stimulation was performed for 20h using hole hemagglutinin proteins from the influenza A virus strains H1N1 and H3N2 and influenza B virus strains Victoria and Yamagata directly issued from the vaccine manufacturing process (kindly provided by our Vital consortium partner Sanofi) at a final concentration of 1 \u0026micro;g/mL. IFN-γ was detected with biotinylated α-IFN-γ (Mabtech, Sweden), Streptavidin-Alkaline phosphatase (ALP) (Mabtech, Sweden), and NBT-BCIP substrate (Moss Inc., USA). IFN-γ spot forming units (SFU)/4x10\u003csup\u003e5\u003c/sup\u003e PBMCs were counted with a CTL Analyzer (ImmunoSpot, USA). Unspecific SFC counts (unstimulated control) were subtracted, and samples with \u0026gt;\u0026thinsp;25 SFC in the negative control were excluded. Vaccine responders were defined as individuals with a\u0026thinsp;\u0026ge;\u0026thinsp;5 spot increase between Day 0 and Day 7. We generated a Flu-specific T cell score for each participant by assigning a value (1, 2, 3 or 4) based on the quartiles of the response for each influenza strain (i.e. an Elispot value in the lowest quartile equals 1, and an Elispot value in the highest quartile equals 4), and calculating the mean value of responses to the four strains. Hemagglutination inhibition (HI) titers of antibodies against the H3N2 (A/Kansas/14/2017) strain were measured by Viroclinics (Rotterdam, the Netherlands) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eEx vivo characterization of influenza-specific CD4 and CD8 T cells\u003c/h3\u003e\n\u003cp\u003ePBMCs were thawed and rested for 2 h in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (all reagents from Thermo Fisher Scientific). Cells were then incubated in the absence or presence of whole protein directly issued from the vaccine manufacturing process (kindly provided by our Vital consortium partner Sanofi), each at a final concentration of 1 \u0026micro;g/mL. After 1 h, protein transport was blocked using GolgiPlug (1 \u0026micro;L/mL, BD Biosciences) and GolgiStop (0.7 \u0026micro;L/mL, BD Biosciences), and cells were cultured for a further 12h. Cells were washed and stained with directly conjugated antibodies specific for CCR7 (clone G043H7, BioLegend), CD3 (clone REA613, Miltenyi Biotec), CD4 (clone REA623, Miltenyi Biotec), CD8 (clone REA734, Miltenyi Biotec), CD45RA (clone REA1047, Miltenyi Biotec), and CXCR5 (clone RF8B2, BD Biosciences). Nonviable events were excluded using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Cells were then fixed/permeabilized using an Inside Stain Kit (Miltenyi Biotec) and stained with directly conjugated antibodies specific for CD40L/CD154 (clone REA238, Miltenyi Biotec), IFN-γ (clone REA600, Miltenyi Biotec), TNF (clone MAb11, BioLegend), IL-2 (clone REA689, Miltenyi Biotec), and IL-21 (clone 3A3-N2, Miltenyi Biotec). Nonviable events were excluded using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Data were acquired using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo version 10.8.1 (FlowJo LLC). Combinatorial analysis of influenza-specific CD4\u003csup\u003e+\u003c/sup\u003e T cell functionality were performed with SPICE version 6 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://niaid.github.io/spice/\u003c/span\u003e\u003cspan address=\"https://niaid.github.io/spice/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e47\u003c/sup\u003e and polyfunctionality indexes calculated using the Funky cells web software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://funkycells.com/main/index.php\u003c/span\u003e\u003cspan address=\"https://funkycells.com/main/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of immune ageing markers in naive and memory CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells\u003c/h2\u003e\u003cp\u003eT cells were phenotyped \u003cem\u003eex vivo\u003c/em\u003e via flow cytometry using directly conjugated antibodies specific for CCR7 (clone 3D12, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone SK1, BD Biosciences), CD27 (clone O323, BioLegend), and CD45RA (clone HI100, eBioscience). Nonviable events were excluded using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Data were acquired using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo version 10.8.1 (FlowJo LLC). For the measurement of senescence-associated β-galactosidase activity, PBMCs were thawed and cultured for 1 h in RPMI 1640 medium containing 1 mM sodium pyruvate, 1 mM nonessential amino acids, 1 mM L-glutamine, 1% penicillin/streptomycin, 25 mM HEPES, and 0.1 \u0026micro;M bafilomycin (all reagents from Thermo Fisher Scientific). Cells were then incubated for a further 2 h in the presence of SA-β-Gal Fluorescent Substrate (33 \u0026micro;M, Cell Signaling Technology). For the measurement proliferation using Cell Proliferation Dye (CPD), PBMCs were labeled with CPD eFluor450 (Thermo Fisher Scientific) and stimulated for 4 days with plate-bound anti-CD3 (clone OKT3; Thermo Fisher Scientific). Proliferation was measured using flow cytometry to quantify the dilution of CPD in CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell subsets based on CCR7 and CD45RA expression. Data were acquired using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo version 10.8.1 (FlowJo LLC).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eEthics\u003c/h2\u003e\u003cp\u003eThe VITAL study was approved by the Medical Research Ethics Committee Utrecht (EU Clinical Trials Registration 2019\u0026ndash;000836\u0026ndash;24) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.clinicaltrialsregister.eu/ctr-search/trial/2019-000836-24/NL\u003c/span\u003e\u003cspan address=\"https://www.clinicaltrialsregister.eu/ctr-search/trial/2019-000836-24/NL\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This study was performed according to Good Clinical Practice, the Declaration of Helsinki, and written informed consent was obtained from all participants.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eSimple group comparisons were performed using the Mann\u0026ndash;Whitney U test or the chi-square test, and correlations were assessed using Spearman\u0026rsquo;s rank test. All basic statistical analyses were performed using Prism software version 9 (GraphPad).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of interest:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing financial interests\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis work was supported by the University of Bordeaux (Senior IdEx Chair) and and the European Union (VITAL study). The VITAL project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No. 806776 and the Dutch Ministry of Health, Welfare and Sport. The JU receives support from the European Union\u0026rsquo;s Horizon 2020 research and innovation program and EFPIA- members. PSC is a laureate of the University of Bordeaux doctorate school program.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePSC, JvB, DvB, BW and VA designed the study and experiments. JvB and DvB wrote the medical ethical application, and performed the clinical trial. PSC, MH and GA performed experiments. PSC, MH, GA, LB, BW, and VA analyzed data. EB was responsible for the data management. PSC, BW and VA wrote the paper. All authors critically revised the manuscript before publication.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the VITAL EFFPIA partners, namely Jim Janimak, Wivine Burny and Ellen Oe from GSK, and Daniel Larocque from Sanofi for productive discussions and for kindly providing influenza vaccine antigens. We are indebted to the VITAL PBMC preparation team. We are grateful to Martin Larsen for assistance with polyfunctionality analyses, to Atika Zouine, Jean-Michel Griffon and Vincent Pitard for technical assistance at the Flow cytometry facility, CNRS UMS 3427, INSERM US 005, Univ. Bordeaux, F-33000 Bordeaux, France, and all the participants of the VITAL cohort.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePetrova, V. N. \u0026amp; Russell, C. A. The evolution of seasonal influenza viruses. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e 16, 47\u0026ndash;60, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrmicro.2017.118\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro.2017.118\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRolfes, M. A. \u003cem\u003eet al.\u003c/em\u003e Effects of Influenza Vaccination in the United States During the 2017\u0026ndash;2018 Influenza Season. \u003cem\u003eClin Infect Dis\u003c/em\u003e 69, 1845\u0026ndash;1853, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/cid/ciz075\u003c/span\u003e\u003cspan address=\"10.1093/cid/ciz075\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIuliano, A. D. \u003cem\u003eet al.\u003c/em\u003e Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. \u003cem\u003eLancet\u003c/em\u003e 391, 1285\u0026ndash;1300, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(17)33293-2\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(17)33293-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrohskopf, L. A. \u003cem\u003eet al.\u003c/em\u003e Prevention and Control of Seasonal Influenza with Vaccines: Recommendations of the Advisory Committee on Immunization Practices - United States, 2020-21 Influenza Season. \u003cem\u003eMMWR Recomm Rep\u003c/em\u003e 69, 1\u0026ndash;24, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.15585/mmwr.rr6908a1\u003c/span\u003e\u003cspan address=\"10.15585/mmwr.rr6908a1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMosmann, T. R., McMichael, A. J., LeVert, A., McCauley, J. W. \u0026amp; Almond, J. W. Opportunities and challenges for T cell-based influenza vaccines. \u003cem\u003eNat Rev Immunol\u003c/em\u003e 24, 736\u0026ndash;752, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41577-024-01030-8\u003c/span\u003e\u003cspan address=\"10.1038/s41577-024-01030-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi, W., Wohlwend, J., Wu, M. \u0026amp; Barzilay, R. Influenza vaccine strain selection with an AI-based evolutionary and antigenicity model. \u003cem\u003eNat Med\u003c/em\u003e, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41591-025-03917-y\u003c/span\u003e\u003cspan address=\"10.1038/s41591-025-03917-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBelongia, E. A. \u003cem\u003eet al.\u003c/em\u003e Variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies. \u003cem\u003eLancet Infect Dis\u003c/em\u003e 16, 942\u0026ndash;951, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1473-3099(16)00129-8\u003c/span\u003e\u003cspan address=\"10.1016/S1473-3099(16)00129-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRondy, M. \u003cem\u003eet al.\u003c/em\u003e Effectiveness of influenza vaccines in preventing severe influenza illness among adults: A systematic review and meta-analysis of test-negative design case-control studies. \u003cem\u003eJ Infect\u003c/em\u003e 75, 381\u0026ndash;394, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jinf.2017.09.010\u003c/span\u003e\u003cspan address=\"10.1016/j.jinf.2017.09.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAllen, J. D. \u0026amp; Ross, T. M. H3N2 influenza viruses in humans: Viral mechanisms, evolution, and evaluation. \u003cem\u003eHum Vaccin Immunother\u003c/em\u003e 14, 1840\u0026ndash;1847, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/21645515.2018.1462639\u003c/span\u003e\u003cspan address=\"10.1080/21645515.2018.1462639\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSasaki, S. \u003cem\u003eet al.\u003c/em\u003e Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies. \u003cem\u003eJ Clin Invest\u003c/em\u003e 121, 3109\u0026ndash;3119, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/JCI57834\u003c/span\u003e\u003cspan address=\"10.1172/JCI57834\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhurana, S., Frasca, D., Blomberg, B. \u0026amp; Golding, H. AID activity in B cells strongly correlates with polyclonal antibody affinity maturation in-vivo following pandemic 2009-H1N1 vaccination in humans. \u003cem\u003ePLoS Pathog\u003c/em\u003e 8, e1002920, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.ppat.1002920\u003c/span\u003e\u003cspan address=\"10.1371/journal.ppat.1002920\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMosterin Hopping, A. \u003cem\u003eet al.\u003c/em\u003e The confounded effects of age and exposure history in response to influenza vaccination. \u003cem\u003eVaccine\u003c/em\u003e 34, 540\u0026ndash;546, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2015.11.058\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2015.11.058\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrasca, D. \u003cem\u003eet al.\u003c/em\u003e Intrinsic defects in B cell response to seasonal influenza vaccination in elderly humans. \u003cem\u003eVaccine\u003c/em\u003e 28, 8077\u0026ndash;8084, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2010.10.023\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2010.10.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHenry, C. \u003cem\u003eet al.\u003c/em\u003e Influenza Virus Vaccination Elicits Poorly Adapted B Cell Responses in Elderly Individuals. \u003cem\u003eCell Host Microbe\u003c/em\u003e 25, 357\u0026ndash;366 e356, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.chom.2019.01.002\u003c/span\u003e\u003cspan address=\"10.1016/j.chom.2019.01.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkbar, A. N., Henson, S. M. \u0026amp; Lanna, A. Senescence of T Lymphocytes: Implications for Enhancing Human Immunity. \u003cem\u003eTrends Immunol\u003c/em\u003e 37, 866\u0026ndash;876, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.it.2016.09.002\u003c/span\u003e\u003cspan address=\"10.1016/j.it.2016.09.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMittelbrunn, M. \u0026amp; Kroemer, G. Hallmarks of T cell aging. \u003cem\u003eNat Immunol\u003c/em\u003e 22, 687\u0026ndash;698, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41590-021-00927-z\u003c/span\u003e\u003cspan address=\"10.1038/s41590-021-00927-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLefebvre, J. S., Masters, A. R., Hopkins, J. W. \u0026amp; Haynes, L. Age-related impairment of humoral response to influenza is associated with changes in antigen specific T follicular helper cell responses. \u003cem\u003eSci Rep\u003c/em\u003e 6, 25051, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/srep25051\u003c/span\u003e\u003cspan address=\"10.1038/srep25051\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHill, D. L. \u003cem\u003eet al.\u003c/em\u003e Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans. \u003cem\u003eElife\u003c/em\u003e 10, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7554/eLife.70554\u003c/span\u003e\u003cspan address=\"10.7554/eLife.70554\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrant, E. J., Quinones-Parra, S. M., Clemens, E. B. \u0026amp; Kedzierska, K. Human influenza viruses and CD8(+) T cell responses. \u003cem\u003eCurr Opin Virol\u003c/em\u003e 16, 132\u0026ndash;142, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.coviro.2016.01.016\u003c/span\u003e\u003cspan address=\"10.1016/j.coviro.2016.01.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcElhaney, J. E. \u003cem\u003eet al.\u003c/em\u003e T cell responses are better correlates of vaccine protection in the elderly. \u003cem\u003eJ Immunol\u003c/em\u003e 176, 6333\u0026ndash;6339, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.176.10.6333\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.176.10.6333\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcElhaney, J. E. \u003cem\u003eet al.\u003c/em\u003e Granzyme B: Correlates with protection and enhanced CTL response to influenza vaccination in older adults. \u003cem\u003eVaccine\u003c/em\u003e 27, 2418\u0026ndash;2425, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2009.01.136\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2009.01.136\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcElhaney, J. E., Kuchel, G. A., Zhou, X., Swain, S. L. \u0026amp; Haynes, L. T-Cell Immunity to Influenza in Older Adults: A Pathophysiological Framework for Development of More Effective Vaccines. \u003cem\u003eFront Immunol\u003c/em\u003e 7, 41, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2016.00041\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2016.00041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLorenzo, E. C., Bartley, J. M. \u0026amp; Haynes, L. The impact of aging on CD4(+) T cell responses to influenza infection. \u003cem\u003eBiogerontology\u003c/em\u003e 19, 437\u0026ndash;446, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10522-018-9754-8\u003c/span\u003e\u003cspan address=\"10.1007/s10522-018-9754-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Baarle, D. \u003cem\u003eet al.\u003c/em\u003e Preventing infectious diseases for healthy ageing: The VITAL public-private partnership project. \u003cem\u003eVaccine\u003c/em\u003e 38, 5896\u0026ndash;5904, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2020.07.005\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2020.07.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan der Heiden, M. \u003cem\u003eet al.\u003c/em\u003e Multiple vaccine comparison in the same adults reveals vaccine-specific and age-related humoral response patterns: an open phase IV trial. \u003cem\u003eNat Commun\u003c/em\u003e 15, 6603, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-024-50760-9\u003c/span\u003e\u003cspan address=\"10.1038/s41467-024-50760-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWertheimer, A. M. \u003cem\u003eet al.\u003c/em\u003e Aging and cytomegalovirus infection differentially and jointly affect distinct circulating T cell subsets in humans. \u003cem\u003eJ Immunol\u003c/em\u003e 192, 2143\u0026ndash;2155, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.1301721\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.1301721\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan den Berg, S. P. H. \u003cem\u003eet al.\u003c/em\u003e Quantification of T-cell dynamics during latent cytomegalovirus infection in humans. \u003cem\u003ePLoS Pathog\u003c/em\u003e 17, e1010152, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.ppat.1010152\u003c/span\u003e\u003cspan address=\"10.1371/journal.ppat.1010152\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBreznik, J. A. \u003cem\u003eet al.\u003c/em\u003e Cytomegalovirus Seropositivity in Older Adults Changes the T Cell Repertoire but Does Not Prevent Antibody or Cellular Responses to SARS-CoV-2 Vaccination. \u003cem\u003eJ Immunol\u003c/em\u003e 209, 1892\u0026ndash;1905, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.2200369\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.2200369\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHerati, R. S. \u003cem\u003eet al.\u003c/em\u003e Circulating CXCR5\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;response predicts influenza vaccine antibody responses in young adults but not elderly adults. \u003cem\u003eJ Immunol\u003c/em\u003e 193, 3528\u0026ndash;3537, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.1302503\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.1302503\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrasca, D., Blomberg, B. B., Garcia, D., Keilich, S. R. \u0026amp; Haynes, L. Age-related factors that affect B cell responses to vaccination in mice and humans. \u003cem\u003eImmunol Rev\u003c/em\u003e 296, 142\u0026ndash;154, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/imr.12864\u003c/span\u003e\u003cspan address=\"10.1111/imr.12864\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLefebvre, J. S. \u003cem\u003eet al.\u003c/em\u003e Vaccine efficacy and T helper cell differentiation change with aging. \u003cem\u003eOncotarget\u003c/em\u003e 7, 33581\u0026ndash;33594, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.9254\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.9254\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLinton, P. J., Haynes, L., Klinman, N. R. \u0026amp; Swain, S. L. Antigen-independent changes in naive CD4 T cells with aging. \u003cem\u003eJ Exp Med\u003c/em\u003e 184, 1891\u0026ndash;1900, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1084/jem.184.5.1891\u003c/span\u003e\u003cspan address=\"10.1084/jem.184.5.1891\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1996).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrasca, D., Diaz, A., Romero, M., Landin, A. M. \u0026amp; Blomberg, B. B. Cytomegalovirus (CMV) seropositivity decreases B cell responses to the influenza vaccine. \u003cem\u003eVaccine\u003c/em\u003e 33, 1433\u0026ndash;1439, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2015.01.071\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2015.01.071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNicoli, F. \u003cem\u003eet al.\u003c/em\u003e Primary immune responses are negatively impacted by persistent herpesvirus infections in older people: results from an observational study on healthy subjects and a vaccination trial on subjects aged more than 70 years old. \u003cem\u003eEBioMedicine\u003c/em\u003e 76, 103852, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ebiom.2022.103852\u003c/span\u003e\u003cspan address=\"10.1016/j.ebiom.2022.103852\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNicoli, F., Karrer, U. \u0026amp; Appay, V. Impact of CMV infection on heterologous vaccine responsiveness. \u003cem\u003ePhil. Trans. R. Soc. B\u003c/em\u003e 380, In Press, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rstb.2024.0407\u003c/span\u003e\u003cspan address=\"10.1098/rstb.2024.0407\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDerhovanessian, E. \u003cem\u003eet al.\u003c/em\u003e Latent infection with cytomegalovirus is associated with poor memory CD4 responses to influenza A core proteins in the elderly. \u003cem\u003eJ Immunol\u003c/em\u003e 193, 3624\u0026ndash;3631, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.1303361\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.1303361\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan den Berg, S. P. H. \u003cem\u003eet al.\u003c/em\u003e Latent CMV Infection Is Associated With Lower Influenza Virus-Specific Memory T-Cell Frequencies, but Not With an Impaired T-Cell Response to Acute Influenza Virus Infection. \u003cem\u003eFront Immunol\u003c/em\u003e 12, 663664, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2021.663664\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2021.663664\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoutsakos, M. \u003cem\u003eet al.\u003c/em\u003e Human CD8(+) T cell cross-reactivity across influenza A, B and C viruses. \u003cem\u003eNat Immunol\u003c/em\u003e 20, 613\u0026ndash;625, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41590-019-0320-6\u003c/span\u003e\u003cspan address=\"10.1038/s41590-019-0320-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDiazGranados, C. A. \u003cem\u003eet al.\u003c/em\u003e Efficacy of high-dose versus standard-dose influenza vaccine in older adults. \u003cem\u003eN Engl J Med\u003c/em\u003e 371, 635\u0026ndash;645, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMoa1315727\u003c/span\u003e\u003cspan address=\"10.1056/NEJMoa1315727\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCouch, R. B. \u003cem\u003eet al.\u003c/em\u003e Superior antigen-specific CD4\u0026thinsp;+\u0026thinsp;T-cell response with AS03-adjuvantation of a trivalent influenza vaccine in a randomised trial of adults aged 65 and older. \u003cem\u003eBMC Infect Dis\u003c/em\u003e 14, 425, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1471-2334-14-425\u003c/span\u003e\u003cspan address=\"10.1186/1471-2334-14-425\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDomnich, A. \u003cem\u003eet al.\u003c/em\u003e Effectiveness of MF59-adjuvanted seasonal influenza vaccine in the elderly: A systematic review and meta-analysis. \u003cem\u003eVaccine\u003c/em\u003e 35, 513\u0026ndash;520, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2016.12.011\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2016.12.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFox, A. \u003cem\u003eet al.\u003c/em\u003e Enhanced Influenza Vaccines Extend A(H3N2) Antibody Reactivity in Older Adults but Prior Vaccination Effects Persist. \u003cem\u003eClin Infect Dis\u003c/em\u003e, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/cid/ciaf060\u003c/span\u003e\u003cspan address=\"10.1093/cid/ciaf060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEssink, B. J. \u003cem\u003eet al.\u003c/em\u003e A randomised phase 2 immunogenicity and safety study of a MF59-adjuvanted quadrivalent subunit inactivated cell-derived influenza vaccine (aQIVc) in adults aged 50 years and older. \u003cem\u003eVaccine\u003c/em\u003e 51, 126791, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2025.126791\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2025.126791\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Beek, J. \u003cem\u003eet al.\u003c/em\u003e Influenza-like Illness Incidence Is Not Reduced by Influenza Vaccination in a Cohort of Older Adults, Despite Effectively Reducing Laboratory-Confirmed Influenza Virus Infections. \u003cem\u003eJ Infect Dis\u003c/em\u003e 216, 415\u0026ndash;424, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/infdis/jix268\u003c/span\u003e\u003cspan address=\"10.1093/infdis/jix268\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKorndewal, M. J. \u003cem\u003eet al.\u003c/em\u003e Cytomegalovirus infection in the Netherlands: seroprevalence, risk factors, and implications. \u003cem\u003eJ Clin Virol\u003c/em\u003e 63, 53\u0026ndash;58, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jcv.2014.11.033\u003c/span\u003e\u003cspan address=\"10.1016/j.jcv.2014.11.033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSamson, L. D. \u003cem\u003eet al.\u003c/em\u003e In-depth immune cellular profiling reveals sex-specific associations with frailty. \u003cem\u003eImmun Ageing\u003c/em\u003e 17, 20, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12979-020-00191-z\u003c/span\u003e\u003cspan address=\"10.1186/s12979-020-00191-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoederer, M., Nozzi, J. L. \u0026amp; Nason, M. C. SPICE: exploration and analysis of post-cytometric complex multivariate datasets. \u003cem\u003eCytometry A\u003c/em\u003e 79, 167\u0026ndash;174, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cyto.a.21015\u003c/span\u003e\u003cspan address=\"10.1002/cyto.a.21015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLarsen, M. \u003cem\u003eet al.\u003c/em\u003e Evaluating cellular polyfunctionality with a novel polyfunctionality index. \u003cem\u003ePLoS One\u003c/em\u003e 7, e42403 (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Influenza, vaccination, immunogenicity, T cells, older adults, antigen design","lastPublishedDoi":"10.21203/rs.3.rs-7818000/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7818000/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImmune defences decline with age, rendering older adults vulnerable to infectious diseases such as influenza. Vaccination remains the most effective means of preventing severe illness and death caused by influenza, although its efficacy is diminished in older adults. Vaccine-specific antibody responses and effectiveness are generally lowest against the influenza A(H3N2) strain, and particularly in individuals over 65 years of age. The mechanisms underlying this age-related decline in vaccine responsiveness remains unclear, prompting the present investigation into the quantity and quality of influenza specific T-cell responses upon vaccination. Frequencies of T cells specific to two influenza A strains H1N1 and H3N2, and two influenza B strains Victoria and Yamagata were measured in adults before and after quadrivalent inactivated influenza vaccination, stratified by age, under (n\u0026thinsp;=\u0026thinsp;100) or over (n\u0026thinsp;=\u0026thinsp;120) 65 years. Polyfunctionality of vaccine induced CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells and immune ageing markers were also assessed in a selection of responders (n\u0026thinsp;=\u0026thinsp;34). Older individuals showed significantly reduced H3N2-specific CD4⁺ T cell frequencies (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) and polyfunctionality (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04) which correlated with lower H3N2 hemagglutination inhibition antibody titers (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.42, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008). Cytomegalovirus seropositivity was associated with diminished influenza specific CD8⁺ T cell responses in the older age group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01). These findings highlight both quantitative and qualitative deficiencies of influenza-specific memory T cells in older vaccinees, which could explain suboptimal humoral responses with advanced age, notably against H3N2. This underscores the need for vaccines designed to boost cellular immunity in this vulnerable population, potentially through improved H3N2 antigen design or alternative vaccine platforms promoting stronger T cell induction.\u003c/p\u003e\u003cp\u003eEU Clinical Trials Registration\u003c/p\u003e\u003cp\u003e2019\u0026ndash;000836\u0026ndash;24\u003c/p\u003e","manuscriptTitle":"Influenza vaccine-specific T cell responses are impaired in older adults","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 05:31:17","doi":"10.21203/rs.3.rs-7818000/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-06T23:21:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T14:38:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-17T15:00:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327771905388180761365943073191870542697","date":"2025-12-08T06:01:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"241390347220314501254024660520927436468","date":"2025-11-25T09:13:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-07T05:16:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-06T17:03:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-21T07:41:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-10-09T13:17:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"88b31d33-8bfe-4d57-9da6-de1bb1ee4dbe","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58083611,"name":"Health sciences/Diseases"},{"id":58083612,"name":"Biological sciences/Immunology"},{"id":58083613,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-13T16:02:24+00:00","versionOfRecord":{"articleIdentity":"rs-7818000","link":"https://doi.org/10.1038/s41541-026-01437-5","journal":{"identity":"npj-vaccines","isVorOnly":false,"title":"npj Vaccines"},"publishedOn":"2026-04-10 15:58:58","publishedOnDateReadable":"April 10th, 2026"},"versionCreatedAt":"2025-11-18 05:31:17","video":"","vorDoi":"10.1038/s41541-026-01437-5","vorDoiUrl":"https://doi.org/10.1038/s41541-026-01437-5","workflowStages":[]},"version":"v1","identity":"rs-7818000","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7818000","identity":"rs-7818000","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}