Quantifying the impact of pre-vaccination titre and vaccination history on influenza vaccine immunogenicity.

Epidemiological studies suggest that heterogeneity in influenza vaccine antibody response is associated with host factors, including pre-vaccination immune status, age, gender, and vaccination history. However, the pattern of reported associations varies between studies. To better understand the underlying influences on antibody responses, we combined host factors and vaccine-induced in-host antibody kinetics from a cohort study conducted across multiple seasons with a unified analysis framework. We developed a flexible individual-level Bayesian model to estimate associations and interactions between host factors, including pre-vaccine HAI titre, age, sex, vaccination history and study setting, and vaccine-induced HAI titre antibody boosting and waning. We applied the model to derive population-level and individual effects of post-vaccine antibody kinetics for vaccinating and circulating strains for A(H1N1) and A(H3N2) influenza subtypes. We found that post-vaccine HAI titre dynamics were significantly influenced by pre-vaccination HAI titre and vaccination history and that lower pre-vaccination HAI titre results in longer durations of seroprotection (HAI titre equal to 1:40 or higher). Consequently, for A(H1N1), our inference finds that the expected duration of seroprotection post-vaccination was 171 (95% Posterior Predictive Interval[PPI] 128-220) and 159 (95% PPI 120-200) days longer for those who are infrequently vaccinated (<2 vaccines in last five years) compared to those who are frequently vaccinated (2 or more vaccines in the last five years) at pre-vaccination HAI titre values of 1:10 and 1:20 respectively. In addition, we found significant differences in the empirical distributions that describe the individual-level duration of seroprotection for A(H1N1) circulating strains. In future, studies that rely on serological endpoints should include the impact of pre-vaccine HAI titre and prior vaccination status on seropositivity and seroconversion estimates, as these significantly influence an individual's post-vaccination antibody kinetics.

Drift and shape-new insights into human immunity against influenza virus neuraminidase.

Influenza virus hemagglutinin mediates infection by binding sialic acids, whereas neuraminidase cleaves sialic acids to release progeny virions. Both are targets of protective antibodies, but influenza vaccine strain selection and antigen dose are based on hemagglutinin alone. Virus characterization using first infection ferret sera indicates that escape from hemagglutination inhibiting (HI) antibodies occurs more frequently and is not coordinated with escape from neuraminidase inhibiting (NI) antibodies. A key question addressed by Daulagala et al. (P. Daulagala, B. R. Mann, K. Leung, E. H. Y. Lau, et al., mBio 14:e00084-23, 2023, https://doi.org/10.1128/mbio.00084-23) is how this translates to humans who encounter multiple influenza viruses throughout life. Their cross-sectional study, using sera from a wide age range of participants and H1N1 viruses spanning 1977-2015, indicates that NI antibodies are more broadly cross-reactive than HI antibodies. Both HI and NI titers were highest against strains encountered in childhood indicating that both are shaped by priming exposures. The study further supports the development of NA-optimized vaccines.

Superior immunogenicity of mRNA over adenoviral vectored COVID-19 vaccines reflects B cell dynamics independent of anti-vector immunity: Implications for future pandemic vaccines.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. The aim of this study was to validate whether immunogenicity differs for adenoviral vectored (AdV) versus mRNA vaccines against SARS-CoV-2, and to investigate how anti-vector immunity and B cell dynamics modulate immunogenicity. We enrolled SARS-CoV-2 infection-naïve health care workers who had received two doses of either AdV AZD1222 (n = 184) or mRNA BNT162b2 vaccine (n = 274) between April and October 2021. Blood was collected at least once, 10-48 days after vaccine dose 2 for antibody and B cell analyses. Median ages were 42 and 39 years, for AdV and mRNA vaccinees, respectively. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p < 0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine, which reflected greater B cell expansion and targeting of the RBD rather than an attenuating effect of anti-vector antibodies. ClinicalTrials.gov Identifier: NCT05110911.

Comparative B cell and antibody responses induced by adenoviral vectored and mRNA vaccines against COVID-19.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. However, adenoviral vectored (AdV) vaccines may be less immunogenic than mRNA vaccines against SARS-CoV-2. We assessed anti-spike and anti-vector immunity among infection-naïve Health Care Workers (HCW) following two doses of AdV (AZD1222) versus mRNA (BNT162b2) vaccine. 183 AdV and 274 mRNA vaccinees enrolled between April and October 2021. Median ages were 42 and 39 years, respectively. Blood was collected at least once, 10-48 days after vaccine dose 2. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p<0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human Adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine due to greater B cell expansion and targeting of the RBD. Pre-existing AdV vector cross-reactive antibodies were boosted following AdV vaccination but had no detectable effect on immunogenicity.

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study.

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.

Does repeated influenza vaccination attenuate effectiveness? A systematic review and meta-analysis.

BACKGROUND: Influenza vaccines require annual readministration; however, several reports have suggested that repeated vaccination might attenuate the vaccine's effectiveness. We aimed to estimate the reduction in vaccine effectiveness associated with repeated influenza vaccination. METHODS: In this systematic review and meta-analysis, we searched MEDLINE, EMBASE, and CINAHL Complete databases for articles published from Jan 1, 2016, to June 13, 2022, and Web of Science for studies published from database inception to June 13, 2022. For studies published before Jan 1, 2016, we consulted published systematic reviews. Two reviewers (EJ-G and EJR) independently screened, extracted data using a data collection form, assessed studies' risk of bias using the Risk Of Bias In Non-Randomized Studies of Interventions (ROBINS-I) and evaluated the weight of evidence by Grading of Recommendations Assessment, Development, and Evaluation (GRADE). We included observational studies and randomised controlled trials that reported vaccine effectiveness against influenza A(H1N1)pdm09, influenza A(H3N2), or influenza B using four vaccination groups: current season; previous season; current and previous seasons; and neither season (reference). For each study, we calculated the absolute difference in vaccine effectiveness (ΔVE) for current season only and previous season only versus current and previous season vaccination to estimate attenuation associated with repeated vaccination. Pooled vaccine effectiveness and ∆VE were calculated by season, age group, and overall. This study is registered with PROSPERO, CRD42021260242. FINDINGS: We identified 4979 publications, selected 681 for full review, and included 83 in the systematic review and 41 in meta-analyses. ΔVE for vaccination in both seasons compared with the current season was -9% (95% CI -16 to -1, I2=0%; low certainty) for influenza A(H1N1)pdm09, -18% (-26 to -11, I2=7%; low certainty) for influenza A(H3N2), and -7% (-14 to 0, I2=0%; low certainty) for influenza B, indicating lower protection with consecutive vaccination. However, for all types, A subtypes and B lineages, vaccination in both seasons afforded better protection than not being vaccinated. INTERPRETATION: Our estimates suggest that, although vaccination in the previous year attenuates vaccine effectiveness, vaccination in two consecutive years provides better protection than does no vaccination. The estimated effects of vaccination in the previous year are concerning and warrant additional investigation, but are not consistent or severe enough to support an alternative vaccination regimen at this time. FUNDING: WHO and the US National Institutes of Health.

Opposing Effects of Prior Infection versus Prior Vaccination on Vaccine Immunogenicity against Influenza A(H3N2) Viruses.

Prior vaccination can alternately enhance or attenuate influenza vaccine immunogenicity and effectiveness. Analogously, we found that vaccine immunogenicity was enhanced by prior A(H3N2) virus infection among participants of the Ha Nam Cohort, Viet Nam, but was attenuated by prior vaccination among Australian Health Care Workers (HCWs) vaccinated in the same year. Here, we combined these studies to directly compare antibody titers against 35 A(H3N2) viruses spanning 1968-2018. Participants received licensed inactivated vaccines containing A/HongKong/4801/2014 (H3N2). The analysis was limited to participants aged 18-65 Y, and compared those exposed to A(H3N2) viruses circulating since 2009 by infection (Ha Nam) or vaccination (HCWs) to a reference group who had no recent A(H3N2) infection or vaccination (Ha Nam). Antibody responses were compared by fitting titer/titer-rise landscapes across strains, and by estimating titer ratios to the reference group of 2009-2018 viruses. Pre-vaccination, titers were lowest against 2009-2014 viruses among the reference (no recent exposure) group. Post-vaccination, titers were, on average, two-fold higher among participants with prior infection and two-fold lower among participants with 3-5 prior vaccinations compared to the reference group. Titer rise was negligible among participants with 3-5 prior vaccinations, poor among participants with 1-2 prior vaccinations, and equivalent or better among those with prior infection compared to the reference group. The enhancing effect of prior infection versus the incrementally attenuating effect of prior vaccinations suggests that these exposures may alternately promote and constrain the generation of memory that can be recalled by a new vaccine strain.

Influenza virus infection history shapes antibody responses to influenza vaccination.

Studies of successive vaccination suggest that immunological memory against past influenza viruses may limit responses to vaccines containing current strains. The impact of memory induced by prior infection is rarely considered and is difficult to ascertain, because infections are often subclinical. This study investigated influenza vaccination among adults from the Ha Nam cohort (Vietnam), who were purposefully selected to include 72 with and 28 without documented influenza A(H3N2) infection during the preceding 9 years (Australian New Zealand Clinical Trials Registry 12621000110886). The primary outcome was the effect of prior influenza A(H3N2) infection on hemagglutinin-inhibiting antibody responses induced by a locally available influenza vaccine administered in November 2016. Baseline and postvaccination sera were titrated against 40 influenza A(H3N2) strains spanning 1968-2018. At each time point (baseline, day 14 and day 280), geometric mean antibody titers against 2008-2018 strains were higher among participants with recent infection (34 (29-40), 187 (154-227) and 86 (72-103)) than among participants without recent infection (19 (17-22), 91 (64-130) and 38 (30-49)). On days 14 and 280, mean titer rises against 2014-2018 strains were 6.1-fold (5.0- to 7.4-fold) and 2.6-fold (2.2- to 3.1-fold) for participants with recent infection versus 4.8-fold (3.5- to 6.7-fold) and 1.9-fold (1.5- to 2.3-fold) for those without. One of 72 vaccinees with recent infection versus 4 of 28 without developed symptomatic A(H3N2) infection in the season after vaccination (P = 0.021). The range of A(H3N2) viruses recognized by vaccine-induced antibodies was associated with the prior infection strain. These results suggest that recall of immunological memory induced by prior infection enhances antibody responses to inactivated influenza vaccine and is important to attain protective antibody titers.

Trend in Sensitivity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Serology One Year After Mild and Asymptomatic Coronavirus Disease 2019 (COVID-19): Unpacking Potential Bias in Seroprevalence Studies.

A key aim of serosurveillance during the coronavirus disease 2019 (COVID-19) pandemic has been to estimate the prevalence of prior infection, by correcting crude seroprevalence against estimated test performance for polymerase chain reaction (PCR)-confirmed COVID-19. We show that poor generalizability of sensitivity estimates to some target populations may lead to substantial underestimation of case numbers.

Influenza A(H1N1)pdm09 But Not A(H3N2) Virus Infection Induces Durable Seroprotection: Results From the Ha Nam Cohort.

BACKGROUND: The extent to which influenza recurrence depends upon waning immunity from prior infection is undefined. We used antibody titers of Ha-Nam cohort participants to estimate protection curves and decay trajectories. METHODS: Households (270) participated in influenza-like-illness (ILI) surveillance and provided blood at intervals spanning laboratory-confirmed virus transmission. Sera were tested in hemagglutination inhibition assay. Infection was defined as influenza virus-positive ILI and/or seroconversion. Median protective titers were estimated using scaled-logistic regression to model pretransmission titer against infection status in that season, limiting analysis to households with infection(s). Titers were modelled against month since infection using mixed-effects linear regression to estimate decay and when titers fell below protection thresholds. RESULTS: From December 2008-2012, 295 and 314 participants were infected with H1N1pdm09-like and A/Perth/16/09-like (H3N2Pe09) viruses, respectively. The proportion protected rose more steeply with titer for H1N1pdm09 than for H3N2Pe09, and estimated 50% protection titers were 19.6 and 37.3, respectively. Postinfection titers started higher against H3N2Pe09 but decayed more steeply than against H1N1pdm09. Seroprotection was estimated to be sustained against H1N1pdm09 but to wane by 8-months for H3N2Pe09. CONCLUSIONS: Estimates indicate that infection induces durable seroprotection against H1N1pdm09 but not H3N2Pe09, which could in part account for the younger age of A(H1N1) versus A(H3N2) cases.

Immune imprinting and SARS-CoV-2 vaccine design.

Reformulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines with variant strains is being pursued to combat the global surge in infections. We hypothesize that this may be suboptimal due to immune imprinting from earlier vaccination or infection with the original SARS-CoV-2 strain. New strategies may be needed to improve efficacy of SARS-CoV-2 variant vaccines.

Within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children.

The evolution of influenza viruses is fundamentally shaped by within-host processes. However, the within-host evolutionary dynamics of influenza viruses remain incompletely understood, in part because most studies have focused on infections in healthy adults based on single timepoint data. Here, we analyzed the within-host evolution of 82 longitudinally sampled individuals, mostly young children, infected with A/H1N1pdm09 or A/H3N2 viruses between 2007 and 2009. For A/H1N1pdm09 infections during the 2009 pandemic, nonsynonymous minority variants were more prevalent than synonymous ones. For A/H3N2 viruses in young children, early infection was dominated by purifying selection. As these infections progressed, nonsynonymous variants typically increased in frequency even when within-host virus titers decreased. Unlike the short-lived infections of adults where de novo within-host variants are rare, longer infections in young children allow for the maintenance of virus diversity via mutation-selection balance creating potentially important opportunities for within-host virus evolution.

Impact of prior vaccination on antibody response and influenza-like illness among Australian healthcare workers after influenza vaccination in 2016.

BACKGROUND: Epidemiological studies suggest that influenza vaccine effectiveness decreases with repeated administration. We examined antibody responses to influenza vaccination among healthcare workers (HCWs) by prior vaccination history and determined the incidence of influenza infection. METHODS: HCWs were vaccinated with the 2016 Southern Hemisphere quadrivalent influenza vaccine. Serum samples were collected pre-vaccination, 21-28 days and 7 months post-vaccination. Influenza antibody titres were measured at each time-point using the haemagglutination inhibition (HI) assay. Immunogenicity was compared by prior vaccination history. RESULTS: A total of 157 HCWs completed the study. The majority were frequently vaccinated, with only 5 reporting no prior vaccinations since 2011. Rises in titres for all vaccine strains among vaccine-naïve HCWs were significantly greater than rises observed for HCWs who received between 1 and 5 prior vaccinations (p < 0.001, respectively). Post-vaccination GMTs against influenza A but not B strains decreased as the number of prior vaccinations increased from 1 to 5. There was a significant decline in GMTs post-season for both B lineages. Sixty five (41%) HCWs reported at least one influenza-like illness episode, with 6 (4%) identified as influenza positive. CONCLUSIONS: Varying serological responses to influenza vaccination were observed among HCWs by prior vaccination history, with vaccine-naïve HCWs demonstrating greater post-vaccination responses against A(H3N2).

Distinguishing naive- from memory-derived human B cells during acute responses.

OBJECTIVES: A fundamental question in influenza research is whether antibody titre decline upon successive exposure to variant strains is consequent to recall of cross-reactive memory B cells that competitively inhibit naive B-cell responses. In connection, it is not clear whether naive and memory B cells remain phenotypically distinct acutely after activation such that they may be distinguished ex vivo. METHODS: Here, we first compared the capacity of anti-Ig and Toll-like-receptor (TLR) 7/8 and TLR9 agonists (R848 and CpG) to augment human B-cell differentiation induced by IL-21 and sCD40L. The conditions that induced optimal differentiation were then used to compare the post-activation phenotype of sort-purified naive and memory B-cell subsets by FACS and antibody-secreting cell (ASC) ELISPOT. RESULTS: Sort-purified naive and memory B cells underwent robust plasmablast and ASC formation when stimulated with R848, but not CpG, and co-cultured with monocytes. This coincided with increased IL-1ß and IL-6 production when B cells were co-cultured with monocytes and stimulated with R848, but not CpG. Naive B cells underwent equivalent ASC generation, but exhibited less class-switch and modulation of CD27, CD38 and CD20 expression than memory B cells after stimulation with R848 and monocytes for 6 days. CONCLUSION: Stimulation with R848, IL-21 and sCD40L in the presence of monocytes induces robust differentiation and ASC generation from both naive and memory B-cells. However, naive and memory B cells retain key phenotypic differences after activation that may facilitate ex vivo discrimination and better characterisation of acute responses to variant antigens.

Recalling the Future: Immunological Memory Toward Unpredictable Influenza Viruses.

Persistent and durable immunological memory forms the basis of any successful vaccination protocol. Generation of pre-existing memory B cell and T cell pools is thus the key for maintaining protective immunity to seasonal, pandemic and avian influenza viruses. Long-lived antibody secreting cells (ASCs) are responsible for maintaining antibody levels in peripheral blood. Generated with CD4+ T help after naïve B cell precursors encounter their cognate antigen, the linked processes of differentiation (including Ig class switching) and proliferation also give rise to memory B cells, which then can change rapidly to ASC status after subsequent influenza encounters. Given that influenza viruses evolve rapidly as a consequence of antibody-driven mutational change (antigenic drift), the current influenza vaccines need to be reformulated frequently and annual vaccination is recommended. Without that process of regular renewal, they provide little protection against "drifted" (particularly H3N2) variants and are mainly ineffective when a novel pandemic (2009 A/H1N1 "swine" flu) strain suddenly emerges. Such limitation of antibody-mediated protection might be circumvented, at least in part, by adding a novel vaccine component that promotes cross-reactive CD8+ T cells specific for conserved viral peptides, presented by widely distributed HLA types. Such "memory" cytotoxic T lymphocytes (CTLs) can rapidly be recalled to CTL effector status. Here, we review how B cells and follicular T cells are elicited following influenza vaccination and how they survive into a long-term memory. We describe how CD8+ CTL memory is established following influenza virus infection, and how a robust CTL recall response can lead to more rapid virus elimination by destroying virus-infected cells, and recovery. Exploiting long-term, cross-reactive CTL against the continuously evolving and unpredictable influenza viruses provides a possible mechanism for preventing a disastrous pandemic comparable to the 1918-1919 H1N1 "Spanish flu," which killed more than 50 million people worldwide.

Limited Phenotypic and Functional Plasticity of Influenza Virus-Specific Memory CD8+ T Cells during Activation in an Alternative Cytokine Environment.

Naive CD8+ T cells show phenotypic, functional, and epigenetic plasticity, enabling differentiation into distinct cellular states. However, whether memory CD8+ T cells demonstrate similar flexibility upon recall is poorly understood. We investigated the potential of influenza A virus (IAV)-specific memory CD8+ T cells from mice to alter their phenotype and function in response to reactivation in the presence of IL-4 and anti-IFN-γ Ab (type 2 conditions). Compared with naive CD8+ T cells, only a small proportion of IAV-specific memory T cells exhibited phenotypic and functional plasticity after clonal activation under type 2 conditions. The potential for modulation of cell-surface phenotype (CD8α expression) was associated with specific epigenetic changes at the Cd8a locus, was greater in central memory T cells than effector memory T cells, and was observed in endogenous memory cells of two TCR specificities. Using a novel technique for intracellular cytokine staining of small clonal populations, we showed that IAV-specific memory CD8+ T cells reactivated under type 2 conditions displayed robust IFN-γ expression and, unlike naive CD8+ T cells activated under type 2 conditions, produced little IL-4 protein. Secondary activation of memory cells under type 2 conditions increased GATA-3 levels with minimal change in T-bet levels. These data suggest that a small population of memory cells, especially central memory T cells, exhibits plasticity; however, most IAV-specific memory CD8+ T cells resist reprogramming upon reactivation and retain the functional state established during priming.

Extending the Breadth of Influenza Vaccines: Status and Prospects for a Universal Vaccine.

Despite the widespread use of seasonal influenza vaccines, there is urgent need for a universal influenza vaccine to provide broad, long-term protection. A number of factors underpin this urgency, including threats posed by zoonotic and pandemic influenza A viruses, suboptimal effectiveness of seasonal influenza vaccines, and concerns surrounding the effects of annual vaccination. In this article, we discuss approaches that are being investigated to increase influenza vaccine breadth, which are near-term, readily achievable approaches to increase the range of strains recognized within a subtype, or longer-term more challenging approaches to produce a truly universal influenza vaccine. Adjuvanted and neuraminidase-optimized vaccines are emerging as the most feasible and promising approaches to extend protection to cover a broader range of strains within a subtype. The goal of developing a universal vaccine has also been advanced with the design of immunogenic influenza HA-stem constructs that induce broadly neutralizing antibodies. However, these constructs are not yet sufficiently immunogenic to induce lasting universal immunity in humans. Advances in understanding how T cells mediate protection, and how viruses are packaged, have facilitated the rationale design and delivery of replication-incompetent virus vaccines that induce broad protection mediated by lung-resident memory T cells. While the lack of clear mechanistic correlates of protection, other than haemagglutination-inhibiting antibodies, remains an impediment to further advancing novel influenza vaccines, the pressing need for such a vaccine is supporting development of highly innovative and effective strategies.

Age-Related Decline in Primary CD8+ T Cell Responses Is Associated with the Development of Senescence in Virtual Memory CD8+ T Cells.

Age-associated decreases in primary CD8+ T cell responses occur, in part, due to direct effects on naive CD8+ T cells to reduce intrinsic functionality, but the precise nature of this defect remains undefined. Aging also causes accumulation of antigen-naive but semi-differentiated "virtual memory" (TVM) cells, but their contribution to age-related functional decline is unclear. Here, we show that TVM cells are poorly proliferative in aged mice and humans, despite being highly proliferative in young individuals, while conventional naive T cells (TN cells) retain proliferative capacity in both aged mice and humans. Adoptive transfer experiments in mice illustrated that naive CD8 T cells can acquire a proliferative defect imposed by the aged environment but age-related proliferative dysfunction could not be rescued by a young environment. Molecular analyses demonstrate that aged TVM cells exhibit a profile consistent with senescence, marking an observation of senescence in an antigenically naive T cell population.

Exposure of Human CD8+ T Cells to Type-2 Cytokines Impairs Division and Differentiation and Induces Limited Polarization.

Effector CD8+ T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8+ cells (Tc2) are detected in type-2 (T2) cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8+ T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8+ T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8+ naïve T cells (TN) in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of TN in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes, and lowered expression of type-1 cytokines, Prf1, Gzmb, T-BET, and Prdm1. However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of TN in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28) and lymph-node homing receptors (CCR7) and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8+ central memory T cells (TCM) were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8+ TN. In summary, while activation of TN in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division are arrested, and reactivation of TCM is reduced in T2 conditions. Taken together, this suggests that exposure to T2 cytokines during activation may not be sufficient to generate and retain human Tc2 cells.