CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

Newborn and child-like molecular signatures in older adults stem from TCR shifts across human lifespan.

CD8+ T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8+ T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M158-66 (A2/M158) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M158+CD8+ T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8+ T cells, which was linked to highly functional public T cell receptor (TCR)αß signatures. Suboptimal TCRαß signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8+ T cell responses toward viral infections.

SARS-CoV-2-specific T cell memory with common TCRαß motifs is established in unvaccinated children who seroconvert after infection.

As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4+ and CD8+ T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαß repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαß motifs in unvaccinated seroconverted children after their first virus encounter.

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.

Increasing HbA1c is associated with reduced CD8+ T cell functionality in response to influenza virus in a TCR-dependent manner in individuals with diabetes mellitus.

Diabetes mellitus is on the rise globally and is a known susceptibility factor for severe influenza virus infections. However, the mechanisms by which diabetes increases the severity of an influenza virus infection are yet to be fully defined. Diabetes mellitus is hallmarked by high glucose concentrations in the blood. We hypothesized that these high glucose concentrations affect the functionality of CD8+ T cells, which play a key role eliminating virus-infected cells and have been shown to decrease influenza disease severity. To study the effect of hyperglycemia on CD8+ T cell function, we stimulated peripheral blood mononuclear cells (PBMCs) from donors with and without diabetes with influenza A virus, anti-CD3/anti-CD28-coated beads, PMA and ionomycin (PMA/I), or an influenza viral peptide pool. After stimulation, cells were assessed for functionality [as defined by expression of IFN-γ, TNF-α, macrophage inflammatory protein (MIP)-1ß, and lysosomal-associated membrane protein-1 (CD107a)] using flow cytometry. Our results showed that increasing HbA1c correlated with a reduction in TNF-α production by CD8+ T cells in response to influenza stimulation in a TCR-specific manner. This was not associated with any changes to CD8+ T cell subsets. We conclude that hyperglycemia impairs CD8+ T cell function to influenza virus infection, which may be linked with the increased risk of severe influenza in patients with diabetes.

mRNA-1273 vaccinated inflammatory bowel disease patients receiving TNF inhibitors develop broad and robust SARS-CoV-2-specific CD8+ T cell responses.

SARS-CoV-2-specific CD8+ T cells recognize conserved viral peptides and in the absence of cross-reactive antibodies form an important line of protection against emerging viral variants as they ameliorate disease severity. SARS-CoV-2 mRNA vaccines induce robust spike-specific antibody and T cell responses in healthy individuals, but their effectiveness in patients with chronic immune-mediated inflammatory disorders (IMIDs) is less well defined. These patients are often treated with systemic immunosuppressants, which may negatively affect vaccine-induced immunity. Indeed, TNF inhibitor (TNFi)-treated inflammatory bowel disease (IBD) patients display reduced ability to maintain SARS-CoV-2 antibody responses post-vaccination, yet the effects on CD8+ T cells remain unclear. Here, we analyzed the impact of IBD and TNFi treatment on mRNA-1273 vaccine-induced CD8+ T cell responses compared to healthy controls in SARS-CoV-2 experienced and inexperienced patients. CD8+ T cells were analyzed for their ability to recognize 32 SARS-CoV-2-specific epitopes, restricted by 10 common HLA class I allotypes using heterotetramer combinatorial coding. This strategy allowed in-depth ex vivo profiling of the vaccine-induced CD8+ T cell responses using phenotypic and activation markers. mRNA vaccination of TNFi-treated and untreated IBD patients induced robust spike-specific CD8+ T cell responses with a predominant central memory and activated phenotype, comparable to those in healthy controls. Prominent non-spike-specific CD8+ T cell responses were observed in SARS-CoV-2 experienced donors prior to vaccination. Non-spike-specific CD8+ T cells persisted and spike-specific CD8+ T cells notably expanded after vaccination in these patient cohorts. Our data demonstrate that regardless of TNFi treatment or prior SARS-CoV-2 infection, IBD patients benefit from vaccination by inducing a robust spike-specific CD8+ T cell response.

T cell activation markers CD38 and HLA-DR indicative of non-seroconversion in anti-CD20-treated patients with multiple sclerosis following SARS-CoV-2 mRNA vaccination.

BACKGROUND: Messenger RNA (mRNA) vaccines provide robust protection against SARS-CoV-2 in healthy individuals. However, immunity after vaccination of patients with multiple sclerosis (MS) treated with ocrelizumab (OCR), a B cell-depleting anti-CD20 monoclonal antibody, is not yet fully understood. METHODS: In this study, deep immune profiling techniques were employed to investigate the immune response induced by SARS-CoV-2 mRNA vaccines in untreated patients with MS (n=21), OCR-treated patients with MS (n=57) and healthy individuals (n=30). RESULTS: Among OCR-treated patients with MS, 63% did not produce detectable levels of antibodies (non-seroconverted), and those who did have lower spike receptor-binding domain-specific IgG responses compared with healthy individuals and untreated patients with MS. Before vaccination, no discernible immunological differences were observed between non-seroconverted and seroconverted OCR-treated patients with MS. However, non-seroconverted patients received overall more OCR infusions, had shorter intervals since their last OCR infusion and displayed higher OCR serum concentrations at the time of their initial vaccination. Following two vaccinations, non-seroconverted patients displayed smaller B cell compartments but instead exhibited more robust activation of general CD4+ and CD8+ T cell compartments, as indicated by upregulation of CD38 and HLA-DR surface expression, when compared with seroconverted patients. CONCLUSION: These findings highlight the importance of optimising treatment regimens when scheduling SARS-CoV-2 vaccination for OCR-treated patients with MS to maximise their humoral and cellular immune responses. This study provides valuable insights for optimising vaccination strategies in OCR-treated patients with MS, including the identification of CD38 and HLA-DR as potential markers to explore vaccine efficacy in non-seroconverting OCR-treated patients with MS.

Cutting Edge: SARS-CoV-2 Infection Induces Robust Germinal Center Activity in the Human Tonsil.

Understanding the generation of immunity to SARS-CoV-2 in lymphoid tissues draining the site of infection has implications for immunity to SARS-CoV-2. We performed tonsil biopsies under local anesthesia in 19 subjects who had recovered from SARS-CoV-2 infection 24-225 d previously. The biopsies yielded >3 million cells for flow cytometric analysis in 17 subjects. Total and SARS-CoV-2 spike-specific germinal center B cells, and T follicular helper cells, were readily detectable in human tonsils early after SARS-CoV-2 infection, as assessed by flow cytometry. Responses were higher in samples within 2 mo of infection but still detectable in some subjects out to 7 mo following infection. We conclude the tonsils are a secondary lymphoid organ that develop germinal center responses to SARS-CoV-2 infection and could play a role in the long-term development of immunity.

Robust and prototypical immune responses toward influenza vaccines in the high-risk group of Indigenous Australians.

Morbidity and mortality rates from seasonal and pandemic influenza occur disproportionately in high-risk groups, including Indigenous people globally. Although vaccination against influenza is recommended for those most at risk, studies on immune responses elicited by seasonal vaccines in Indigenous populations are largely missing, with no data available for Indigenous Australians and only one report published on antibody responses in Indigenous Canadians. We recruited 78 Indigenous and 84 non-Indigenous Australians vaccinated with the quadrivalent influenza vaccine into the Looking into InFluenza T cell immunity - Vaccination cohort study and collected blood to define baseline, early (day 7), and memory (day 28) immune responses. We performed in-depth analyses of T and B cell activation, formation of memory B cells, and antibody profiles and investigated host factors that could contribute to vaccine responses. We found activation profiles of circulating T follicular helper type-1 cells at the early stage correlated strongly with the total change in antibody titers induced by vaccination. Formation of influenza-specific hemagglutinin-binding memory B cells was significantly higher in seroconverters compared with nonseroconverters. In-depth antibody characterization revealed a reduction in immunoglobulin G3 before and after vaccination in the Indigenous Australian population, potentially linked to the increased frequency of the G3m21* allotype. Overall, our data provide evidence that Indigenous populations elicit robust, broad, and prototypical immune responses following immunization with seasonal inactivated influenza vaccines. Our work strongly supports the recommendation of influenza vaccination to protect Indigenous populations from severe seasonal influenza virus infections and their subsequent complications.

Suboptimal SARS-CoV-2-specific CD8+ T cell response associated with the prominent HLA-A*02:01 phenotype.

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269-277 and A2/Orf1ab3183-3191 Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S269+CD8+ and A2/Orf1ab3183+CD8+ populations indicated that A2/S269+CD8+ T cells were detected at comparable frequencies (∼1.3 × 10-5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (∼2.5 × 10-6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein-Barr virus (EBV)-specific (A2/BMLF1280) (∼1.38 × 10-4) populations. Phenotyping A2/S269+CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269+CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269+CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19.

HLA-B*27:05 alters immunodominance hierarchy of universal influenza-specific CD8+ T cells.

Seasonal influenza virus infections cause 290,000-650,000 deaths annually and severe morbidity in 3-5 million people. CD8+ T-cell responses towards virus-derived peptide/human leukocyte antigen (HLA) complexes provide the broadest cross-reactive immunity against human influenza viruses. Several universally-conserved CD8+ T-cell specificities that elicit prominent responses against human influenza A viruses (IAVs) have been identified. These include HLA-A*02:01-M158-66 (A2/M158), HLA-A*03:01-NP265-273, HLA-B*08:01-NP225-233, HLA-B*18:01-NP219-226, HLA-B*27:05-NP383-391 and HLA-B*57:01-NP199-207. The immunodominance hierarchies across these universal CD8+ T-cell epitopes were however unknown. Here, we probed immunodominance status of influenza-specific universal CD8+ T-cells in HLA-I heterozygote individuals expressing two or more universal HLAs for IAV. We found that while CD8+ T-cell responses directed towards A2/M158 were generally immunodominant, A2/M158+CD8+ T-cells were markedly diminished (subdominant) in HLA-A*02:01/B*27:05-expressing donors following ex vivo and in vitro analyses. A2/M158+CD8+ T-cells in non-HLA-B*27:05 individuals were immunodominant, contained optimal public TRBV19/TRAV27 TCRαß clonotypes and displayed highly polyfunctional and proliferative capacity, while A2/M158+CD8+ T cells in HLA-B*27:05-expressing donors were subdominant, with largely distinct TCRαß clonotypes and consequently markedly reduced avidity, proliferative and polyfunctional efficacy. Our data illustrate altered immunodominance patterns and immunodomination within human influenza-specific CD8+ T-cells. Accordingly, our work highlights the importance of understanding immunodominance hierarchies within individual donors across a spectrum of prominent virus-specific CD8+ T-cell specificities prior to designing T cell-directed vaccines and immunotherapies, for influenza and other infectious diseases.

Human Mucosal-Associated Invariant T Cells in Older Individuals Display Expanded TCRαß Clonotypes with Potent Antimicrobial Responses.

Mucosal-associated invariant T (MAIT) cells are important for immune responses against microbial infections. Although known to undergo marked numerical changes with age in humans, our understanding of how MAIT cells are altered during different phases across the human life span is largely unknown. Although also abundant in the tissues, our study focuses on MAIT cell analyses in blood. Across the human life span, we show that naive-like MAIT cells in umbilical cord blood switch to a central/effector memory-like profile that is sustained into older age. Whereas low-grade levels of plasma cytokine/chemokine were apparent in older donors (>65 y old), surprisingly, they did not correlate with the ex vivo MAIT hyperinflammatory cytokine profile observed in older adults. Removal of MAIT cells from older individuals and an aged environment resulted in the reversal of the baseline effector molecule profile comparable with MAIT cells from younger adults. An upregulated basal inflammatory profile accounted for reduced Escherichia coli-specific responses in aged MAIT cells compared with their young adult counterparts when fold change in expression levels of GzmB, CD107a, IFN-γ, and TNF was examined. However, the magnitude of antimicrobial MR1-dependent activation remained as potent and polyfunctional as with younger adults. Paired TCRαß analyses of MAIT cells revealed large clonal expansions in older adults and tissues that rivalled, remarkably, the TCRαß repertoire diversity of virus-specific CD8+ T cells. These data suggest that MAIT cells in older individuals, although associated with large clonal TCRαß expansions and increased baseline inflammatory potential, demonstrate plasticity and provide potent antimicrobial immunity.

Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M158-66 Epitope-Specific CD8+ T Lymphocytes.

Influenza virus-specific CD8+ T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M158-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M158-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M158-66-specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M158-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity.IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8+ T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8+ T lymphocytes in affording protection more accurately. Improving our insight into the interaction between influenza viruses and virus-specific CD8+ T lymphocyte immunity may help to advance our understanding of influenza virus epidemiology, aid in risk assessment of potentially pandemic influenza virus strains, and benefit the design of vaccines that induce more broadly protective immunity.

H1N1pdm09 Influenza Virus and Its Descendants Lack Extra-epitopic Amino Acid Residues Associated With Reduced Recognition by M158-66-Specific CD8+ T Cells.

Extra-epitopic amino acid residues affect recognition of human influenza A viruses (IAVs) by CD8+ T-lymphocytes (CTLs) specific for the highly conserved HLA-A*0201 restricted M158-66 epitope located in the matrix 1 (M1) protein. These residues are absent in the M1 protein of the 2009-pandemic IAV (H1N1pdm09). Consequently, stimulation with M1 protein of H1N1pdm09 IAV resulted in stronger activation and lytic activity of M158-66-specific CTLs than stimulation with seasonal H3N2 IAVs. During >6 years of circulation in the human population, descendants of the H1N1pdm09 virus had accumulated 4 other amino acid substitutions. However, these did not affect M158-66-specific CTL activation.

Human CD8+ T Cells Damage Noninfected Epithelial Cells during Influenza Virus Infection In Vitro.

During severe influenza A virus (IAV) infections, a large amount of damage to the pulmonary epithelium is the result of the antiviral immune response. Specifically, whilst CD8+ T cells are important for killing IAV-infected cells, during a severe IAV infection, they can damage uninfected epithelial cells. At present, the mechanisms by which this occurs are unclear. Here, we used a novel in vitro coculture model of human NCl-H441 cells and CD8+ T cells to provide a new insight into how CD8+ T cells may affect uninfected epithelial cells during severe IAV infections. Using this model, we show that human IAV-specific CD8+ T cells produce soluble factors that reduce the barrier integrity of noninfected epithelial cells (referred to as "bystander damage"). We show that this bystander damage is the result of a combination of TNF-α and IFN-γ. This bystander damage occurred in the absence of widespread epithelial cell death and was instead associated with decreased expression of epithelial cell ion channels and pumps. Together, these data suggest that ameliorating the function of epithelial cell ion channels and pumps may help reduce immunopathology during severe IAV infections.

Differential Recognition of Influenza A Viruses by M158-66 Epitope-Specific CD8+ T Cells Is Determined by Extraepitopic Amino Acid Residues.

UNLABELLED: Natural influenza A virus infections elicit both virus-specific antibody and CD4(+) and CD8(+) T cell responses. Influenza A virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M158-66) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M158-66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8(+) T cell recognition of the M158-66 epitope. These data indicate that human influenza A viruses can impair recognition by M158-66-specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. IMPORTANCE: Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8(+) cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes, mutations occur that allow influenza A viruses to evade recognition by CTLs. However, the immunodominant HLA-A*0201-restricted M158-66 epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M158-66 epitope that influence the recognition of the epitope. These results provide novel insights into the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

Increased Protein Degradation Improves Influenza Virus Nucleoprotein-Specific CD8+ T Cell Activation In Vitro but Not in C57BL/6 Mice.

Due to antigenic drift of influenza viruses, seasonal influenza vaccines need to be updated annually. These vaccines are based on predictions of strains likely to circulate in the next season. However, vaccine efficacy is greatly reduced in the case of a mismatch between circulating and vaccine strains. Furthermore, novel antigenically distinct influenza viruses are introduced into the human population from animal reservoirs occasionally and may cause pandemic outbreaks. To dampen the impact of seasonal and pandemic influenza, vaccines that induce broadly protective and long-lasting immunity are preferred. Because influenza virus-specific CD8+ T cells are directed mainly against relatively conserved internal proteins, like nucleoprotein (NP), they are highly cross-reactive and afford protection against infection with antigenically distinct influenza virus strains, so-called heterosubtypic immunity. Here, we used modified vaccinia virus Ankara (MVA) as a vaccine vector for the induction of influenza virus NP-specific CD8+ T cells. To optimize the induction of CD8+ T cell responses, we made several modifications to NP, aiming at retaining the protein in the cytosol or targeting it to the proteasome. We hypothesized that these strategies would increase antigen processing and presentation and thus improve the induction of CD8+ T cell responses. We showed that NP with increased degradation rates improved CD8+ T cell activation in vitro if the amount of antigen was limited or if CD8+ T cells were of low functional avidity. However, after immunization of C57BL/6 mice, no differences were detected between modified NP and wild-type NP (NPwt), since NPwt already induced optimal CD8+ T cell responses. IMPORTANCE: Due to the continuous antigenic drift of seasonal influenza viruses and the threat of a novel pandemic, there is a great need for the development of novel influenza vaccines that offer broadly protective immunity against multiple subtypes. CD8+ T cells can provide immunity against multiple subtypes of influenza viruses by the recognition of relatively conserved internal antigens. In this study, we aimed at optimizing the CD8+ T cell response to influenza A virus by making modifications to influenza A virus nucleoprotein (NP) expressed from the modified vaccinia virus Ankara (MVA) vaccine vector. These modifications resulted in increased antigen degradation, thereby producing elevated levels of peptides that can be presented on major histocompatibility complex (MHC) class I molecules to CD8+ T cells. Although we were unable to increase the NP-specific immune response in the mouse strain used, this approach may have benefits for vaccine development using less-immunogenic proteins.

Human Influenza A Virus-Specific CD8+ T-Cell Response Is Long-lived.

Animal and human studies have demonstrated the importance of influenza A virus (IAV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) in heterosubtypic cross-protective immunity. Using peripheral blood mononuclear cells obtained intermittently from healthy HLA-typed blood donors between 1999 and 2012, we were able to demonstrate that IAV-specific CTLs are long-lived. Intercurrent IAV infections transiently increase the frequency of functionally distinct subsets of IAV-specific CTLs, in particular effector and effector memory T cells.

Influenza B virus-specific CD8+ T-lymphocytes strongly cross-react with viruses of the opposing influenza B lineage.

Influenza B viruses fall in two antigenically distinct lineages (B/Victoria/2/1987 and B/Yamagata/16/1988 lineage) that co-circulate with influenza A viruses of the H3N2 and H1N1 subtypes during seasonal epidemics. Infections with influenza B viruses contribute considerably to morbidity and mortality in the human population. Influenza B virus neutralizing antibodies, elicited by natural infections or vaccination, poorly cross-react with viruses of the opposing influenza B lineage. Therefore, there is an increased interest in identifying other correlates of protection which could aid the development of broadly protective vaccines. blast analysis revealed high sequence identity of all viral proteins. With two online epitope prediction algorithms, putative conserved epitopes relevant for study subjects used in the present study were predicted. The cross-reactivity of influenza B virus-specific polyclonal CD8+ cytotoxic T-lymphocyte (CTL) populations obtained from HLA-typed healthy study subjects, with intra-lineage drift variants and viruses of the opposing lineage, was determined by assessing their in vitro IFN-γ response and lytic activity. Here, we show for the first time, to the best of our knowledge, that CTLs directed to viruses of the B/Victoria/2/1987 lineage cross-react with viruses of the B/Yamagata/16/1988 lineage and vice versa.

Human cytotoxic T lymphocytes directed to seasonal influenza A viruses cross-react with the newly emerging H7N9 virus.

In February 2013, zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported, a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population, there is interest in identifying other correlates of protection, such as cross-reactive CD8(+) T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8(+) T cells are known to recognize conserved internal proteins of influenza A viruses predominantly, but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here, we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8(+) T cells, obtained from HLA-typed study subjects, with the novel H7N9 virus. The cross-reactivity of CD8(+) T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that, apart from recognition of individual H7N9 variant epitopes, CD8(+) T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8(+) T cells may afford some protection against infection with the new virus.