Human mucosal-associated invariant T cells contribute to antiviral influenza immunity via IL-18–dependent activation

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes known to elicit potent immunity to a broad range of bacteria, mainly via the rapid production of inflammatory cytokines. Whether MAIT cells contribute to antiviral immunity is less clear. Here we asked whether MAIT cells produce cytokines/chemokines during severe human influenza virus infection. Our analysis in patients hospitalized with avian H7N9 influenza pneumonia showed that individuals who recovered had higher numbers of CD161+Vα7.2+ MAIT cells in peripheral blood compared with those who succumbed, suggesting a possible protective role for this lymphocyte population. To understand the mechanism underlying MAIT cell activation during influenza, we cocultured influenza A virus (IAV)-infected human lung epithelial cells (A549) and human peripheral blood mononuclear cells in vitro, then assayed them by intracellular cytokine staining. Comparison of influenza-induced MAIT cell activation with the profile for natural killer cells (CD56+CD3−) showed robust up-regulation of IFNγ for both cell populations and granzyme B in MAIT cells, although the individual responses varied among healthy donors. However, in contrast to the requirement for cell-associated factors to promote NK cell activation, the induction of MAIT cell cytokine production was dependent on IL-18 (but not IL-12) production by IAV-exposed CD14+ monocytes. Overall, this evidence for IAV activation via an indirect, IL-18–dependent mechanism indicates that MAIT cells are protective in influenza, and also possibly in any human disease process in which inflammation and IL-18 production occur

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 TCRαβ 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 TCRαβ 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

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)αβ signatures. Suboptimal TCRαβ 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

Human CD8+ T cell cross-reactivity across influenza A, B and C viruses

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8+ T cells confer cross-protection against IAV strains, however the responses of CD8+ T cells to IBV and ICV are understudied. We investigated the breadth of CD8+ T cell cross-recognition and provide evidence of CD8+ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8+ T cell epitopes from IBVs that were protective in mice and found memory CD8+ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8+ T cells displayed tissue-resident memory phenotypes. Notably, CD38+Ki67+CD8+ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8+ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines

Immune cellular networks underlying recovery from influenza virus infection in acute hospitalized patients

How innate and adaptive immune responses work in concert to resolve influenza disease is yet to be fully investigated in one single study. Here, we utilize longitudinal samples from patients hospitalized with acute influenza to understand these immune responses. We report the dynamics of 18 important immune parameters, related to clinical, genetic and virological factors, in influenza patients across different severity levels. Influenza disease correlates with increases in IL-6/IL-8/MIP-1α/β cytokines and lower antibody responses. Robust activation of circulating T follicular helper cells correlates with peak antibody-secreting cells and influenza heamaglutinin-specific memory B-cell numbers, which phenotypically differs from vaccination-induced B-cell responses. Numbers of influenza-specific CD8+ or CD4+ T cells increase early in disease and retain an activated phenotype during patient recovery. We report the characterisation of immune cellular networks underlying recovery from influenza infection which are highly relevant to other infectious diseases

Tracking human CD8+ and γδ T cell receptor repertoire dynamics to understand the impact on immune responses towards influenza viruses

© 2018 Dr. Sneha Ashok SantSeasonal IAV epidemics cause severe morbidity and mortality, resulting in up to 250,000 -600,000 deaths worldwide annually, especially in the young, elderly, immunocompromised, pregnant and those with co-morbidities. Given the segmented nature of the viral genome and a rapid mutational rate, newly-emerging influenza viruses have the potential to cause pandemics. Current seasonal vaccination regimens elicit neutralizing antibodies (nAbs), which require yearly updates to account for the antigenic evolution of influenza viruses. Alternative strategies such as the development of a universal vaccine that can provide broad protection by eliciting immune responses across different strains of influenza viruses are ugently needed. CD8+ T cells recognize internal conserved segments of the viral protein and thus have the potential to provide cross-strain immunity. The efficient immune response of a CD8+ T cell is dictated by the recognition of peptide-MHC complex by its T cell receptor (TCR). However, the response magnitude varies with age and immunogenetics. Similarly, innate γδ T cells are potently activated by stress-induced ligands, independently of classical antigen-presenting molecules and could provide immediate effector function for novel influenza immunotherapies and vaccines. However, much of human γδ T cell biology remains understudied. Therefore, this PhD aimed to determine TCR dynamics of both γδ T cells and influenza-specific CD8+ T cells across different age groups, anatomical locations and influenza infection. Aim 1 explored the diversity of the human γδ T cell TCR repertoire and how γδ T cells are actived by influenza viruses. We implemented a single-cell RT-PCR for paired analysis of the TCRγ and TCRδ chains and developed an in vitro infection model of influenza-infected lung epithelial cells co-cultured with peripheral blood mononuclear cells (PBMCs). We performed a thorough repertoire analysis of ex vivo γδ T cells from cord blood, young adult, elderly adult and human tissues (spleen, lung and lymph node). Our analyses found diverse and private γδ T cells in cord blood and spleen, while those in young adults and lungs were highly focused towards invariant γ9δ2 TCRs. Elderly adult γδ T cells displayed expansion of private or the invariant γ9δ2 clonotypes. Using the in vitro infection model of influenza, we next investigated γδ TCRs which produced IFNγ during an in vitro influenza infection and PBMC co-culture. Our results demonstrated that the majority of responding γδ T cells harbored γ9δ2 TCRs. We observed heterogeneity in the influenza response between cord blood, young adult and elderly adults. γδ T cells within cord blood and the elderly adults had minimal IFN- production in the absence of γ9δ2 TCRs. Thus, this study provided an understanding on how γδ T cells contribute to immune protection during influenza infection and which TCRs are important to elicit across all age groups vulnerable to influenza virus infection. In Aim 2 and 3, we tracked the repertoire dynamics of influenza specific CD8+ T cells across age groups and tissue locations. Since HLA-A*02:01-restricted M158-66 viral peptide has high sequence conservation and elicits immunodominant CD8+ responses, we focused our analyses on HLA-A*02:01-M158+CD8+ TCRs. A robust response elicited by HLA-A*02:01-M158+CD8+ TCRs is governed by the presence of the public TCR signature, TRBV19/TRAV27 (CDR3 motif “GGSQGNL”/“SIRSYEQ”). Our study demonstrated the loss of this public TCR and presence of large private clonotypes in the HLA-A*02:01-M158+CD8+ TCRs isolated from the elderly donors, as compared to young adults who maintained high frequencies of public TCRs. Our study showed, for the first time, HLA-A*02:01-M158+CD8+ T cells were present in human tissues (spleen, lung and lymph nodes) obtained from young adults. Furthermore, lung tissue-resident HLA-A*02:01-M158+CD8+ T cells and those isolated from spleen and lymph nodes displayed a prominent presence of public TCRs. Overall, we showed a loss of public TCRs with aging and we speculate that this is a mechanism underlying reduced immune responsiveness during influenza infection with aging. Moreover, the presence of public TCRs in distal tissues could provide a reservoir to replenish “optimum” TCRs at the site of infection. The magnitude of antigen-specific CD8+ T cells can be influenced by the different Human Leukocyte Antigens (HLAs) expressed by an individual, thus contributing to the phenomenon of CD8+ T cell immunodominance hierarchy. Using the known highly conserved immunodominant epitopes restricted by 6 HLAs that have broad coverage towards influenza viruses across different ethnicities, we compared the response magnitude of these epitope-specific CD8+ T cells to that of HLA-A*02:01-M158+CD8+ T cells. We showed that individuals co-expressing HLA-B*27:05 and HLA-A*02:01 had higher magnitude of B*27:05-NP383+CD8+ T cell responses compared to that of A*02:01-M158+CD8+ T cells. Our findings showed that B27/NP383+CD8+ T cells had higher functional capacity as compared to A02/M158+CD8+ T cells. Moreover, TCRs of A02/M158+CD8+ T cells from heterozygous donors showed a reduction or complete loss of the public TCR present in A*02:01+B*27:05- individuals. This suggested that the reduction in the observed magnitude of response was partly attributed to changes within the A02/M158 TCR repertiore. Overall, this PhD contributes to our understanding of innate and adaptive T cell compartments during influenza virus infection. It provides evidence that influenza-specifc γδ and CD8+ T cell immune responses are affected by age, HLA genotype and alterations in the TCR repertoire. These findings form an important foundation for future research developing universal vaccines against influenza viruses and immunotherapies against viral infections or cancer

The role of CD27 in anti-viral T-cell immunity

CD27 is a co-stimulatory immune-checkpoint receptor, constitutively expressed on a broad range of T-cells (αβ and γδ), NK-cells and B-cells. Ligation of CD27 with CD70 results in potent co-stimulatory effects. In mice, co-stimulation of CD8+ T-cells through CD27 promotes immune activation and enhances primary, secondary, memory and recall responses towards viral infections. Limited in vitro human studies support mouse experiments and show that CD27 co-stimulation enhances antiviral T-cell immunity. Given the potent co-stimulatory effects of CD27, manipulating CD27 signalling is of interest for viral, autoimmune and anti-tumour immunotherapies. This review focuses on the role of CD27 co-stimulation in anti-viral T-cell immunity and discusses clinical studies utilising the CD27 co-stimulation pathway for anti-viral, anti-tumour and autoimmune immunotherapy

Perturbed CD8+ T cell immunity across universal influenza epitopes in the elderly

Influenza epidemics lead to severe illness, life‐threatening complications, and deaths, especially in the elderly. As CD8+ T cells are associated with rapid recovery from influenza, we investigated the effects of aging on antigen‐specific CD8+ T cells across the universal influenza epitopes in humans. We show that aging is characterized by altered frequencies in T cell subsets, with naive T cells being partially replaced by activated effector/memory populations. Although we observed no striking differences in TCR signaling capacity, T cells in the elderly had increased expression of transcription factors Eomes and T‐bet, and such changes were most apparent in CD8+ T cells. Strikingly, the numbers of antigen‐specific CD8+ T cells across universal influenza epitopes were reduced in the elderly, although their effector/memory phenotypes remained stable. To understand whether diminished numbers of influenza‐specific CD8+ T cells in the elderly resulted from alteration in TCR clonotypes, we dissected the TCRαβ repertoire specific for the prominent HLA‐A*02:01‐restricted‐M158–66 (A2/M158) influenza epitope. We provide the first ex vivo data on paired antigen‐specific TCRαβ clonotypes in the elderly, showing that influenza‐specific A2/M158+ TCRαβ repertoires in the elderly adults varied from those in younger adults, with the main features being a reduction in the frequency of the public TRAV27–TRBV19 TCRαβ clonotype, increased proportion of private TCRαβ signatures, broader use of TRAV and TRBV gene segments, and large clonal expansion of private TCRαβ clonotypes with longer CDR3 loops. Our study supports the development of T cell‐targeted influenza vaccines that would boost the T cell compartment during life and maintain the numbers and optimal TCRαβ signatures in the elderly