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Abortive infection of macrophages serves as a "dead end" for most seasonal influenza A virus (IAV) strains, and it is likely to contribute to effective host defence. Interferon (IFN)-induced transmembrane protein 3 (IFITM3) restricts the early stages of IAV replication in epithelial cells, but IFITM3 restriction of IAV replication in macrophages has not been previously investigated. Herein, macrophages isolated from IFITM3-deficient mice were more susceptible to initial IAV infection, but late-stage viral replication was still controlled through abortive infection. Strikingly, IFNα/β receptor (IFNAR)-deficient macrophages infected with IAV were not only more susceptible to initial infection, but these cells also supported productive viral replication. Significantly, we have established that abortive IAV infection in macrophages is controlled through a type I IFN-dependent mechanism, where late-stage IAV replication can proceed in the absence of type I IFN responses. These findings provide novel mechanistic insight into macrophage-specific processes that potently shut down IAV replication., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The magnitude of T cell responses to infection is a function of the naïve T cell repertoire combined with the context and duration of antigen presentation. Using mass spectrometry, we identify and quantify 21 class 1 MHC-restricted influenza A virus (IAV)-peptides following either direct or cross-presentation. All these peptides, including seven novel epitopes, elicit T cell responses in infected C57BL/6 mice. Directly presented IAV epitopes maintain their relative abundance across distinct cell types and reveal a broad range of epitope abundances. In contrast, cross-presented epitopes are more uniform in abundance. We observe a clear disparity in the abundance of the two key immunodominant IAV antigens, wherein direct infection drives optimal nucleoprotein (NP) 366-374 presentation, while cross-presentation is optimal for acid polymerase (PA) 224-233 presentation. The study demonstrates how assessment of epitope abundance in both modes of antigen presentation is necessary to fully understand the immunogenicity and response magnitude to T cell epitopes.

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.

Tissue-resident memory T cells (Trm) in the lung provide a frontline defence against respiratory pathogens. Vaccination models that lodge CD8 + Trm populations in the lung have been developed, all of which incorporate the local delivery of antigen plus adjuvant into the airways; a necessary approach as local cognate antigen recognition is required for optimal lung Trm development. Although pulmonary delivery of antigen is important for lung Trm development, the impact the co-administered adjuvant has on Trm differentiation is unclear. We show that while altering the adjuvant co-administered with the pulmonary delivered antigen does not impact the size of the lung Trm population, a particular adjuvant, zymosan, when administered into the airways without antigen can drive effector CD8 + T cells to differentiate into lung Trm. Zymosan signalling via dectin-1 receptor was sufficient to promote antigen-independent lung Trm development. When combined with an injectable influenza vaccination regime, intranasal zymosan delivery significantly boosted the size of the influenza virus-specific lung Trm population. Our results highlight that eliciting the appropriate local inflammatory milieu can by-pass the requirement for local antigen recognition in lung Trm development and emphasises that the appropriate selection of adjuvant can greatly improve vaccines that aim to elicit pulmonary Trm.

Influenza A virus is highly contagious, infecting 5-15% of the global population every year. It causes significant morbidity and mortality, particularly among immunocompromised and at-risk individuals. Influenza virus is constantly evolving, undergoing continuous, rapid, and unpredictable mutation, giving rise to novel viruses that can escape the humoral immunity generated by current influenza virus vaccines. Growing evidence indicates that influenza-specific T cells resident along the respiratory tract are highly effective at providing potent and rapid protection against this inhaled pathogen. As these T cells recognize fragments of the virus that are highly conserved and less prone to mutation, they have the potential to provide cross-strain protection against a wide breadth of influenza viruses, including newly emerging strains. In this review, we will discuss how influenza-specific memory T cells in the lung are established and maintained and how we can harness this knowledge to design broadly protective influenza A virus vaccines., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Exosomes are extracellular vesicles secreted by cells that have an important biological function in intercellular communication by transferring biologically active proteins, lipids, and RNAs to neighboring or distant cells. While a role for exosomes in antimicrobial defense has recently emerged, currently very little is known regarding the nature and functional relevance of exosomes generated in vivo , particularly during an active viral infection. Here, we characterized exosomes released into the airways during influenza virus infection. We show that these vesicles dynamically change in protein composition over the course of infection, increasing expression of host proteins with known anti-influenza activity, and viral proteins with the potential to trigger host immune responses. We show that exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigen that can be utilized by antigen presenting cells to drive the induction of a cellular immune response. Moreover, we show that attachment factors for influenza virus, namely α2,3 and α2,6-linked sialic acids, are present on the surface of airway exosomes and these vesicles have the ability to neutralize influenza virus, thereby preventing the virus from binding and entering target cells. These data reveal a novel role for airway exosomes in the antiviral innate immune defense against influenza virus infection. [ABSTRACT FROM AUTHOR]

Interferon-induced transmembrane protein 3 (IFITM3) is a potent antiviral protein that enhances cellular resistance to a variety of pathogens, including influenza virus. Classically defined as an interferon-stimulated gene, expression of IFITM3 on cells is rapidly up-regulated in response to type I and II interferon. Here we found that IFITM3 is rapidly up-regulated by T cells following their activation and this occurred independently of type I and II interferon and the interferon regulatory factors 3 and 7. Up-regulation of IFITM3 on effector T cells protected these cells from virus infection and imparted a survival advantage at sites of virus infection. Our results show that IFITM3 expression on effector T cells is crucial for these cells to mediate their effector function and highlights an interferon independent pathway for the induction of IFITM3 which, if targeted, could be an effective approach to harness the activity of IFITM3 for infection prevention. [ABSTRACT FROM AUTHOR]

CD8+ T cells recognizing antigenic peptides derived from conserved internal viral proteins confer broad protection against distinct influenza viruses. As memory CD8+ T cells change throughout the human lifetime and across tissue compartments, we investigated how T cell receptor (TCR) composition and diversity relate to memory CD8+ T cells across anatomical sites and immunological phases of human life. We used ex vivo peptide-HLA tetramer magnetic enrichment, single-cell multiplex RT-PCR for both the TCR-alpha (TCRα) and TCR-beta (TCRβ) chains, and new TCRdist and grouping of lymphocyte interactions by paratope hotspots (GLIPH) algorithms to compare TCRs directed against the most prominent human influenza epitope, HLA-A*02:01-M158–66 (A2+M158). We dissected memory TCR repertoires directed toward A2+M158 CD8+ T cells within human tissues and compared them to human peripheral blood of young and elderly adults. Furthermore, we compared these memory CD8+ T cell repertoires to A2+M158 CD8+ TCRs during acute influenza disease in patients hospitalized with avian A/H7N9 virus. Our study provides the first ex vivo comparative analysis of paired antigen-specific TCR-α/β clonotypes across different tissues and peripheral blood across different age groups. We show that human A2+M158 CD8+ T cells can be readily detected in human lungs, spleens, and lymph nodes, and that tissue A2+M158 TCRαβ repertoires reflect A2+M158 TCRαβ clonotypes derived from peripheral blood in healthy adults and influenza-infected patients. A2+M158 TCRαβ repertoires displayed distinct features only in elderly adults, with large private TCRαβ clonotypes replacing the prominent and public TRBV19/TRAV27 TCRs. Our study provides novel findings on influenza-specific TCRαβ repertoires within human tissues, raises the question of how we can prevent the loss of optimal TCRαβ signatures with aging, and provides important insights into the rational design of T cell-mediated vaccines and immunotherapies. [ABSTRACT FROM AUTHOR]

BST-2 (tetherin, CD317, HM1.24) restricts virus growth by tethering enveloped viruses to the cell surface. The role of BST-2 during influenza A virus infection (IAV) is controversial. Here, we assessed the capacity of endogenous BST-2 to restrict IAV in primary murine cells. IAV infection increased BST-2 surface expression by primary macrophages, but not alveolar epithelial cells (AEC). BST-2-deficient AEC and macrophages displayed no difference in susceptibility to IAV infection relative to wild type cells. Furthermore, BST-2 played little role in infectious IAV release from either AEC or macrophages. To examine BST-2 during IAV infection in vivo, we infected BST-2-deficient mice. No difference in weight loss or in viral loads in the lungs and/or nasal tissues were detected between BST-2-deficient and wild type animals. This study rules out a major role for endogenous BST-2 in modulating IAV in the mouse model of infection. [ABSTRACT FROM AUTHOR]

Rapid antigen clearance from the nasal mucosa is one of the major challenges in the development of intranasal vaccines. Here, we tested whether intranasal immunization with a chitosan-hydrogel vaccine, with in situ gelling properties, extended antigen retention time within the nasal mucosa. Intranasal immunization with a chitosan-hydrogel vaccine retained antigen within the upper respiratory tract (URT), while intranasal delivery of less viscous vaccines led to antigen accumulation within the lower airways. Interestingly, sustained antigen retention within the URT following chitosan-hydrogel vaccination boosted the number of vaccine-specific, tissue resident memory (Trm) CD8+ T cells that developed within the nasal mucosa. Mice immunized with a chitosan-hydrogel vaccine loaded with influenza virus peptides developed a large pool of influenza-specific CD8+ nasal Trm and these cells were highly protective during an influenza challenge. Our results describe an effective vaccine formulation that can be utilized to boost local immunity in the nasal mucosa. [ABSTRACT FROM AUTHOR]