Publications by authors named "Linda M Wakim"

35 Publications

CD8 T cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph.

Nat Commun 2021 05 18;12(1):2931. Epub 2021 May 18.

Department of Biochemistry and Molecular Biology & Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

Indigenous people worldwide are at high risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. Here, we define CD8 T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identify immunodominant protective CD8 T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2-specific CD8 T cells being cross-reactive between IAV and IBV. Memory CD8 T cells towards these specificities are present in blood (CD27CD45RA phenotype) and tissues (CD103CD69 phenotype) of healthy individuals, and effector CD27CD45RAPD-1CD38CD8 T cells in IAV/IBV patients. Our data show influenza-specific CD8 T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8 T cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease.
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http://dx.doi.org/10.1038/s41467-021-23212-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132304PMC
May 2021

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

Immunity 2021 05 15;54(5):1066-1082.e5. Epub 2021 Apr 15.

Department of Infectious Diseases, Austin Hospital, Heidelberg, VIC 3084, Australia; Department of Medicine and Radiology, The University of Melbourne, Parkville, VIC 3000, Australia; Data Analytics Research and Evaluation (DARE) Centre, Austin Health and The University of Melbourne, Heidelberg, VIC 3084, Australia.

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/N, A2/S, and A24/S) CD8 T cells specific for the immunodominant B7/N 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 TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/NCD8 T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N-specific CD8 T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.
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http://dx.doi.org/10.1016/j.immuni.2021.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049468PMC
May 2021

Influenza, but not SARS-CoV-2, infection induces a rapid interferon response that wanes with age and diminished tissue-resident memory CD8 T cells.

Clin Transl Immunology 2021 26;10(1):e1242. Epub 2021 Jan 26.

Department of Microbiology and Immunology Peter Doherty Institute for Infection and Immunity The University of Melbourne Melbourne VIC Australia.

Older individuals exhibit a diminished ability to respond to and clear respiratory pathogens and, as such, experience a higher rate of lung infections with a higher mortality rate. It is unclear why respiratory pathogens impact older people disproportionately. Using human lung tissue from donors aged 22-68 years, we assessed how the immune cell landscape in lungs changes throughout life and investigated how these immune cells respond following exposure to influenza virus and SARS-CoV-2, two clinically relevant respiratory viruses. While the frequency of most immune cell subsets profiled in the human lung remained stable with age, memory CD8 T cells declined, with the tissue-resident memory (Trm) CD8 T-cell subset being most susceptible to age-associated attrition. Infection of lung tissue with influenza virus resulted in an age-associated attenuation in the antiviral immune response, with aged donors producing less type I interferon (IFN), GM-CSF and IFNγ, the latter correlated with a reduction of IFNγ-producing memory CD8 T cells. In contrast, irrespective of donor age, exposure of human lung cells to SARS-CoV-2, a pathogen for which all donors were immunologically naïve, did not trigger activation of local immune cells and did not result in the induction of an early IFN response. Our findings show that the attrition of tissue-bound pathogen-specific Trm in the lung that occurs with advanced age, or their absence in immunologically naïve individuals, results in a diminished early antiviral immune response which creates a window of opportunity for respiratory pathogens to gain a greater foothold.
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http://dx.doi.org/10.1002/cti2.1242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837404PMC
January 2021

RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells.

Elife 2020 12 2;9. Epub 2020 Dec 2.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.

The dendritic cell receptor Clec9A facilitates processing of dead cell-derived antigens for cross-presentation and the induction of effective CD8 T cell immune responses. Here, we show that this process is regulated by E3 ubiquitin ligase RNF41 and define a new ubiquitin-mediated mechanism for regulation of Clec9A, reflecting the unique properties of Clec9A as a receptor specialized for delivery of antigens for cross-presentation. We reveal RNF41 is a negative regulator of Clec9A and the cross-presentation of dead cell-derived antigens by mouse dendritic cells. Intriguingly, RNF41 regulates the downstream fate of Clec9A by directly binding and ubiquitinating the extracellular domains of Clec9A. At steady-state, RNF41 ubiquitination of Clec9A facilitates interactions with ER-associated proteins and degradation machinery to control Clec9A levels. However, Clec9A interactions are altered following dead cell uptake to favor antigen presentation. These findings provide important insights into antigen cross-presentation and have implications for development of approaches to modulate immune responses.
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http://dx.doi.org/10.7554/eLife.63452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710356PMC
December 2020

Unresponsiveness to inhaled antigen is governed by conventional dendritic cells and overridden during infection by monocytes.

Sci Immunol 2020 10;5(52)

Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.

The nasal-associated lymphoid tissues (NALTs) are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage. NALTs represent a known site for the deposition of inhaled antigens, but little is known of the mechanisms involved in the induction of immunity within this lymphoid tissue. We find that during the steady state, conventional dendritic cells (cDCs) within the NALTs suppress T cell responses. These cDCs, which are also prevalent within human NALTs (tonsils/adenoids), express a unique transcriptional profile and inhibit T cell proliferation via contact-independent mechanisms that can be diminished by blocking the actions of reactive oxygen species and prostaglandin E Although the prevention of unrestrained immune activation to inhaled antigens appears to be the default function of NALT cDCs, inflammation after localized virus infection recruited monocyte-derived DCs (moDCs) to this region, which diluted out the suppressive DC pool, and permitted local T cell priming. Accommodating for inflammation-induced temporal changes in NALT DC composition and function, we developed an intranasal vaccine delivery system that coupled the recruitment of moDCs with the sustained release of antigen into the NALTs, and we were able to substantially improve T cell responses after intranasal immunization. Thus, homeostasis and immunity to inhaled antigens is tuned by inflammatory signals that regulate the balance between conventional and moDC populations within the NALTs.
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http://dx.doi.org/10.1126/sciimmunol.abb5439DOI Listing
October 2020

Intranasal Delivery of a Chitosan-Hydrogel Vaccine Generates Nasal Tissue Resident Memory CD8 T Cells That Are Protective against Influenza Virus Infection.

Vaccines (Basel) 2020 Oct 1;8(4). Epub 2020 Oct 1.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne 3000, Australia.

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.
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http://dx.doi.org/10.3390/vaccines8040572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712318PMC
October 2020

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

Proc Natl Acad Sci U S A 2020 09 10;117(39):24384-24391. Epub 2020 Sep 10.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia;

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/S and A2/Orf1ab Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/SCD8 and A2/Orf1abCD8 populations indicated that A2/SCD8 T cells were detected at comparable frequencies (∼1.3 × 10) 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), but low when compared to frequencies for influenza-specific (A2/M1) and Epstein-Barr virus (EBV)-specific (A2/BMLF) (∼1.38 × 10) populations. Phenotyping A2/SCD8 T cells from COVID-19 convalescents ex vivo showed that A2/SCD8 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/SCD8 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.
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http://dx.doi.org/10.1073/pnas.2015486117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533701PMC
September 2020

Airway Exosomes Released During Influenza Virus Infection Serve as a Key Component of the Antiviral Innate Immune Response.

Front Immunol 2020 12;11:887. Epub 2020 May 12.

Department of Microbiology and Immunology, The University of Melbourne, The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.

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 , 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.
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http://dx.doi.org/10.3389/fimmu.2020.00887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236881PMC
April 2021

Neutrophils play an ongoing role in preventing bacterial pneumonia by blocking the dissemination of Staphylococcus aureus from the upper to the lower airways.

Immunol Cell Biol 2020 08 8;98(7):577-594. Epub 2020 Jun 8.

Department of Microbiology and Immunology, The University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3000, Australia.

Staphylococcus aureus is found in the nasal cavity of up to 30% of the human population. Persistent nasal carriage of S. aureus is a risk factor for influenza virus-induced secondary bacterial pneumonia. There is limited understanding of the factors that cause S. aureus to shift from the upper to the lower respiratory tract and convert from a commensal organism to an invasive pathogen. Here we show that neutrophils actively prevent S. aureus dissemination. Establishment of a mouse model of localized S. aureus nasal carriage revealed variations in the longevity of persistence of S. aureus isolates. Improved persistence within this site was associated with reduced nasal inflammation, less neutrophil egress into the airways and reduced neutrophil-bacteria association. Neutrophil depletion of mice with localized S. aureus nasal carriage triggered the development of an invasive S. aureus infection. Moreover, utilizing a model of influenza-induced staphylococcal pneumonia we showed that treatment with granulocyte-colony-stimulating factor, a potent enhancer of neutrophil number and function, significantly reduced bacterial loads in the lung and improved disease outcomes. These data reveal that neutrophils play an important and active role in confining S. aureus to the upper respiratory tract and highlight the use of approaches that improve neutrophil function as effective strategies to attenuate morbidity associated with staphylococcal pneumonia.
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http://dx.doi.org/10.1111/imcb.12343DOI Listing
August 2020

Bystander Activation of Pulmonary Trm Cells Attenuates the Severity of Bacterial Pneumonia by Enhancing Neutrophil Recruitment.

Cell Rep 2019 12;29(13):4236-4244.e3

Department of Microbiology and Immunology, The University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia. Electronic address:

Tissue-resident memory T (Trm) cells are described as having a "sensing and alarming" function, meaning they can rapidly release cytokines in response to local cognate antigen recognition, which in turn, draws circulating immune cells into the tissue. Here, we show noncognate, bystander activation can also trigger the sensing and alarming function of pulmonary CD8 Trm cells. Virus-specific CD8 Trm cells lodged in the lung parenchyma, but not memory CD8 T cells located in the vasculature, rapidly synthesize interferon γ (IFN-γ) following the inhalation of heat-killed bacteria or bacterial products, a process driven by interleukin-12 (IL-12)/IL-18 exposure. We show that a respiratory bacterial infection leads to bystander activation of lung Trm cells that boosts neutrophil recruitment into the airways and attenuates the severity of bacterial pneumonia. These data reveal that lung Trm cells have innate-like properties, enabling amplification of inflammation and participation in noncognate responses to bacterial infections.
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http://dx.doi.org/10.1016/j.celrep.2019.11.103DOI Listing
December 2019

IFITM3 and type I interferons are important for the control of influenza A virus replication in murine macrophages.

Virology 2020 01 5;540:17-22. Epub 2019 Nov 5.

Department of Microbiology and Immunology, The University of Melbourne, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia; WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia.

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.
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http://dx.doi.org/10.1016/j.virol.2019.11.003DOI Listing
January 2020

Quantification of epitope abundance reveals the effect of direct and cross-presentation on influenza CTL responses.

Nat Commun 2019 06 28;10(1):2846. Epub 2019 Jun 28.

Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia.

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) presentation, while cross-presentation is optimal for acid polymerase (PA) 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.
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http://dx.doi.org/10.1038/s41467-019-10661-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599079PMC
June 2019

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

Nat Immunol 2019 05 18;20(5):613-625. Epub 2019 Feb 18.

Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.

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, CD38Ki67CD8 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.
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http://dx.doi.org/10.1038/s41590-019-0320-6DOI Listing
May 2019

Zymosan by-passes the requirement for pulmonary antigen encounter in lung tissue-resident memory CD8 T cell development.

Mucosal Immunol 2019 03 21;12(2):403-412. Epub 2019 Jan 21.

Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3000, Australia.

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.
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http://dx.doi.org/10.1038/s41385-018-0124-2DOI Listing
March 2019

Rapid interferon independent expression of IFITM3 following T cell activation protects cells from influenza virus infection.

PLoS One 2019 16;14(1):e0210132. Epub 2019 Jan 16.

Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210132PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334895PMC
September 2019

Memory T Cell Dynamics in the Lung during Influenza Virus Infection.

J Immunol 2019 01;202(2):374-381

Department of Microbiology and Immunology, University of Melbourne, at Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia

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.
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http://dx.doi.org/10.4049/jimmunol.1800979DOI Listing
January 2019

Single-Cell Approach to Influenza-Specific CD8 T Cell Receptor Repertoires Across Different Age Groups, Tissues, and Following Influenza Virus Infection.

Front Immunol 2018 27;9:1453. Epub 2018 Jun 27.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia.

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 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-M1 (A2M1). We dissected memory TCR repertoires directed toward A2M1 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 A2M1 CD8 TCRs during acute influenza disease in patients hospitalized with avian A/H7N9 virus. Our study provides the first comparative analysis of paired antigen-specific TCR-α/β clonotypes across different tissues and peripheral blood across different age groups. We show that human A2M1 CD8 T cells can be readily detected in human lungs, spleens, and lymph nodes, and that tissue A2M1 TCRαβ repertoires reflect A2M1 TCRαβ clonotypes derived from peripheral blood in healthy adults and influenza-infected patients. A2M1 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.
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http://dx.doi.org/10.3389/fimmu.2018.01453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030351PMC
June 2018

Harnessing the Power of T Cells: The Promising Hope for a Universal Influenza Vaccine.

Vaccines (Basel) 2018 Mar 26;6(2). Epub 2018 Mar 26.

HKU Pasteur Research Pole, School of Public Health, University of Hong Kong, Hong Kong 999077, China.

Next-generation vaccines that utilize T cells could potentially overcome the limitations of current influenza vaccines that rely on antibodies to provide narrow subtype-specific protection and are prone to antigenic mismatch with circulating strains. Evidence from animal models shows that T cells can provide heterosubtypic protection and are crucial for immune control of influenza virus infections. This has provided hope for the design of a universal vaccine able to prime against diverse influenza virus strains and subtypes. However, multiple hurdles exist for the realisation of a universal T cell vaccine. Overall primary concerns are: extrapolating human clinical studies, seeding durable effective T cell resident memory (Trm), population human leucocyte antigen (HLA) coverage, and the potential for T cell-mediated immune escape. Further comprehensive human clinical data is needed during natural infection to validate the protective role T cells play during infection in the absence of antibodies. Furthermore, fundamental questions still exist regarding the site, longevity and duration, quantity, and phenotype of T cells needed for optimal protection. Standardised experimental methods, and eventually simplified commercial assays, to assess peripheral influenza-specific T cell responses are needed for larger-scale clinical studies of T cells as a correlate of protection against influenza infection. The design and implementation of a T cell-inducing vaccine will require a consensus on the level of protection acceptable in the community, which may not provide sterilizing immunity but could protect the individual from severe disease, reduce the length of infection, and potentially reduce transmission in the community. Therefore, increasing the standard of care potentially offered by T cell vaccines should be considered in the context of pandemic preparedness and zoonotic infections, and in combination with improved antibody vaccine targeting methods. Current pandemic vaccine preparedness measures and ongoing clinical trials under-utilise T cell-inducing vaccines, reflecting the myriad questions that remain about how, when, where, and which T cells are needed to fight influenza virus infection. This review aims to bring together basic fundamentals of T cell biology with human clinical data, which need to be considered for the implementation of a universal vaccine against influenza that harnesses the power of T cells.
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http://dx.doi.org/10.3390/vaccines6020018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027237PMC
March 2018

Circulating T cells, serological memory, and tissue compartmentalization shape human influenza-specific B cell immunity.

Sci Transl Med 2018 02;10(428)

Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, Parkville, Victoria 3010, Australia.

Immunization with the inactivated influenza vaccine (IIV) remains the most effective strategy to combat seasonal influenza infections. IIV activates B cells and T follicular helper (T) cells and thus engenders antibody-secreting cells and serum antibody titers. However, the cellular events preceding generation of protective immunity in humans are inadequately understood. We undertook an in-depth analysis of B cell and T cell immune responses to IIV in 35 healthy adults. Using recombinant hemagglutinin (rHA) probes to dissect the quantity, phenotype, and isotype of influenza-specific B cells against A/California09-H1N1, A/Switzerland-H3N2, and B/Phuket, we showed that vaccination induced a three-pronged B cell response comprising a transient CXCR5CXCR3 antibody-secreting B cell population, CD21CD27 memory B cells, and CD21CD27 B cells. Activation of circulating T cells correlated with the development of both CD21 and CD21 memory B cells. However, preexisting antibodies could limit increases in serum antibody titers. IIV had no marked effect on CD8, mucosal-associated invariant T, γδ T, and natural killer cell activation. In addition, vaccine-induced B cells were not maintained in peripheral blood at 1 year after vaccination. We provide a dissection of rHA-specific B cells across seven human tissue compartments, showing that influenza-specific memory (CD21CD27) B cells primarily reside within secondary lymphoid tissues and the lungs. Our study suggests that a rational design of universal vaccines needs to consider circulating T cells, preexisting serological memory, and tissue compartmentalization for effective B cell immunity, as well as to improve targeting cellular T cell immunity.
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http://dx.doi.org/10.1126/scitranslmed.aan8405DOI Listing
February 2018

Influenza-specific lung-resident memory T cells are proliferative and polyfunctional and maintain diverse TCR profiles.

J Clin Invest 2018 02 8;128(2):721-733. Epub 2018 Jan 8.

Department of Microbiology and Immunology, The University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

The human lung harbors a large population of resident memory T cells (Trm cells). These cells are perfectly positioned to mediate rapid protection against respiratory pathogens such as influenza virus, a highly contagious respiratory pathogen that continues to be a major public health burden. Animal models show that influenza-specific lung CD8+ Trm cells are indispensable for crossprotection against pulmonary infection with different influenza virus strains. However, it is not known whether influenza-specific CD8+ Trm cells present within the human lung have the same critical role in modulating the course of the disease. Here, we showed that human lung contains a population of CD8+ Trm cells that are highly proliferative and have polyfunctional progeny. We observed that different influenza virus-specific CD8+ T cell specificities differentiated into Trm cells with varying efficiencies and that the size of the influenza-specific CD8+ T cell population persisting in the lung directly correlated with the efficiency of differentiation into Trm cells. To our knowledge, we provide the first ex vivo dissection of paired T cell receptor (TCR) repertoires of human influenza-specific CD8+ Trm cells. Our data reveal diverse TCR profiles within the human lung Trm cells and a high degree of clonal sharing with other CD8+ T cell populations, a feature important for effective T cell function and protection against the generation of viral-escape mutants.
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http://dx.doi.org/10.1172/JCI96957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785253PMC
February 2018

Resident memory CD8 T cells in the upper respiratory tract prevent pulmonary influenza virus infection.

Sci Immunol 2017 Jun;2(12)

Department of Microbiology and Immunology, University of Melbourne, at Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.

Nasal epithelial tissue of the upper respiratory tract is the first site of contact by inhaled pathogens such as influenza virus. We show that this region is key to limiting viral spread to the lower respiratory tract and associated disease pathology. Immunization of the upper respiratory tract leads to the formation of local tissue-resident memory CD8 T cells (Trm cells). Unlike Trm cells in the lung, these cells develop independently of local cognate antigen recognition and transforming growth factor-β signaling and persist with minimal decay, representing a long-term protective population. Repertoire characterization revealed unexpected differences between lung and nasal tissue Trm cells, the composition of which was shaped by the developmental need for lung, but not nasal, Trm cells to recognize antigen within their local tissue. We show that influenza-specific Trm cells in the nasal epithelia can block the transmission of influenza virus from the upper respiratory tract to the lung and, in doing so, prevent the development of severe pulmonary disease. Our findings reveal the protective capacity and longevity of upper respiratory tract Trm cells and highlight the potential of targeting these cells to augment protective responses induced to respiratory viral vaccines.
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http://dx.doi.org/10.1126/sciimmunol.aam6970DOI Listing
June 2017

Nasal-associated lymphoid tissues (NALTs) support the recall but not priming of influenza virus-specific cytotoxic T cells.

Proc Natl Acad Sci U S A 2017 05 1;114(20):5225-5230. Epub 2017 May 1.

Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia;

The lymphoid tissue that drains the upper respiratory tract represents an important induction site for cytotoxic T lymphocyte (CTL) immunity to airborne pathogens and intranasal vaccines. Here, we investigated the role of the nasal-associated lymphoid tissues (NALTs), which are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage, in the initial priming and recall expansion of CD8 T cells following an upper respiratory tract infection with a pathogenic influenza virus and immunization with a live attenuated influenza virus vaccine. Whereas NALTs served as the induction site for the recall expansion of memory CD8 T cells following influenza virus infection or vaccination, they failed to support activation of naïve CD8 T cells. Strikingly, NALTs, unlike other lymphoid tissues, were not routinely surveyed during the steady state by circulating T cells. The selective recruitment of memory T cells into these lymphoid structures occurred in response to infection-induced elevation of the chemokine CXCL10, which attracted CXCR3 memory CD8 T cells. These results have significant implications for intranasal vaccines, which deliver antigen to mucosal-associated lymphoid tissue and aim to elicit protective CTL-mediated immunity.
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http://dx.doi.org/10.1073/pnas.1620194114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441821PMC
May 2017

When input does not match output, lung-resident memory T cells decay.

Authors:
Linda M Wakim

Immunol Cell Biol 2017 04 14;95(4):321-322. Epub 2017 Mar 14.

Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

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http://dx.doi.org/10.1038/icb.2017.14DOI Listing
April 2017

Respiratory DC Use IFITM3 to Avoid Direct Viral Infection and Safeguard Virus-Specific CD8+ T Cell Priming.

PLoS One 2015 23;10(11):e0143539. Epub 2015 Nov 23.

Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.

Respiratory dendritic cells (DC) play a pivotal role in the initiation of adaptive immune responses to influenza virus. To do this, respiratory DCs must ferry viral antigen from the lung to the draining lymph node without becoming infected and perishing en route. We show that respiratory DCs up-regulate the expression of the antiviral molecule, interferon-induced transmembrane protein 3 (IFITM3) in response to influenza virus infection, in a manner dependent on type I interferon signaling and the transcription factors IRF7 and IRF3. Failure of respiratory DCs to up-regulate IFITM3 following influenza virus infection resulted in impaired trafficking to the draining LN and consequently in impaired priming of an influenza-specific CD8+ T cell response. The impaired trafficking of IFITM3-deficient DC correlated with an increased susceptibility of these DC to influenza virus infection. This work shows that the expression of IFITM3 protects respiratory DCs from influenza virus infection, permitting migration from lung to LN and optimal priming of a virus specific T-cell response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143539PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657952PMC
June 2016

Endogenous Murine BST-2/Tetherin Is Not a Major Restriction Factor of Influenza A Virus Infection.

PLoS One 2015 13;10(11):e0142925. Epub 2015 Nov 13.

Department of Biochemistry and Molecular Biology, The University of Melbourne, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Rd, Parkville, Victoria, 3010, Australia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142925PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643895PMC
June 2016

Enhanced survival of lung tissue-resident memory CD8⁺ T cells during infection with influenza virus due to selective expression of IFITM3.

Nat Immunol 2013 Mar 27;14(3):238-45. Epub 2013 Jan 27.

Division of Inflammation, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.

Infection with influenza virus results in the deposition of anti-influenza CD8(+) resident memory T cells (T(RM) cells) in the lung. As a consequence of their location in the lung mucosal tissue, these cells are exposed to cytopathic pathogens over the life of the organism and may themselves be susceptible to infection. Here we found that lung T(RM) cells selectively maintained expression of the interferon-induced transmembrane protein IFITM3, a protein that confers broad resistance to viral infection. Lung T(RM) cells that lacked IFITM3 expression were more susceptible to infection than were their normal counterparts and were selectively lost during a secondary bout of infection. Thus, lung T(RM) cells were programmed to retain IFITM3 expression, which facilitated their survival and protection from viral infection during subsequent exposures.
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http://dx.doi.org/10.1038/ni.2525DOI Listing
March 2013

The molecular signature of tissue resident memory CD8 T cells isolated from the brain.

J Immunol 2012 Oct 24;189(7):3462-71. Epub 2012 Aug 24.

Department of Immunology, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.

Tissue resident memory (Trm) CD8 T cells represent a newly described memory T cell population. We have previously characterized a population of Trm cells that persists within the brain after acute virus infection. Although capable of providing marked protection against a subsequent local challenge, brain Trm cells do not undergo recall expansion after dissociation from the tissue. Furthermore, these Trm cells do not depend on the same survival factors as the circulating memory T cell pool as assessed either in vivo or in vitro. To gain greater insight into this population of cells, we compared the gene expression profiles of Trm cells isolated from the brain with those of circulating memory T cells isolated from the spleen after an acute virus infection. Trm cells displayed altered expression of genes involved in chemotaxis, expressed a distinct set of transcription factors, and overexpressed several inhibitory receptors. Cumulatively, these data indicate that Trm cells are a distinct memory T cell population disconnected from the circulating memory T cell pool and display a unique molecular signature that likely results in optimal survival and function within their local environment.
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http://dx.doi.org/10.4049/jimmunol.1201305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3884813PMC
October 2012

Cross-dressed dendritic cells drive memory CD8+ T-cell activation after viral infection.

Nature 2011 Mar;471(7340):629-32

Department of Immunology, Howard Hughes Medical Institute, University of Washington, Box 357370, Seattle, Washington 98195, USA.

After an infection, cytotoxic T lymphocyte precursors proliferate and become effector cells by recognizing foreign peptides in the groove of major histocompatibility complex (MHC) class I molecules expressed by antigen-presenting cells (APCs). Professional APCs specialized for T-cell activation acquire viral antigen either by becoming infected themselves (direct presentation) or by phagocytosis of infected cells, followed by transfer of antigen to the cytosol, processing and MHC class I loading in a process referred to as cross-presentation. An alternative way, referred to as 'cross-dressing', by which an uninfected APC could present antigen was postulated to be by the transfer of preformed peptide-MHC complexes from the surface of an infected cell to the APC without the need of further processing. Here we show that this mechanism exists and boosts the antiviral response of mouse memory CD8(+) T cells. A number of publications have demonstrated sharing of peptide-loaded MHC molecules in vitro. Our in vitro experiments demonstrate that cross-dressing APCs do not acquire peptide-MHC complexes in the form of exosomes released by donor cells. Rather, the APCs and donor cells have to contact each other for the transfer to occur. After a viral infection, we could isolate cross-dressed APCs able to present viral antigen in vitro. Furthermore, using the diphtheria toxin system to selectively eliminate APCs that could only acquire viral peptide-MHC complexes by cross-dressing, we show that such presentation can promote the expansion of resting memory T cells. Notably, naive T cells were excluded from taking part in the response. Cross-dressing is a mechanism of antigen presentation used by dendritic cells that may have a significant role in activating previously primed CD8(+) T cells.
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http://dx.doi.org/10.1038/nature09863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423191PMC
March 2011

Memory T cells persisting within the brain after local infection show functional adaptations to their tissue of residence.

Proc Natl Acad Sci U S A 2010 Oct 5;107(42):17872-9. Epub 2010 Oct 5.

Department of Immunology, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.

The brain is not routinely surveyed by lymphocytes and is defined as an immuno-privileged site. However, viral infection of the brain results in the infiltration and long-term persistence of pathogen-specific CD8(+) T cells. These cells survive without replenishment from the circulation and are referred to as resident memory T cells (Trm). Brain Trm selectively express the integrin CD103, the expression of which is dependent on antigen recognition within the tissue. After clearance of virus, CD8(+) T cells persist in tight clusters, presumably at prior infection hot spots. Antigen persistence is not a prerequisite for T-cell retention, as suggested by the failure to detect viral genomes in the T-cell clusters. Furthermore, we show that an intracranial dendritic cell immunization regimen, which allows the transient introduction of antigen, also results in the generation of memory T cells that persist long term in the brain. Brain Trm die rapidly on isolation from the tissue and fail to undergo recall expansion after adoptive transfer into the bloodstream of antigen-challenged recipients. These ex vivo defects imply a dependency on the local milieu for function and survival. Cumulatively, this work shows that Trm are a specialized population of memory T cells that can be deposited in tissues previously thought to be beyond routine immune surveillance.
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http://dx.doi.org/10.1073/pnas.1010201107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2964240PMC
October 2010

From the thymus to longevity in the periphery.

Curr Opin Immunol 2010 Jun 6;22(3):274-8. Epub 2010 Apr 6.

Department of Immunology, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.

An important attribute of the adaptive immune system is the ability to remember a prior encounter with a pathogen; an ability termed immunological memory. Bigger, better, and stronger responses are mounted upon a secondary encounter with the pathogen potentially resulting in clearance of the infection before the development of disease. We will review recent advances in the field of memory CD8(+) T cell differentiation focusing on both intrinsic and extrinsic factors that govern the development of T cell memory.
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http://dx.doi.org/10.1016/j.coi.2010.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904522PMC
June 2010