Publications by authors named "Liyen Loh"

51 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

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

Nat Commun 2021 05 11;12(1):2691. Epub 2021 May 11.

Department of Biochemistry and Genetics, La Trobe Institute For Molecular Science, La Trobe University, Bundoora, VIC, Australia.

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.
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http://dx.doi.org/10.1038/s41467-021-23018-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113517PMC
May 2021

FOXO1 constrains activation and regulates senescence in CD8 T cells.

Cell Rep 2021 Jan;34(4):108674

Division of Biological Sciences, Molecular Biology Section, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0377, USA; Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0377, USA. Electronic address:

Naive and memory T cells are maintained in a quiescent state, yet capable of rapid response and differentiation to antigen challenge via molecular mechanisms that are not fully understood. In naive cells, the deletion of Foxo1 following thymic development results in the increased expression of multiple AP-1 family members, rendering T cells less able to respond to antigenic challenge. Similarly, in the absence of FOXO1, post-infection memory T cells exhibit the characteristics of extended activation and senescence. Age-based analysis of human peripheral T cells reveals that levels of FOXO1 and its downstream target, TCF7, are inversely related to host age, whereas the opposite is found for AP-1 factors. These characteristics of aging also correlate with the formation of T cells manifesting features of cellular senescence. Our work illustrates a role for FOXO1 in the active maintenance of stem-like properties in T cells at the timescales of acute infection and organismal life span.
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http://dx.doi.org/10.1016/j.celrep.2020.108674DOI Listing
January 2021

Thymic iNKT single cell analyses unmask the common developmental program of mouse innate T cells.

Nat Commun 2020 12 7;11(1):6238. Epub 2020 Dec 7.

Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Most T lymphocytes leave the thymus as naïve cells with limited functionality. However, unique populations of innate-like T cells differentiate into functionally distinct effector subsets during their development in the thymus. Here, we profiled >10,000 differentiating thymic invariant natural killer T (iNKT) cells using single-cell RNA sequencing to produce a comprehensive transcriptional landscape that highlights their maturation, function, and fate decisions at homeostasis. Our results reveal transcriptional profiles that are broadly shared between iNKT and mucosal-associated invariant T (MAIT) cells, illustrating a common core developmental program. We further unmask a mutual requirement for Hivep3, a zinc finger transcription factor and adapter protein. Hivep3 is expressed in early precursors and regulates the post-selection proliferative burst, differentiation and functions of iNKT cells. Altogether, our results highlight the common requirements for the development of innate-like T cells with a focus on how Hivep3 impacts the maturation of these lymphocytes.
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http://dx.doi.org/10.1038/s41467-020-20073-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721697PMC
December 2020

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

PLoS Pathog 2020 08 4;16(8):e1008714. Epub 2020 Aug 4.

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

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.
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http://dx.doi.org/10.1371/journal.ppat.1008714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7428290PMC
August 2020

Publisher Correction: Metabolic characteristics of CD8 T cell subsets in young and aged individuals are not predictive of functionality.

Nat Commun 2020 07 9;11(1):3517. Epub 2020 Jul 9.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-17441-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347533PMC
July 2020

Metabolic characteristics of CD8 T cell subsets in young and aged individuals are not predictive of functionality.

Nat Commun 2020 06 5;11(1):2857. Epub 2020 Jun 5.

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

Virtual memory T (T) cells are antigen-naïve CD8 T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (T) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of T cells and their altered functionality with age, here we investigate T cell metabolism and its association with longevity and functionality. Elevated SRC is a feature of T, but not T, cells and it increases with age in both subsets. The elevated SRC observed in aged mouse T cells and human CD8 T cells from older individuals is associated with a heightened sensitivity to IL-15. We conclude that elevated SRC is a feature of T, but not T, cells, is driven by physiological levels of IL-15, and is not indicative of enhanced functionality in CD8 T cells.
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http://dx.doi.org/10.1038/s41467-020-16633-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275080PMC
June 2020

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

Commun Biol 2020 05 8;3(1):223. Epub 2020 May 8.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia.

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections.
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http://dx.doi.org/10.1038/s42003-020-0955-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210108PMC
May 2020

Hypersensitivities following allergen antigen recognition by unconventional T cells.

Allergy 2020 10 13;75(10):2477-2490. Epub 2020 Apr 13.

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

Conventional T cells recognise protein-derived antigens in the context of major histocompatibility complex (MHC) class Ia and class II molecules and provide anti-microbial and anti-tumour immunity. Conventional T cells have also been implicated in type IV (also termed delayed-type or T cell-mediated) hypersensitivity reactions in response to protein-derived allergen antigens. In addition to conventional T cells, subsets of unconventional T cells exist, which recognise non-protein antigens in the context of monomorphic MHC class I-like molecules. These include T cells that are restricted to the cluster of differentiation 1 (CD1) family members, known as CD1-restricted T cells, and mucosal-associated invariant T cells (MAIT cells) that are restricted to the MHC-related protein 1 (MR1). Compared with conventional T cells, much less is known about the immune functions of unconventional T cells and their role in hypersensitivities. Here, we review allergen antigen presentation by MHC-I-like molecules, their recognition by unconventional T cells, and the potential role of unconventional T cells in hypersensitivities. We also speculate on possible scenarios of allergen antigen presentation by MHC-I-like molecules to unconventional T cells, the hallmarks of such responses, and the expected frequencies of hypersensitivities within the human population.
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http://dx.doi.org/10.1111/all.14279DOI Listing
October 2020

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

J Immunol 2020 03 27;204(5):1119-1133. Epub 2020 Jan 27.

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

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 -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 TCRαβ analyses of MAIT cells revealed large clonal expansions in older adults and tissues that rivalled, remarkably, the TCRαβ repertoire diversity of virus-specific CD8 T cells. These data suggest that MAIT cells in older individuals, although associated with large clonal TCRαβ expansions and increased baseline inflammatory potential, demonstrate plasticity and provide potent antimicrobial immunity.
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http://dx.doi.org/10.4049/jimmunol.1900774DOI Listing
March 2020

Study of MAIT Cell Activation in Viral Infections In Vivo.

Methods Mol Biol 2020 ;2098:261-281

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

MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.
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http://dx.doi.org/10.1007/978-1-0716-0207-2_17DOI Listing
January 2021

Influenza A Virus-Infected Lung Epithelial Cell Co-Culture with Human Peripheral Blood Mononuclear Cells.

Methods Mol Biol 2020 ;2098:141-147

Respiratory Medicine Unit, Nuffield Department of Medicine Experimental Medicine, University of Oxford, Oxfordshire, UK.

Sensing of influenza A virus (IAV) infection by pattern recognition receptors can occur by either direct infection of lung epithelial cells or uptake of virus-infected cells by innate cells such as dendritic cells/monocytes. This triggers a series of downstream events including activation of the inflammasome, the production of cytokines, chemokines, and the upregulation of stress-induced ligands that can lead to the activation of innate cells. These cells include innate lymphocytes such as MAIT, NKT, NK, and γδ T cells. Here we describe a method used to allow activation of human innate lymphocytes in co-culture with an IAV-infected human lung epithelial cell line (A549) to measure ex vivo effector functions (TNF and IFNγ) in a mixed culture environment. We describe (1) infection of the human lung epithelial cell line, (2) co-culture with PBMC, and (3) measurement of activation using intracellular cytokine staining.
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http://dx.doi.org/10.1007/978-1-0716-0207-2_9DOI Listing
January 2021

Characterization of Human Mucosal-associated Invariant T (MAIT) Cells.

Curr Protoc Immunol 2019 12;127(1):e90

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

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells restricted by the major histocompatibility complex (MHC) class I-like molecule MHC-related protein 1 (MR1). MAIT cells are found throughout the body, especially in human blood and liver. Unlike conventional T cells, which are stimulated by peptide antigens presented by MHC molecules, MAIT cells recognize metabolite antigens derived from an intermediate in the microbial biosynthesis of riboflavin. MAIT cells mediate protective immunity to infections by riboflavin-producing microbes via the production of cytokines and cytotoxicity. The discovery of stimulating MAIT cell antigens allowed for the development of an analytical tool, the MR1 tetramer, that binds specifically to the MAIT T cell receptor (TCR) and is becoming the gold standard for identification of MAIT cells by flow cytometry. This article describes protocols to characterize the phenotype of human MAIT cells in blood and tissues by flow cytometry using fluorescently labeled human MR1 tetramers alongside antibodies specific for MAIT cell markers. © 2019 by John Wiley & Sons, Inc. The main protocols include: Basic Protocol 1: Determining the frequency and steady-state surface phenotype of human MAIT cells Basic Protocol 2: Determining the activation phenotype of human MAIT cells in blood Basic Protocol 3: Characterizing MAIT cell TCRs using TCR-positive reporter cell lines Alternate protocols are provided for determining the absolute number, transcription factor phenotype, and TCR usage of human MAIT cells; and determining activation phenotype by staining for intracellular markers, measuring secreted cytokines, and measuring fluorescent dye dilution due to proliferation. Additional methods are provided for determining the capacity of MAIT cells to produce cytokine independently of antigen using plate-bound or bead-immobilized CD3/CD28 stimulation; and determining the MR1-Ag dependence of MAIT cell activation using MR1-blocking antibody or competitive inhibition. For TCR-positive reporter cell lines, methods are also provided for evaluating the MAIT TCR-mediated MR1-Ag response, determining the capacity of the reporter lines to produce cytokine independently of antigen, determining the MR1-Ag dependence of the reporter lines, and evaluating the MR1-Ag response of the reporter lines using IL-2 secretion. Support Protocols describe the preparation of PBMCs from human blood, the preparation of single-cell suspensions from tissue, the isolation of MAIT cells by FACS and MACS, cloning MAIT TCRα and β chain genes and MR1 genes for transduction, generating stably and transiently transfected cells lines, generating a stable MR1 knockout antigen-presenting cell line, and generating monocyte-derived dendritic cells.
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http://dx.doi.org/10.1002/cpim.90DOI Listing
December 2019

Human γδ T-cell receptor repertoire is shaped by influenza viruses, age and tissue compartmentalisation.

Clin Transl Immunology 2019 23;8(9):e1079. Epub 2019 Sep 23.

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

Background: Although γδ T cells comprise up to 10% of human peripheral blood T cells, questions remain regarding their role in disease states and T-cell receptor (TCR) clonal expansions. We dissected anti-viral functions of human γδ T cells towards influenza viruses and defined influenza-reactive γδ TCRs in the context of γδ-TCRs across the human lifespan.

Methods: We performed Cr-killing assay and single-cell time-lapse live video microscopy to define mechanisms underlying γδ T-cell-mediated killing of influenza-infected targets. We assessed cytotoxic profiles of γδ T cells in influenza-infected patients and IFN-γ production towards influenza-infected lung epithelial cells. Using single-cell RT-PCR, we characterised paired TCRγδ clonotypes for influenza-reactive γδ T cells in comparison with TCRs from healthy neonates, adults, elderly donors and tissues.

Results: We provide the first visual evidence of γδ T-cell-mediated killing of influenza-infected targets and show distinct features to those reported for CD8 T cells. γδ T cells displayed poly-cytotoxic profiles in influenza-infected patients and produced IFN-γ towards influenza-infected cells. These IFN-γ-producing γδ T cells were skewed towards the γ9δ2 TCRs, particularly expressing the public GV9-TCRγ, capable of pairing with numerous TCR-δ chains, suggesting their significant role in γδ T-cell immunity. Neonatal γδ T cells displayed extensive non-overlapping TCRγδ repertoires, while adults had enriched γ9δ2-pairings with diverse CDR3γδ regions. Conversely, the elderly showed distinct γδ-pairings characterised by large clonal expansions, a profile also prominent in adult tissues.

Conclusion: Human TCRγδ repertoire is shaped by age, tissue compartmentalisation and the individual's history of infection, suggesting that these somewhat enigmatic γδ T cells indeed respond to antigen challenge.
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http://dx.doi.org/10.1002/cti2.1079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756999PMC
September 2019

Broad CD8 T cell cross-recognition of distinct influenza A strains in humans.

Nat Commun 2018 12 21;9(1):5427. Epub 2018 Dec 21.

Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3010, Australia.

Newly-emerged and vaccine-mismatched influenza A viruses (IAVs) result in a rapid global spread of the virus due to minimal antibody-mediated immunity. In that case, established CD8 T-cells can reduce disease severity. However, as mutations occur sporadically within immunogenic IAV-derived T-cell peptides, understanding of T-cell receptor (TCRαβ) cross-reactivity towards IAV variants is needed for a vaccine design. Here, we investigate TCRαβ cross-strain recognition across IAV variants within two immunodominant human IAV-specific CD8 T-cell epitopes, HLA-B*37:01-restricted NP (B37-NP) and HLA-A*01:01-restricted NP (A1-NP). We find high abundance of cross-reactive TCRαβ clonotypes recognizing distinct IAV variants. Structures of the wild-type and variant peptides revealed preserved conformation of the bound peptides. Structures of a cross-reactive TCR-HLA-B37-NP complex suggest that the conserved conformation of the variants underpins TCR cross-reactivity. Overall, cross-reactive CD8 T-cell responses, underpinned by conserved epitope structure, facilitates recognition of distinct IAV variants, thus CD8 T-cell-targeted vaccines could provide protection across different IAV strains.
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http://dx.doi.org/10.1038/s41467-018-07815-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303473PMC
December 2018

Multiplex Droplet Digital PCR Assay for Quantification of Human T-Cell Leukemia Virus Type 1 Subtype c DNA Proviral Load and T Cells from Blood and Respiratory Exudates Sampled in a Remote Setting.

J Clin Microbiol 2019 02 30;57(2). Epub 2019 Jan 30.

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

During human T-cell leukemia virus type 1 (HTLV-1) infection, the frequency of cells harboring an integrated copy of viral cDNA, the proviral load (PVL), is the main risk factor for progression of HTLV-1-associated diseases. Accurate quantification of provirus by droplet digital PCR (ddPCR) is a powerful diagnostic tool with emerging uses for monitoring viral expression. Current ddPCR techniques quantify HTLV-1 PVL in terms of whole genomic cellular material, while the main targets of HTLV-1 infection are CD4 and CD8 T cells. Our understanding of HTLV-1 proliferation and the amount of viral burden present in different compartments is limited. Recently a sensitive ddPCR assay was applied to quantifying T cells by measuring loss of germ line T-cell receptor genes as method of distinguishing non-T-cell from recombined T-cell DNA. In this study, we demonstrated and validated novel applications of the duplex ddPCR assay to quantify T cells from various sources of human genomic DNA (gDNA) extracted from frozen material (peripheral blood mononuclear cells [PBMCs], bronchoalveolar lavage fluid, and induced sputum) from a cohort of remote Indigenous Australians and then compared the T-cell measurements by ddPCR to the prevailing standard method of flow cytometry. The HTLV-1 subtype c (HTLV-1c) PVL was then calculated in terms of extracted T-cell gDNA from various compartments. Because HTLV-1c preferentially infects CD4 T cells, and the amount of viral burden correlates with HTLV-1c disease pathogenesis, application of this ddPCR assay to accurately measure HTLV-1c-infected T cells can be of greater importance for clinical diagnostics and prognostics as well as monitoring therapeutic applications.
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http://dx.doi.org/10.1128/JCM.01063-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355533PMC
February 2019

The CD4CD8 MAIT cell subpopulation is a functionally distinct subset developmentally related to the main CD8 MAIT cell pool.

Proc Natl Acad Sci U S A 2018 12 15;115(49):E11513-E11522. Epub 2018 Nov 15.

Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 14186 Stockholm, Sweden;

Mucosa-associated invariant T (MAIT) cells are unconventional innate-like T cells that recognize microbial riboflavin metabolites presented by the MHC class I-like protein MR1. Human MAIT cells predominantly express the CD8α coreceptor (CD8), with a smaller subset lacking both CD4 and CD8 (double-negative, DN). However, it is unclear if these two MAIT cell subpopulations distinguished by CD8α represent functionally distinct subsets. Here, we show that the two MAIT cell subsets express divergent transcriptional programs and distinct patterns of classic T cell transcription factors. Furthermore, CD8 MAIT cells have higher levels of receptors for IL-12 and IL-18, as well as of the activating receptors CD2, CD9, and NKG2D, and display superior functionality following stimulation with riboflavin-autotrophic as well as riboflavin-auxotrophic bacterial strains. DN MAIT cells display higher RORγt/T-bet ratio, and express less IFN-γ and more IL-17. Furthermore, the DN subset displays enrichment of an apoptosis gene signature and higher propensity for activation-induced apoptosis. During development in human fetal tissues, DN MAIT cells are more mature and accumulate over gestational time with reciprocal contraction of the CD8 subset. Analysis of the T cell receptor repertoire reveals higher diversity in CD8 MAIT cells than in DN MAIT cells. Finally, chronic T cell receptor stimulation of CD8 MAIT cells in an in vitro culture system supports the accumulation and maintenance of the DN subpopulation. These findings define human CD8 and DN MAIT cells as functionally distinct subsets and indicate a derivative developmental relationship.
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http://dx.doi.org/10.1073/pnas.1812273115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298106PMC
December 2018

MAIT cells contribute to protection against lethal influenza infection in vivo.

Nat Commun 2018 11 9;9(1):4706. Epub 2018 Nov 9.

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

Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. MAIT cells are also activated during human viral infections, yet it remains unknown whether MAIT cells play a significant protective or even detrimental role during viral infections in vivo. Using murine experimental challenge with two strains of influenza A virus, we show that MAIT cells accumulate and are activated early in infection, with upregulation of CD25, CD69 and Granzyme B, peaking at 5 days post-infection. Activation is modulated via cytokines independently of MR1. MAIT cell-deficient MR1 mice show enhanced weight loss and mortality to severe (H1N1) influenza. This is ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient RAG2γC mice. Thus, MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.
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http://dx.doi.org/10.1038/s41467-018-07207-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226485PMC
November 2018

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

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

Cell Rep 2018 06;23(12):3512-3524

Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia; Department of Microbiology and Immunology, The Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia. Electronic address:

Age-associated decreases in primary CD8 T cell responses occur, in part, due to direct effects on naive CD8 T cells to reduce intrinsic functionality, but the precise nature of this defect remains undefined. Aging also causes accumulation of antigen-naive but semi-differentiated "virtual memory" (T) cells, but their contribution to age-related functional decline is unclear. Here, we show that T cells are poorly proliferative in aged mice and humans, despite being highly proliferative in young individuals, while conventional naive T cells (T cells) retain proliferative capacity in both aged mice and humans. Adoptive transfer experiments in mice illustrated that naive CD8 T cells can acquire a proliferative defect imposed by the aged environment but age-related proliferative dysfunction could not be rescued by a young environment. Molecular analyses demonstrate that aged T cells exhibit a profile consistent with senescence, marking an observation of senescence in an antigenically naive T cell population.
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http://dx.doi.org/10.1016/j.celrep.2018.05.057DOI Listing
June 2018

Enumeration, functional responses and cytotoxic capacity of MAIT cells in newly diagnosed and relapsed multiple myeloma.

Sci Rep 2018 03 7;8(1):4159. Epub 2018 Mar 7.

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

Mucosal-associated invariant T (MAIT) cells are T cells that recognise vitamin-B derivative Ag presented by the MHC-related-protein 1 (MR1) antigen-presenting molecule. While MAIT cells are highly abundant in humans, their role in tumour immunity remains unknown. Here we have analysed the frequency and function of MAIT cells in multiple myeloma (MM) patients. We show that MAIT cell frequency in blood is reduced compared to healthy adult donors, but comparable to elderly healthy control donors. Furthermore, there was no evidence that MAIT cells accumulated at the disease site (bone marrow) of these patients. Newly diagnosed MM patient MAIT cells had reduced IFNγ production and CD27 expression, suggesting an exhausted phenotype, although IFNγ-producing capacity is restored in relapsed/refractory patient samples. Moreover, immunomodulatory drugs Lenalidomide and Pomalidomide, indirectly inhibited MAIT cell activation. We further show that cell lines can be pulsed with vitamin-B derivative Ags and that these can be presented via MR1 to MAIT cells in vitro, to induce cytotoxic activity comparable to that of natural killer (NK) cells. Thus, MAIT cells are reduced in MM patients, which may contribute to disease in these individuals, and moreover, MAIT cells may represent new immunotherapeutic targets for treatment of MM and other malignancies.
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http://dx.doi.org/10.1038/s41598-018-22130-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841305PMC
March 2018

Clonally diverse CD38HLA-DRCD8 T cells persist during fatal H7N9 disease.

Nat Commun 2018 02 26;9(1):824. Epub 2018 Feb 26.

Shanghai Public Health Clinical Center & Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology of Ministry of Education/Health, Shanghai Medical College, Fudan University, 201508, Shangai, China.

Severe influenza A virus (IAV) infection is associated with immune dysfunction. Here, we show circulating CD8 T-cell profiles from patients hospitalized with avian H7N9, seasonal IAV, and influenza vaccinees. Patient survival reflects an early, transient prevalence of highly activated CD38HLA-DRPD-1 CD8 T cells, whereas the prolonged persistence of this set is found in ultimately fatal cases. Single-cell T cell receptor (TCR)-αβ analyses of activated CD38HLA-DRCD8 T cells show similar TCRαβ diversity but differential clonal expansion kinetics in surviving and fatal H7N9 patients. Delayed clonal expansion associated with an early dichotomy at a transcriptome level (as detected by single-cell RNAseq) is found in CD38HLA-DRCD8 T cells from patients who succumbed to the disease, suggesting a divergent differentiation pathway of CD38HLA-DRCD8 T cells from the outset during fatal disease. Our study proposes that effective expansion of cross-reactive influenza-specific TCRαβ clonotypes with appropriate transcriptome signatures is needed for early protection against severe influenza disease.
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http://dx.doi.org/10.1038/s41467-018-03243-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827521PMC
February 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

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

J Leukoc Biol 2018 02 29;103(2):321-339. Epub 2017 Dec 29.

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

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-M1 (A2/M1 ) influenza epitope. We provide the first ex vivo data on paired antigen-specific TCRαβ clonotypes in the elderly, showing that influenza-specific A2/M1 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.
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http://dx.doi.org/10.1189/jlb.5MA0517-207RDOI Listing
February 2018

A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage.

Nat Immunol 2016 Nov 26;17(11):1300-1311. Epub 2016 Sep 26.

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

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.
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http://dx.doi.org/10.1038/ni.3565DOI Listing
November 2016

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

Proc Natl Acad Sci U S A 2016 09 19;113(36):10133-8. Epub 2016 Aug 19.

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

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.
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http://dx.doi.org/10.1073/pnas.1610750113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018778PMC
September 2016

Avian Influenza Viruses, Inflammation, and CD8(+) T Cell Immunity.

Front Immunol 2016 1;7:60. Epub 2016 Mar 1.

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

Avian influenza viruses (AIVs) circulate naturally in wild aquatic birds, infect domestic poultry, and are capable of causing sporadic bird-to-human transmissions. AIVs capable of infecting humans include a highly pathogenic AIV H5N1, first detected in humans in 1997, and a low pathogenic AIV H7N9, reported in humans in 2013. Both H5N1 and H7N9 cause severe influenza disease in humans, manifested by acute respiratory distress syndrome, multi-organ failure, and high mortality rates of 60% and 35%, respectively. Ongoing circulation of H5N1 and H7N9 viruses in wild birds and poultry, and their ability to infect humans emphasizes their epidemic and pandemic potential and poses a public health threat. It is, thus, imperative to understand the host immune responses to the AIVs so we can control severe influenza disease caused by H5N1 or H7N9 and rationally design new immunotherapies and vaccines. This review summarizes our current knowledge on AIV epidemiology, disease symptoms, inflammatory processes underlying the AIV infection in humans, and recent studies on universal pre-existing CD8(+) T cell immunity to AIVs. Immune responses driving the host recovery from AIV infection in patients hospitalized with severe influenza disease are also discussed.
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http://dx.doi.org/10.3389/fimmu.2016.00060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4771736PMC
March 2016

Recovery from severe H7N9 disease is associated with diverse response mechanisms dominated by CD8⁺ T cells.

Nat Commun 2015 May 13;6:6833. Epub 2015 May 13.

Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology of Ministry of Education/Health, Shanghai Medical College, Fudan University, Shanghai 201508, China.

The avian origin A/H7N9 influenza virus causes high admission rates (>99%) and mortality (>30%), with ultimately favourable outcomes ranging from rapid recovery to prolonged hospitalization. Using a multicolour assay for monitoring adaptive and innate immunity, here we dissect the kinetic emergence of different effector mechanisms across the spectrum of H7N9 disease and recovery. We find that a diversity of response mechanisms contribute to resolution and survival. Patients discharged within 2-3 weeks have early prominent H7N9-specific CD8(+) T-cell responses, while individuals with prolonged hospital stays have late recruitment of CD8(+)/CD4(+) T cells and antibodies simultaneously (recovery by week 4), augmented even later by prominent NK cell responses (recovery >30 days). In contrast, those who succumbed have minimal influenza-specific immunity and little evidence of T-cell activation. Our study illustrates the importance of robust CD8(+) T-cell memory for protection against severe influenza disease caused by newly emerging influenza A viruses.
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http://dx.doi.org/10.1038/ncomms7833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4479016PMC
May 2015

Role of education and differentiation in determining the potential of natural killer cells to respond to antibody-dependent stimulation.

AIDS 2014 Nov;28(18):2781-6

aDepartment of Microbiology and Immunology, Peter Doherty Institute, University of Melbourne bBurnet Institute, Melbourne cMelbourne Sexual Health Centre, Carlton, Victoria, Australia.

Antibody-dependent activation of natural killer (NK) cells might facilitate protective outcomes in the context of HIV exposure or infection. Antibody-dependent activation is heightened in NK cells educated by interactions between killer immunoglobulin-like receptors (KIRs) and their major histocompatibility complex class I ligands during ontogeny. Differentiated NK cells, defined as CD57, also exhibit enhanced antibody-dependent responsiveness. Although KIRs are more frequently expressed on CD57 NK cells, the presented data suggest education and differentiation make independent contributions to NK cell anti-HIV envelope antibody-dependent activation.
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http://dx.doi.org/10.1097/QAD.0000000000000489DOI Listing
November 2014

Universal immunity to influenza must outwit immune evasion.

Front Microbiol 2014 12;5:285. Epub 2014 Jun 12.

Centre for Influenza Research and School of Public Health, The University of Hong Kong Hong Kong, China.

Although an influenza vaccine has been available for 70 years, influenza virus still causes seasonal epidemics and worldwide pandemics. Currently available vaccines elicit strain-specific antibody (Ab) responses to the surface haemagglutinin (HA) and neuraminidase (NA) proteins, but these can be ineffective against serologically-distinct viral variants and novel subtypes. Thus, there is a great need for cross-protective or "universal" influenza vaccines to overcome the necessity for annual immunization against seasonal influenza and to provide immunity to reduce the severity of infection with pandemic or outbreak viruses. It is well established that natural influenza infection can provide cross-reactive immunity that can reduce the impact of infection with distinct influenza type A strains and subtypes, including H1N1, H3N2, H2N2, H5N1, and H7N9. The key to generating universal influenza immunity through vaccination is to target functionally-conserved regions of the virus, which include epitopes on the internal proteins for cross-reactive T cell immunity or on the HA stem for broadly reactive Ab responses. In the wake of the 2009 H1N1 pandemic, broadly neutralizing antibodies (bnAbs) have been characterized and isolated from convalescent and vaccinated individuals, inspiring development of new vaccination techniques to elicit such responses. Induction of influenza-specific T cell responses through vaccination has also been recently examined in clinical trials. Strong evidence is available from human and animal models of influenza to show that established influenza-specific T cell memory can reduce viral shedding and symptom severity. However, the published evidence also shows that CD8(+) T cells can efficiently select immune escape mutants early after influenza virus infection. Here, we discuss universal immunity to influenza viruses mediated by both cross-reactive T cells and Abs, the mechanisms of immune evasion in influenza, and propose how to counteract commonly occurring immune-escape variants.
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http://dx.doi.org/10.3389/fmicb.2014.00285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054793PMC
June 2014