Memory CD4 T Cells Direct Protective Responses to Influenza Virus in the Lungs through Helper-Independent Mechanisms

Memory CD4 T cells specific for influenza virus are generated from natural infection and vaccination, persist long-term, and recognize determinants in seasonal and pandemic influenza virus strains. However, the protective potential of these long-lived influenza virus-specific memory CD4 T cells is not clear, including whether CD4 T-cell helper or effector functions are important in secondary antiviral responses. Here we demonstrate that memory CD4 T cells specific for H1N1 influenza virus directed protective responses to influenza virus challenge through intrinsic effector mechanisms, resulting in enhanced viral clearance, recovery from sublethal infection, and full protection from lethal challenge. Mice with influenza virus hemagglutinin (HA)-specific memory CD4 T cells or polyclonal influenza virus-specific memory CD4 T cells exhibited protection from influenza virus challenge that occurred in the presence of CD8-depleting antibodies in B-cell-deficient mice and when CD4 T cells were transferred into lymphocyte-deficient RAG2^−/− mice. Moreover, the presence of memory CD4 T cells mobilized enhanced T-cell recruitment and immune responses in the lung. Neutralization of gamma interferon (IFN-γ) production in vivo abrogated memory CD4 T-cell-mediated protection from influenza virus challenge by HA-specific memory T cells and heterosubtypic protection by polyclonal memory CD4 T cells. Our results indicate that memory CD4 T cells can direct enhanced protection from influenza virus infection through mobilization of immune effectors in the lung, independent of their helper functions. These findings have important implications for the generation of universal influenza vaccines by promoting long-lived protective CD4 T-cell responses.Influenza virus poses substantial threats to world health due to the emergence of new pandemic strains through viral mutation and reassortment, including the 2009 H1N1 pandemic strain. Developing effective vaccines that can provide immune-mediated protection to multiple influenza virus strains remains a major challenge, as current vaccines generate neutralizing antibodies directed against the highly variable hemagglutinin (HA) and neuraminidase (NA) surface viral glycoproteins (18). These vaccines are only partially effective at protecting individuals from succumbing to seasonal strains and are largely ineffective at protecting individuals from new pandemics. In contrast, T lymphocytes have the potential to provide long-term cross-strain protection, through their recognition of invariant viral determinants (3, 9), generation of effector responses to coordinate both cellular and humoral immunity, and development of memory populations that persist for decades (34). In humans, influenza virus-specific CD4 and CD8 T cells recognize internal polymerase, matrix, and nucleoprotein components of influenza virus which are conserved in multiple strains (3). Influenza virus-specific memory T cells generated from virus exposure and vaccines can be detected readily in the peripheral blood of healthy older children and adults (16, 30). Elucidating the protective capacities of memory T cells in antiviral immunity and their underlying mechanisms is therefore crucial to understanding clinical responses to influenza and to developing strategies to boost T-cell-mediated immunity for the next emerging pandemic.The potent cytolytic responses of virus-specific CD8 T cells and their roles in antiviral primary and secondary responses have been well established (58); however, considerably less is known about the function of memory CD4 T cells in antiviral immunity. Memory CD4 T cells have the potential to play more diverse roles in coordinating secondary responses than those of memory CD8 T cells via their ability to “help” or promote cellular and humoral immunity, and also through direct effector functions. Compared to CD8 T-cell responses, memory CD4 T-cell responses in humans were found to recognize a more diverse array of influenza virus-specific epitopes (46-48) and to exhibit cross-reactivities with new pandemic strains, including avian H5N1 and 2009 H1N1 “swine flu” strains (23, 28, 36, 48). In addition, antiviral memory CD4 T cells generated as a result of influenza vaccination (22) were found to persist longer than CD8 T cells in vivo following smallpox vaccination (29). These findings suggest that memory CD4 T-cell responses could be potential targets for boosting long-term cellular immunity following vaccination, although their protective capacity remains undefined.The role of CD4 T cells in anti-influenza virus immunity has been elucidated mainly for primary responses, and less is known about the protective potential and mechanisms underlying memory CD4 T-cell-directed secondary responses. In primary influenza virus infection, CD4 T cells promote antibody production by B cells necessary for complete viral clearance (2, 17, 19, 39, 40, 57) and also promote the generation of memory CD8 T cells (4). Whether memory CD4 T cells have a similar helper-intensive role in promoting B cells and CD8 T cells in secondary influenza responses or whether effector responses predominate is not known. In this study, we investigated the mechanisms by which memory CD4 T cells mediate secondary responses and promote recovery from influenza virus infection in the clinically relevant scenario of a persisting CD4 T-cell response but no preexisting antibody response to a new influenza virus strain. We demonstrate that both influenza virus HA-specific and polyclonal influenza virus-specific memory CD4 T cells direct rapid lung viral clearance and protect from lethality via secondary antiviral responses in the absence of CD8 T cells, B cells, or any lymphocytes. Unlike primary responses to influenza virus, which can mediate protection independent of gamma interferon (IFN-γ), memory CD4 T-cell-mediated protection in the lung is dependent on secreted IFN-γ and is associated with localized interactions with lung airways and foci of T-cell-directed responses. Our findings reveal that memory CD4 T cells drive antiviral protection in the lung through a qualitatively distinct mechanism and have important implications for exploiting the protective role of persisting memory CD4 T cells in vaccines and immunotherapies.     

Memory T Cells Generated by Prior Exposure to Influenza Cross React with the Novel H7N9 Influenza Virus and Confer Protective Heterosubtypic Immunity

Influenza virus is a source of significant health and economic burden from yearly epidemics and sporadic pandemics. Given the potential for the emerging H7N9 influenza virus to cause severe respiratory infections and the lack of exposure to H7 and N9 influenza viruses in the human population, we aimed to quantify the H7N9 cross-reactive memory T cell reservoir in humans and mice previously exposed to common circulating influenza viruses. We identified significant cross-reactive T cell populations in humans and mice; we also found that cross-reactive memory T cells afforded heterosubtypic protection by reducing morbidity and mortality upon lethal H7N9 challenge. In context with our observation that PR8-primed mice have limited humoral cross-reactivity with H7N9, our data suggest protection from H7N9 challenge is indeed mediated by cross-reactive T cell populations established upon previous priming with another influenza virus. Thus, pre-existing cross-reactive memory T cells may limit disease severity in the event of an H7N9 influenza virus pandemic.     

Suppression by Thimerosal of Ex-Vivo CD4+ T Cell Response to Influenza Vaccine and Induction of Apoptosis in Primary Memory T Cells

Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine. Thimerosal was included in the multidose non-adjuvanted pandemic 2009 H1N1 vaccine Panenza. In the context of the analysis of the ex-vivo T cell responses directed against influenza vaccine, we discovered the in vitro toxicity Panenza, due to its content in thimerosal. Because thimerosal may skew the immune response to vaccines, we investigated in detail the ex-vivo effects of thimerosal on the fate and functions of T cells in response to TCR ligation. We report that ex-vivo exposure of quiescent or TCR-activated primary human T cells to thimerosal induced a dose-dependent apoptotic cell death associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, cytochrome c release from the mitochondria and caspase-3 activation. Moreover, exposure to non-toxic concentrations of thimerosal induced cell cycle arrest in G0/G1 phase of TCR-activated T cells, and inhibition of the release of proinflammatory cytokines such as IFN gamma, IL-1 beta, TNF alpha, IL-2, as well as the chemokine MCP1. No shift towards Th2 or Th17 cells was detected. Overall these results underline the proapoptotic effect of thimerosal on primary human lymphocytes at concentrations 100 times less to those contained in the multidose vaccine, and they reveal the inhibitory effect of this preservative on T-cell proliferation and functions at nanomolar concentrations.     

The Recent Establishment of North American H10 Lineage Influenza Viruses in Australian Wild Waterfowl and the Evolution of Australian Avian Influenza Viruses

Influenza A H10N7 virus with a hemagglutinin gene of North American origin was detected in Australian chickens and poultry abattoir workers in New South Wales, Australia, in 2010 and in chickens in Queensland, Australia, on a mixed chicken and domestic duck farm in 2012. We investigated their genomic origins by sequencing full and partial genomes of H10 viruses isolated from wild aquatic birds and poultry in Australia and analyzed them with all available avian influenza virus sequences from Oceania and representative viruses from North America and Eurasia. Our analysis showed that the H10N7 viruses isolated from poultry were similar to those that have been circulating since 2009 in Australian aquatic birds and that their initial transmission into Australia occurred during 2007 and 2008. The H10 viruses that appear to have developed endemicity in Australian wild aquatic birds were derived from several viruses circulating in waterfowl along various flyways. Their hemagglutinin gene was derived from aquatic birds in the western states of the United States, whereas the neuraminidase was closely related to that from viruses previously detected in waterfowl in Japan. The remaining genes were derived from Eurasian avian influenza virus lineages. Our analysis of virological data spanning 40 years in Oceania indicates that the long-term evolutionary dynamics of avian influenza viruses in Australia may be determined by climatic changes. The introduction and long-term persistence of avian influenza virus lineages were observed during periods with increased rainfall, whereas bottlenecks and extinction were observed during phases of widespread decreases in rainfall. These results extend our understanding of factors affecting the dynamics of avian influenza and provide important considerations for surveillance and disease control strategies.     

Establishment of a Feline T-Lymphoblastoid Cell Line Susceptible to Feline Immunodeficiency Viruses

A feline T-lymphoblastoid cell line susceptible to feline immunodeficiency viruses (FIV) was established and designated as Yu-1 cells. Yu-1 cells continued to grow over one year with more than 100 successive passages in the presence of human recombinant interleukin-2. Surface antigens of Yu-1 cells were feline Pan-T+, CD4+, and CD8. Susceptibility of Yu-1 cells to FIV strains were higher than that of the primary culture of the feline peripheral blood mononuclear cells, indicating that this cell line should be useful for isolation, propagation, and neutralization test of FIV.     

T细胞记忆的理论研究

基于CD8⁺ T记忆细胞的线性和逆线性分化假说分别建立了数学模型,并研究了各种T细胞亚类的动力学.发现在优化剂量抗原入侵的条件下,两个模型均能产生记忆,并可较好地模拟实验结果.通过进一步模拟发现CD8⁺ T细胞记忆与抗原的存在紧密相关,再次证实了抗原在维持T细胞记忆中的作用.另外还讨论了记忆细胞寿命的问题.认为逆线性假说具有更强的反应性和记忆性.     

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.     

Recombinant Yellow Fever Viruses Elicit CD8+ T Cell Responses and Protective Immunity against Trypanosoma cruzi

Chagas’ disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF) 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2). The cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. The replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc) was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-γ) producing-cells against the YF virus. Also, it was able to prime a CD8⁺ T cell directed against the transgenic T. cruzi epitope (TEWETGQI) which expanded significantly as measured by T cell-specific production of IFN-γ before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. The superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-γ-producing T CD8⁺ cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general.