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Influenza hemagglutinin (HA)-specific T-Cell-Receptor (TCR) transgenic mice sustain the influenza virus infection of an inoculum size which is fatal in wild type mice. There is a prominent IL-17 response of the HA-specific TCR transgenic CD4+ T cells in late phase of infection in the transgenic mice. We have demonstrated that the late phase IL-17 response is a de novo response of naïve T cells under guidance of the Th1 cells with influenza viral neuraminidase-activated TGF-β. There is also a late phase up-regulation of epidermal growth factor receptor (EGFR) on the transgenic CD4+ T cells in the lungs. EGFR inhibition with Gefitinib resulted in impaired sustainability of the infection of the transgenic mice. They suffered from profound body weight loss and severe disease with significant mortality. IL-17-induced EGFR-mediated signaling cascade has been documented in skin stem cells that are involved in wound healing. We will further dissect the role of this cascade in alleviation of lung inflammation and recovery from the disease during the late phase of acute influenza virus infection.

The 6.5 CD4+ T cell receptor (TCR) transgenic mice have a monotonous CD4+ TCR which responds to the hemagglutinin (HA) antigen of PR8 influenza virus. Naive 6.5 CD4+ T cells adoptively transferred into wild type mice were activated by PR8 influenza virus with Th1 response. With PR8 influenza virus infection, all CD4+ T cells in 6.5 mice were activated and they were predominantly Th1 in phenotype at the early phase of infection and Th17 at the late phase. Th1 effector responses made infected mice sick and even succumb to death. The 6.5 mice were sicker at the early phase but recovered with better survival at last. IL-17KO 6.5 mice cannot display Th17 responses and lost the survival benefit as well. IL-17 supplement at the late phase of infection enhanced survival of wild type mice with PR8 influenza virus infection. With consecutive two adoptive transfers of naive 6.5 CD4+ T cells, 6.5 CD4+ T cells of the first transfer evolved into Th1 cells but 6.5 CD4+ T cells of the second transfer demonstrated a de novoTh17 response. Thymectomy or aging in 6.5 mice limited the thymic output of naive 6.5 CD4+ T cells and diminished the Th17 response. Viral neuraminidase activated TGF-β in the Th1 6.5 CD4+ T cells at the early phase. T-bet dominance was altered with excessive ROR-γt activation in the following responding 6.5 CD4+ T cells. They have substantial IL-17 with restrained IFN-γ production. We demonstrated a Th1 guided evolution of protective Th17 response in severe influenza.

Bacterial superinfection aggravates the disease of influenza virus infection. Streptococcus pneumoniae is a frequently encountered bacterial pathogen. Superinfection with neuraminidase (Nan) C deficient wild type pneumococcus, which expresses NanA and NanB only, suppressed antigen-specific anti-influenza immunity, with impaired viral clearance. There was augmented TGF-β activation in the lung-infiltrating influenza hemagglutinin (HA)-specific CD4+ T cells. Mice suffered from exacerbated disease and died with higher virus loads in the lung. We modified this wild type strain with NanC insertion. This modified strain expresses all neuraminidases A, B and C. Superinfection with this NanC inserted pneumococcus reverted the immune-suppressive effect of the wild type pneumococcus with NanA and NanB only. There were augmented inflammatory cytokines IFN-γ and TNF-α production and suppressed TGF-β activation in the lung-infiltrating influenza HA-specific CD4+ T cells. The pro-inflammatory response enhanced viral clearance. Even the virus eradication is enhanced, the mice actually suffered from even more severe disease. Our results demonstrated the important role of NanC-mediated inflammation in the disease of severe influenza with pneumococcal superinfection.

Naïve CD4+ T cells respond in influenza with T-bet expression, a transcription factor up-regulated with Th1 commitment. We report concomitant LAG-3 expression on these Th1 committed cells with evolution into regulatory T cells. LAG-3 expression increases with activation as revealed by CFSE cell division study. Cells with moderate LAG-3 expression produce IFN-γ and contribute to inflammation and disease. They no longer produce IFN-γ with higher LAG-3 expression upon further activation. High LAG-3 cells suppress and decouple effector function from the preserved proliferation of CD8+ T cells. They restrain inflammation with facilitated viral clearance and ameliorate the disease of severe influenza. Foxp-3+CD4+ T cells suppress both effector and proliferative responses with impaired viral clearance and they are less potent in disease amelioration. Principal component analysis of genome-wide expression further confirmed the stepwise evolution from effector to regulatory T cells. Regulatory genes are up-regulated progressively from LAG-3Neg to LAG-3Med and to LAG-3High cells.

T-bet induced CD4+ T cells are committed to be IFN-γ competent Th1 cells. The Th1 response is essential for virus eradication, but is also a major cause of inflammation and disease aggravation in severe influenza. In our influenza hemagglutinin (HA) Ag-specific mouse experimental system, naïve CD4+ T cells respond to the infection with T-bet expression. However, they suppress cognate CD8+ T cell effector response-mediated inflammation and alleviate the disease. Here we report concomitant LAG-3 expression and IFN-γ competence in Th1 committed CD4+ T cells. LAG-3 expression increases with activation by virus as revealed by CFSE based cell division study. The Th1 committed cells with moderate LAG-3 expression produce IFN-γ and contribute to inflammation and aggravated disease. They can no longer produce IFN-γ with higher LAG-3 expression upon further activation. Th1 committed CD4+ T cells with high LAG-3 expression suppress cognate CD8+ T cells by decoupling their effector function from proliferative response, restrain lung inflammation and facilitate viral clearance. They are better than Foxp-3+HA-specific CD4+ T cells in disease amelioration. The Foxp-3+ cells suppress both effector function and proliferative response of CD8+ T cells. They moderate inflammation but impair viral clearance. Principal component analysis of genome-wide gene expression profiles further confirmed the stepwise evolution. Several regulatory function-associated genes such as Lag3, Pdcd1, Tigit, Ctla2a, Nr4a2, Klrc1, Rgs16, Sytl3, Il21, Il1r2 and Dusp4 are up-regulated graduallly as well from LAG-3Neg to LAG-3Med and LAG-3High cells.

Induced regulatory T cells (iTreg) is an adaptive response in inflammation with disease processes such as infection, autoimmunity or cancer. A small number of iTreg may exert long-lasting regulation. Here we report the LAG-3+ iTreg transfer regulatory activity to the CD4+ T cells they suppress. This infectious propagation of regulatory activity exemplify a dominant immune regulation in vivo. Naïve hemagglutinin (HA) antigen-specific CD4+ T cells are induced to differentiate into IFN-γ competent effector T cells and then LAG-3+ Tregs in HA transgenic mice inherently expressing the cognate HA antigen. The LAG-3+ iTreg suppresses activation of effector function of cognate naïve CD4+ T cells in HA transgenic mice, with silenced IFN-γ machinery including STAT1 phosphorylation and T-bet induction. The LAG-3 iTreg suppressed, IFN-γ disarmed CD4+ T cells still proliferate and acquire LAG-3 expression on their surface in vivo. They become the next generation LAG-3+ iTregs with the ability of suppression of activation of effector function of cognate naïve CD4+ T cells in HA transgenic mice, with the same mechanism of silenced IFN-γ machinery. LAG-3+ iTreg exercises regulation in vivo by suppression of effector function decoupled from reserved proliferative response. The effector function suppressed CD4+ T cells still proliferate upon activation and acquire LAG-3+ iTreg phenotype, a reminiscence of the infectious tolerance proposed in 1970.

Cytokine storm has been postulated as one of the major causes of mortality in patients with severe respiratory viral infections such as influenza. With the help of an influenza Ag- specific mouse experimental system, we report that CD4+ T cells contribute effector cytokines leading to lung inflammation in acute influenza. Although virus can no longer be detected from tissues 14 d postinfection, virus-derived Ag continues to drive a CD4+ T cell response after viral clearance. Ag-specific CD4+ T cells proliferate and evolve into memory CD4+ T cells efficiently, but the production of effector cytokines is seriously hampered during this phase. This decoupling of proliferation and effector cytokine production doesn’t appear in conjunction with increased suppression by regulatory T cells or decreased induction of transcription factors. Rather, GATA-3 and ROR-γt levels are elevated when compared with cells that have effector cytokine production. T-bet dominance over GATA-3 and ROR-γt decreases with the disarmament of effector cytokine production. Importantly, upon reinfection, these decoupled cells produce elevated levels of IFN-γ and were effective in virus eradication. These results provide a mechanism through altered T-bet dominance to dampen the cytokine storm without impeding the generation of memory T cells in influenza virus infection.

Some healthy individuals suffer from severe disease and even mortality with the same strain of influenza virus that causes self-limited illnesses in the majority of people. This fact exemplifies the importance of host factors in influenza disease manifestation. Our C3-HA transgenic mice express hemagglutinin (HA) as a self-antigen that is identical to the HA of PR8 influenza virus. Compared to wild type mice, C3-HA mice incurred severe disease with PR8, but not with HA-mismatched influenza virus. Self-HA in C3-HA mice altered the immune response to the infection and impaired clearance of the influenza virus. However, different mechanisms contribute for exacerbated disease in C3-HA mice with different level of HA. In C3-HALow mice, low-level self-HA modulated HA specific immunity upon infection with less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production of both HA-specific CD4+ and CD8+ T cells on day 4- post infection. The T cell activation status contracted further on day 7- post infection. Impaired immunity resulted in delayed virus clearance leading to severe disease and death. In C3-HAHigh mice, high-level self-HA also modulated impaired immunity and virus clearance on day 4- post infection. In contrast to further reduction on day 7- post infection in C3-HALow mice, the HA specific T cell immunity was higher on day 7- than day 4- post infection in C3-HAHigh mice. The boosted HA specific immunity resulted in severe disease with autoimmunity as a predominant feature in pathology. Our animal model exemplified the complicated interaction between cross-reactive self-antigen and influenza virus with severe disease as the results of antigen level dependent distinct mechanisms.

Streptococcus pneumoniae often causes superinfection in influenza and exacerbates the disease with more severity and even mortality. In our influenza hemagglutinin (HA) antigen-specific transgenic mouse model, mice suffered from severe disease and died with increased virus titer and pathology in the lungs upon serotype-3 Streptococcus pneumoniae superinfection on day 4- post PR8 strain H1N1 influenza virus infection. Same dose viral or bacterial infection caused mild disease in control mice. Streptococcus pneumoniae supreinfection attenuated anti-influenza T cell immunity in the lungs. There was less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production in HA-specific CD4+ T cells on day 2- and 3- post bacterial superinfection, with impaired viral clearance. Streptococcus pneumoniae strains with different combinations of bacterial neuraminidase (NA) demonstrated varied degrees of T cell immunity attenuation. Streptococcus pneumoniae with NA-B only was the most suppressive with up-regulated active TGF-β and down-regulated IFN-γ expression in HA-specific CD4+ T cells, followed by those with both NA-A and NA-B. Streptococcus pneumoniae with both NA-A and NA-B and inserted NA-C was least suppressive. With in vitro experiments, recombinant bacterial NA-A or NA-B augmented TGF-β and attenuated IFN-γ in HA-specific CD4+ T cells in overnight culture with PR8 virus. Recombinant bacterial NA-C mitigated TGF-β and increased IFN-γ in HA-specific CD4+ T cells. Our results demonestrates distinct immune alteration by different bacterial neuraminidases, which may play a significnt role of disease manifestation in influenza with bacterial superinfection.

The immune system responds to influenza virus infection for eradication of the virus but the associated inflammation may cause tissue damage to the host. In our antigen-specific mouse system, naïve influenza hemagglutinin (HA)-specific CD4+ T cells adoptively transferred with infection respond with Th1 phenotype and produce IFN-γ. Some of them secrete IL-17 as well. Second batch HA specific CD4+ T cells, adoptively transferred on day 4 after infection and the first transfer, respond to the infection with Th17 phenotype. They produce IL-17 but not IFN-γ. Both the Th1 and Th17 cells are activated with similar levels of ZAP-70 phosphorylation and T-bet induction. For the Th17 cells, IFN-γ deficiency is associated with excessive ROR-γt induction and altered T-bet dominance. Th17 cells display surface LAG-3 expression, suppress T cell activation in vitro and decrease lung inflammation in vivo. The evolution of Th17 cells is augmented in IL-10 deficiency, with IL-10 knockout mice as the recipients transferred with IL-10 knockout HA specific CD4+ T cells. There is increased neuraminidase-mediated TGF-β activation in this IL-10 deficient environment. Viral neuraminidase mediated TGF-β activation in the first batch of HA specific CD4+ T cells facilitates the Th17 evolution of the 2nd batch of cells. The 6.5 T cell receptor (TCR) transgenic mice of monotonous HA specific TCR repertoire respond to influenza virus infection with Th17 deviation and demonstrate survival benefit. Consecutive waves of naïve HA specific T cells from the thymus in 6.5 mice may evolve with mechanisms similar to consecutive adoptive transfers. Evolution of protective Th17 immunity is facilitated by neuraminidase-mediated TGF-β activation.

Influenza virus infection causes mild and self-limiting illness in most of the healthy individuals, but some healthy people develops complications as a result of the infection and suffers from severe disease and eventually dies. Here we report that pre-existing matched antigens attenuate antigen-specific immunity to the infection. High virus load and associated increase of pathogenic innate response in the lungs exacerbates the disease. The C3-HAHigh and C3-HALow transgenic mice are with inherent expression of Hemagglutinin (HA) antigen from PR8 strain of H1N1 influenza virus. Compared to wild type mice, these mice suffered from severe disease and higher mortality upon infection of wild type PR8 virus but not upon infection of reverse genetically raised PR8 virus with replaced HA from H3N2 influenza virus. Adoptively transferred PR8 virus HA-specific 6.5 CD4+ and Clone-4 CD8+ T cells responded minimally to PR8 virus infection in C3-HA mice. There was less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production of both 6.5 CD4+ and Clone-4 CD8+ T cells. Virus neutralizing antibodies were less as well. The attenuation was associated with increased LAG-3+ but not Foxp-3+ population of HA-specific T cells in the lungs. In contrast to the instigated weak antigen-specific immunity, PR8 virus infection caused neutrophils more efficient for inflammatory IL-6 secretion and robust bronchus infiltration in C3-HA mice. These results suggest that a host factor cross-reactive to the antigens of influenza virus may exacerbate the disease with similar attenuated antigen-specific adaptive immunity accompanied by augmented innate immunity to influenza virus infection.

The host immune system responds to influenza virus infection for eradication of the virus but the immune response associated inflammation causes tissue injury and damage to the host as well. In our antigen-specific mouse model system for influenza virus infection, influenza hemagglutinin antigen-specific CD4+ T cells responded to acute infection with Th1 effector phenotype. A significant proportion of them were also with concurrent expression of LAG-3, CTLA-4, GITR or CD25. Adoptive transfer of purified LAG-3 expressing hemagglutinin antigen-specific CD4+ T cells controlled lung inflammation with acute influenza virus infection, as represented by the activation of co-transferred influenza hemagglutinin antigen-specific CD8+ T cells. Control of the inflammation by LAG-3 expressing antigen-specific Th1 CD4+ T cells was not necessarily associated with impaired capacity of virus eradication. These results reveal, besides effector function for virus eradication, subsets of CD4+ T cells activated by acute influenza virus infection also acquired regulatory function to attenuate inflammation for reducing the damage to the host. These subsets of CD4+ T cells may be employed as the practical handle for management of severe influenza disease.

Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and antigen-specific T cell response in the lung. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8 specific T cell immune response in the lung and cannot prevent infection with PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lung but cannot prevent infection with nH1N1 virus either. Antigen specific T cell immunity is required for sterilizing immunity. We discuss the potential implications of these findings in vaccination strategy for effectual protection comparable to pre-infection.

The host immune system responds to influenza virus infection for eradication of the virus but the immune response associated inflammation causes tissue injury and damage to the host as well. Severe influenza with morbidity and mortality is commonly believed to be the result of overwhelming immune response. In addition to antiviral agent, adjuvant therapy with immune modulating agent is a reasonable approach for severe influenza and has been reported to be effective in clinical series. As the immunity is also critical for virus eradication and recovery, we investigated to try to define the appropriate window for immune modulation in our murine animal model of influenza virus infection. Rapamycin treatment in a wide dose range did not alter the clinical course of severe influenza. In combination with antiviral agent Oseltamivir, rapamycin failed to offer additional benefit beyond treatment with Oseltamivir alone. Oseltamivir barely rescued the mice from severe influenza if the treatment was delayed until 48 hours after infection. Rapamycin did not provide salvage effect in this scenario. In fact, rapamycin suppressed influenza antigen specific CD4+ T cell response and impaired the virus eradication capacity. The virus burden in the lung increased with intense neutrophil infiltration and hyaline deposition in the alveoli. In conclusion, rapamycin cannot be relied upon as a salvage measure in severe influenza with progressive illness under treatment with Oseltamivir. T cell immunity is suppressed but other arms of immunity are recruited with undesirable effect on the disease outcome.

Th1 cells control their activity by producing regulatory IL-10. Here we report that Th1 cell-derived IL-10 facilitates their expansion and in addition, augments Th1 cell production of IFN-γ, TNF-α, and IL-2 during the early phase of influenza. In our antigen-specific mouse experimental system, influenza hemagglutinin-specific CD4+ T cells respond to infection with the induction of T-bet, and produce both IFN-γ and IL-10. In the early phase of infection, an abundance of viral neuraminidase causes TGF-β activation of hemagglutinin-specific CD4+ T cells. CD4+ T cell-derived IL-10 inhibits neuraminidase-driven TGF-β activation and counteracts the virus-mediated immune suppression. As the host eradicates the virus, neuraminidase activity wanes and IL-10 receptors are up-regulated on CD4+ T cells in the late phase of infection. IL-10 then suppresses immune activation and aids in recovery from infection and inflammation. These results reveal a previously unrecognized function of Th1 cell-derived IL-10 in vivo.

Overwhelming inflammation with cytokine storm has been postulated as a major cause of severe influenza disease in human. In our influenza Ag- specific mouse experimental system, we discovered a Th17 deviation of CD4+ T cell response with resultant attenuation of inflammation in the late phase of influenza infection. Naïve hemagglutinin (HA)-specific CD4+ T cells respond to acute influenza virus infection with Th1 phenotype. In the presence of antigen specific Th1 CD4+ T cells, naïve CD4+ T cells adoptively transferred 4 days after the acute infection are activated with deviation to the Th17 phenotype. The second batch of naïve HA-specific CD4+ T cells is activated in an environment enriched with Th1 cytokines and active TGF-β. They proliferate, secrete IL-17 but not IFN-γ and express LAG-3 on the cell surface. They can suppress T cell activation in vitro and decrease lung inflammation in vivo. The 6.5 mice, with monotonous TCR repertoire responsive to influenza HA, reveal a Th17 deviated CD4+ T cell response and significant survival benefit in influenza virus infection. Consecutive waves of naïve CD4+ T cells from the thymus in 6.5 mice are probably activated as subsequent batches of naïve CD4+ T cells, with Th17 deviation which attenuates the inflammation in late phase of influenza virus infection.

Influenza virus invades the host while the host immune response defends to clear the virus. These responses may result in excessive inflammation with tissue injury, which is as detrimental as the invasiveness of the virus. Here we demonstrate a biphasic immune regulation by the host T cell cytokine interleukin-10. During the early phase of infection, viral neuraminidase increases with multiplication of the virus. The abundance of neuraminidase mediates a robust release of the suppressive cytokine TGF-β. Host interleukin-10 interacts with neuraminidase, counteracts the release of TGF-β, and supports the antiviral immunity. Neuraminidase activity wanes with eradication of the virus. In the late phase of the infection, interleukin-10 preferentially binds to up-regulated interleukin-10 receptors and attenuates the immune responses to avoid immune-mediated damage to the host.

IL-10 is regarded as an anti-inflammatory cytokine which prevents damage from immunopathology with exacerbated immune responses. However, severe lung inflammation in fatal human influenza is accompanied with increased levels of IL-10 as well as other pro-inflammatory cytokines. This information raises questions regarding the role IL-10 plays in inflammation associated with acute influenza. We found that IL-10 was produced by CD4+ T cells in synchronization with pro-inflammatory cytokines during an acute influenza virus infection in mice. IL-10 supplementation during the acute phase increased inflammation leading to death, while IL-10 knockout mice displayed decreased lung inflammation and survival benefits. In the absence of IL-10, influenza-specific CD4+ T cells divided less and produced less pro-inflammatory cytokines despite unaffected Th1 polarity, no increase of regulatory T cell populations, and no alteration in the kinetics of viral eradication. Influenza-specific CD4+ T cell derived IL-10 was more important than IL-10 from other sources for the detrimental effect. In this context of acute influenza in mice, IL-10 also impeded the release of mature TGF-β with decreased neuraminidase-mediated desialylation of host immune cells. The abundance of neuraminidase may contribute to the pro-inflammatory role of IL-10 we demonstrated in this context of acute influenza virus infection.

Cytokine storm has been postulated as one of the major causes of mortality in patients with severe respiratory viral infections such as influenza. With the help of an influenza antigen- specific mouse experimental system, we report that CD4+ T cells contribute effector cytokines leading to lung inflammation in acute influenza. Although virus can no longer be detected from tissues 14 days after infection, virus-derived antigen continues to drive a CD4+ T cell response after viral clearance. Antigen specific CD4+ T cells proliferate and evolve into memory CD4+ T cells efficiently, but the production of effector cytokines is seriously hampered during this phase. This decoupling of proliferation and effector cytokine production doesn’t appear in conjunction with increased suppression by regulatory T cells or decreased induction of transcription factors. Rather, GATA-3 and ROR-γt levels are elevated when compared to cells that have effector cytokine production. T-bet dominance over GATA-3 and ROR-γt decreases with the disarmament of effector cytokine production. Importantly, upon re-infection, these decoupled cells produce elevated levels of IFN-γ and were effective in virus eradication. These results provide a mechanism through altered T-bet expression to dampen the cytokine storm without impeding the generation of memory T cells in influenza virus infection.