CD8+ CTL are the main effector cells responsible for resolving viral infections. However, the CTL response to respiratory syncytial virus (RSV) infection in mice facilitates viral clearance at the expense of significant immunopathology. Previous reports have shown a strong correlation between the mechanism of CTL activity and the severity of RSV-induced illness. Furthermore, experiments in perforin knockout mice revealed that antiviral cytokine production temporally correlated with RSV-induced illness. In the current study, we show that TNF-α is the dominant mediator of RSV-associated illness, and it is also important for clearance of virus-infected cells during the early stages of infection. We also demonstrate that IFN-γ plays a protective role in conjunction with perforin/granzyme-mediated killing. Preliminary experiments in gld mice that express nonfunctional Fas ligand (FasL) revealed that RSV-induced illness is significantly reduced in the absence of FasL-mediated killing. Antiviral cytokine production was not elevated in the absence of FasL, suggesting a possible link between FasL and antiviral cytokine activity. This work shows that multiple phenotypic subsets of CD8+ CTLs respond to RSV infection, each with varying capacities for clearance of virus-infected cells and the induction of illness. In addition, the revelation that TNF-α is the principal mediator of RSV-induced illness means that administration of TNF receptor antagonists, in combination with antiviral therapy, may be an effective method to treat RSV infections.

One question that continues to challenge influenza A research is why some strains of virus are so devastating compared to their more mild counterparts. We approached this question from an immunological perspective, investigating the CD8 ؉ T cell response in a mouse model system comparing high-and low-pathological influenza virus infections. Our findings reveal that the early (day 0 to 5) viral titer was not the determining factor in the outcome of disease. Instead, increased numbers of antigen-specific CD8؉ T cells and elevated effector function on a per-cell basis were found in the low-pathological infection and correlated with reduced illness and latertime-point (day 6 to 10) viral titer. High-pathological infection was associated with increased PD-1 expression on influenza virus-specific CD8؉ T cells, and blockade of PD-L1 in vivo led to reduced virus titers and increased CD8 ؉ T cell numbers in high-but not lowpathological infection, though T cell functionality was not restored. These data show that high-pathological acute influenza virus infection is associated with a dysregulated CD8 ؉ T cell response, which is likely caused by the more highly inflamed airway microenvironment during the early days of infection. Therapeutic approaches specifically aimed at modulating innate airway inflammation may therefore promote efficient CD8 ؉ T cell activity. IMPORTANCEWe show that during a severe influenza virus infection, one type of immune cell, the CD8 T cell, is less abundant and less functional than in a more mild infection. This dysregulated T cell phenotype correlates with a lower rate of virus clearance in the severe infection and is partially regulated by the expression of a suppressive coreceptor called PD-1. Treatment with an antibody that blocks PD-1 improves T cell functionality and increases virus clearance.

Cytotoxic T lymphocytes (CTL) play a significant role in the clearance of respiratory syncytial virus (RSV) infection in humans and mice. Identification of class I MHC-restricted CTL epitopes is critical in elucidating mechanisms of CTL responses against viral infections. However, only four H-2d-restricted epitopes have been reported in mice. Because of the diversity of transgenic and knockout mice available to study immune responses, new epitopes in additional strains of mice must be identified. We therefore attempted to discover novel CTL epitopes in C57Bl/6 mice. Our efforts revealed a new H-2D(b)-restricted CTL epitope from the RSV M protein, corresponding to aa 187-195 (NAITNAKII). Also, M187-195-specific CTLs were activated with kinetics similar to the immunodominant BALB/c epitope, M2 82-90. This is the first RSV-specific CTL epitope described in a strain of mice other than BALB/c. Furthermore, identification of this H-2b-restricted CTL epitope provides access to genetically modified H-2b mice for more detailed studies of CTL mechanisms in RSV infection.

Virus-specific cytotoxic T lymphocytes are key effectors for the clearance of virus-infected cells and are required for the normal clearance of respiratory syncytial virus (RSV) in mice. Although perforin/granzymemediated lysis of infected cells is thought to be the major molecular mechanism used by CD8 ؉ cytotoxic T lymphocytes for elimination of virus, its role in RSV has not been reported. Here, we show that viral clearance in perforin knockout (PKO) mice is slightly delayed but that both PKO and wild-type mice clear virus by day 10, suggesting an alternative mechanism of RSV clearance. Effector T cells from the lungs of both groups of mice were shown to lyse Fas (CD95)-overexpressing target cells in greater numbers than target cells expressing low levels of Fas, suggesting that Fas ligand (CD95L)-mediated target cell lysis was occurring in vivo. This cell lysis was associated with a delay in RSV-induced disease in PKO mice compared to the time of disease onset for wild-type controls, which correlated with increased and prolonged production of gamma interferon and tumor necrosis factor alpha levels in PKO mice. We conclude that while perforin is not necessary for the clearance of primary RSV infection, the use of alternative CTL target cell killing mechanisms is less efficient and can lead to enhanced disease.

The introduction of a new influenza strain into human circulation leads to rapid global spread. This review summarizes innate and adaptive immunity to influenza viruses, with an emphasis on T-cell responses that provide cross-protection between distinct subtypes and strains. We discuss antigenic variation within T-cell immunogenic peptides and our understanding of pre-existing immunity towards the pandemic A(H1N1) 2009 strain.

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CD8+ cytotoxic T lymphocytes are key effectors of adaptive immunity for the control of virus infections. Epitope-specific responses are hierarchical and the rules for dominance are not well defined. Here we show that the H2-Kd-restricted RSV M2(82-90) (KdM2(82-90)) epitope dominates the H2-Db-restricted RSV M187-195 (DbM187-195) epitope and influences epitope-specific effector function in the acute and memory phases of the immune response to primary RSV infection in H-2b/d hybrid mice. The hybrid mouse model provides a system to define rules of epitope hierarchy and better understand how antigen presentation and epitope competition affect the phenotype of effector and memory T cells.

Influenza virus infections carry a high public health cost, and pandemics are potentially catastrophic. Though the ferret is generally regarded as the best model for human influenza, no reagents are available for the analysis of cellular immunity. We thus screened monoclonal antibodies (mAbs) made for identifying immune cells in other species to see if any were cross-reactive. Flow cytometric analysis of lymphocytes isolated from blood, spleen, and lung of normal and virus-infected ferrets indicated that several mouse mAbs bound to the corresponding antigens in ferrets. Typing bronchoalveolar lavage populations from pneumonic ferrets with mAb to human CD8 showed the massive CD8+ T cell enrichment characteristic of this infection in mice. The availability of this, and several other mAbs that showed cross-reactivity, should allow us to begin the dissection of cellmediated immunity in the ferret, which, at least from these early results, looks similar to the situation in mice.