Anti-NP IgG1/IgG2a Ab ratios in IFN-?/? mice were significantly higher than the ratios determined in WT mice (Fig. nucleoprotein in IFN-?/? mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN- does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus. Protection against challenge with influenza viruses of the same strain is normally attributed to neutralizing antibodies specific for two viral membrane glycoproteins, hemagglutinin and neuraminidase. Due to periodic antigenic shifts in these two glycoproteins, virus-neutralizing antibodies induced by primary infection fail to protect from secondary infection with a different subtype, and cross-protection between different subtypes of influenza A virus is mediated by heterosubtypic immunity (HSI) (30). Although the precise effector mechanisms for HSI remain undefined, HSI is thought to be mediated by subtype cross-reactive cytotoxic T lymphocytes (CTL) (34, 36, 41, 47). These CTL recognize conserved epitopes of internal proteins, such as nucleoprotein (NP) or matrix protein shared by influenza A virus subtypes. Passive transfer of large numbers of in vitro-activated T cells possessing subtype-specific cytotoxic activity to influenza virus-infected mice can reduce pulmonary influenza virus titers, promote their recovery, and provide protection against infection under certain circumstances (6, 18, 20, 33, 39, 44, 45). More recently, we have reported that antigen-specific CTL responses induced in mediastinal lymph nodes (MLN), a type of mucosa-associated lymphoid tissue (MALT), are associated with host recovery from lethal infection with heterosubtypic influenza A virus (25). The CTL responses d-Atabrine dihydrochloride may control virus infection through direct lysis of infected cells or by secretion of antiviral cytokines such as gamma interferon (IFN-) and tumor necrosis factor alpha (TNF-) (for a review, see reference 13). While the direct lysis of virus-infected cells by CTL is thought to control infection with noncytopathic virus, secretion of antiviral cytokines by CTL is considered more effective in control of infection by cytopathic d-Atabrine dihydrochloride viruses such as influenza virus. IFN- exerts pleiotropic effects, including direct antiviral activity and stimulation of antiviral immune responses. In virally infected cells, IFN- induces synthesis of proteins and enzymes that inhibit viral replication by impairing accumulation of virus-specific mRNA, double-stranded RNA, and proteins (26). In addition, IFN- stimulates antiviral immune responses by upregulating major histocompatibility complex (MHC) molecules on antigen-presenting cells (4), augmenting the proteolysis and peptide transport machinery in antigen-presenting cells (28, 42), and activating immune cells (5, 21). IFN- plays a protective role Tmem10 against infections by vaccinia (12, 15), herpes simplex (32), cytomegalovirus (11), murine hepatitis virus 3 (19), lymphocytic choriomeningitis virus (16), and adenovirus (43). Interestingly, IFN- is not necessary for recovery from primary infection with influenza virus (7) but has been reported to play d-Atabrine dihydrochloride a role in HSI (1). However, the latter study employed immunization as well as challenge protocols distinct from those routinely used for d-Atabrine dihydrochloride studies of HSI (3, 17, 25, 30, 46). Thus, the precise role of IFN- in HSI has not been unambiguously elucidated. Antigen-specific MHC class I-restricted CTL secrete IFN- upon antigenic stimulation (14, 22), and differentiation to CTL effector cells is dependent on the action of IFN- (21, 31). The mutual interaction between antigen-specific CTL responses and IFN- and the association of CTL responses in HSI led d-Atabrine dihydrochloride us to assess this in mice deficient in IFN-. In this report, the effect of IFN- on induction of cellular and humoral immune responses and HSI to lethal influenza challenge was systematically studied using IFN- gene-deleted (IFN-?/?) mice. MATERIALS AND METHODS Viruses. Influenza virus strains A/Udorn/307/72 (H3N2) (henceforth A/Udorn) (a gift from Brian Murphy, National Institutes of Health, Bethesda, Md.) and A/PR/8/34 (H1N1) (a gift from Thomas M. Moran, Mount Sinai School of Medicine, New York, N.Y.) were prepared as previously reported (25). Mouse-adapted virus A/PR/8/34 (H1N1), prepared as lung homogenates of intranasally infected mice, was used for a challenge. Mice. Six-week-old female BALB/c ((CD8) alleles. These Abs can inhibit T-cell-mediated cytolysis in the absence of complement (29). Lung tissue extracts for cytokine analysis. Lung tissue extracts were prepared as described by others (9). Intact lungs were collected for assessment of cytokines characteristic of Th1- or Th2-type responses. Prior to lung removal,.