The Recovery of Mice from Influenza A Virus Infection: Adoptive Transfer of Immunity with Influenza Virus-specific Cytotoxic T Lymphocytes Recognizing a Common Virion Antigen

Mice inoculated intranasally with infectious influenza virus of a given A strain were adoptively transferred 24 h later with preparations of secondary influenza virus-immune T cells generated either in vitro or entirely in vivo. The immune cells were raised during infection with homologous or heterologous A strain influenza viruses or with a type B virus. The greatest antiviral effect, measured by reduction in lung virus level of recipient mice, occurred if homologous viruses were used. Sharing of haemagglutinin specificity was shown to be important, but significant antiviral activity was still expressed if neither haemagglutinin nor neuraminidase antigenic specificities were shared. The antiviral effect was type-specific. Adoptive transfer of type A influenza immune T cells did not express antiviral activity against type B virus, and vice versa. On the basis of earlier work, the effector population in the transferred cells was cytotoxic T cells (Tc). Intranasal reinfection of mice with a heterologous type A virus sharing neither haemagglutinin nor neuraminidase antigenic specificity with the first infecting virus induced enhanced and earlier production of cross-reactive Tc against type A influenza viruses. This was paralleled by significantly lower virus levels in the lungs. The results of this work demonstrate heterotypic cell-mediated immunity in influenza virus infection in mice.     

Influence of type 2 T cell responses on the severity of encephalitis associated with influenza virus infection

The role of type 2 T cell responses on the severity of post-infectious encephalitis was investigated in a mouse model of influenza virus infection. When mice were infected intracerebrally with 3.0 LD(50) of A/NWS33 strain of influenza virus, they all showed clinical signs of encephalitis, and 90% of them died within 10 days of the infection. However, the post-infectious encephalitis was not demonstrated in mice exposed to 0.5 LD50 of the same virus. The mortality rates of mice infected with 0.5 LD(50) of the virus were increased to levels observed in mice exposed to 3.0 LD(50) of influenza virus infection, after the administration of a mixture of interleukin (IL)-4 and IL-10 (2 ng/mouse each; immediately, 1 and 2 days after the infection). In contrast, mortality rates of mice exposed to 3.0 LD(50) of influenza virus were substantially decreased when these mice were treated with a mixture of monoclonal antibodies directed against IL-4 and IL-10. A predominance of type 2 T cell responses was demonstrated in splenic T cells of mice infected with 3.0 LD(50) of influenza virus, although these responses were minimal in mice infected with 0.5 LD(50) of the virus. After the treatment with the mixture of type 2 cytokines, an increase in the type 2 T cell responses in mice exposed to 0.5 LD(50) of the virus was shown. These results indicate that type 2 T cell responses associated with the viral infection play an important role in the severity of post-infectious encephalitis induced in mice by the intracerebral infection of influenza A virus.     

Cell-mediated cytotoxicity following influenza infection and vaccination in humans

Cell-mediated cytotoxic activity in circulating mononuclear cells from 31 volunteers challenged with live influenza A/Victoria virus, and 22 volunteers vaccinated with inactivated influenza vaccine, was examined employing target cells infected with several viruses by means of a ⁵¹ Cr release assay. Effectors from infected volunteers, and from volunteers who manifested four-fold rises in serum HAI antibody after vaccination, demonstrated significantly elevated levels of cytotoxicity against targets infected with the homologous virus. Elevated cytotoxicity was seen by days 3 and 4 after challenge or vaccination and returned to baseline levels by day 9 to 10. In infected volunteers, cytotoxic activity was broadly directed, rising against targets infected with an antigenically distinct virus within the same influenza type (A), against targets infected with a serologically unrelated virus of a different influenza type (B), and also against cells infected with Newcastle disease virus, a paramyxovirus from another species. However, elevated levels of cytotoxicity were not observed when targets infected with herpes simplex virus, a member of an entirely different virus group, or when uninfected target cells were employed. In vaccinated volunteers, the rise in cytotoxicity was more restricted than after infection, since elevated cytotoxic activity was seen only against cells infected with the homologous virus and not against influenza B-infected cells. Fractionation of mononuclear cell populations indicated that effector cell activity is associated with T-cell depleted fractions and can only partially be reduced by depletion of adherent cells. The rapid development, short duration, and broadly directed specificity of this cytotoxic response suggest that it may be involved in early events following acute influenza infection in humans.     

Do L3T4+ T cells act as effector cells in protection against influenza virus infection

This study aimed to analyse the roles of Lyt 2+ and L3T4+ memory T-cell subpopulations in murine influenza infection. Previous work has shown that Lyt 2+ cytotoxic T-cell (Tc) clones can adoptively transfer protection. We therefore wished to see whether L3T4+ (Th) cells could also act as protective effector cells. Donors for adoptive cell transfer were thymectomized mice, depleted in vivo of either Lyt 2+ or L3T4+ T cells with monoclonal antibodies (MAb) and then infected with influenza virus (A/X31). Primed spleen cells, after removal of the B cells, were transferred into irradiated hosts infected simultaneously or persistently with a heterologous influenza virus and the effect on lung virus replication determined. Depletion of L3T4+ T cells suppressed the formation of IgG antibodies after influenza virus infection, indicating significant depletion of T-helper function. Yet Lyt 2+ class I MHC-restricted Tc cells were effectively primed in these mice, albeit to half the normal level. Adoptive transfer of the Lyt 2+ memory T cells cleared virus in a persistent infection within 6 days. Spleen cells selected for L3T4+ T cells cleared virus within 21 days of transfer in a simultaneous infection and reduced viral titres in a persistent infection, but not as effectively as L3T4+-depleted spleen cells. Although no Lyt 2+ cells were detected by fluorescence staining in Lyt 2+-depleted spleens, we could detect low levels of class I MHC-restricted influenza-specific Tc memory cells in host spleens following influenza infection. Therefore, whether the early viral clearance is solely due to L3T4+ T cells is not clear. Lyt 2+ memory T cells appear more efficient in this respect than L3T4+ memory T cells.     

Influenza virus depresses the PFC response of mice by affecting T-cell function

Five influenza virus strains of type A and one strain of type B were used to elicit suppressive effect on the formation of antibody producing cells in response to sheep red blood cells (SRBC) both in vivo and in vitro. Not only live virus but also the formalin inactivated one exerted immunosuppression (IS). Although intranasal infection only could cause disease in mice, it exerted no IS which, in contrast, occurred after intraperitoneal or intravenous injection of the virus. In nude mice these effects were not seen, but they could be elicited when transferred with normal T cells. The antibody response to T cell independent antigen such as DNP was normal. Thus, it was proved that influenza virus induced T cell dependent immunodeficiency. However, T cell enriched population from infected mice when cultured with normal spleen cells could not suppress their PFC response in vitro. This suggested that suppressor T cells were not required for the immunodeficiency caused by influenza virus.     

T cell responses during influenza infection: getting and keeping control

The 2009 influenza pandemic highlighted the threat that type A influenza poses to human health. Thus, there is an urgency to understand the pathobiology of influenza infection and the contribution of the host immune response to virus elimination and the development of lung injury. This review focuses on the T cell arm of the adaptive host immune response to influenza. We assess recent developments in the understanding of how primary influenza virus-specific T cell responses are induced by antigen-presenting cells, the interaction of activated effector T cells with antigen-bearing cells in the infected lungs. Also examined is the contribution of influenza-specific effector T cells to the development and control of lung injury and inflammation during infection.     

Role of IgM antibodies versus B cells in influenza virus-specific immunity

We have compared the role of IgM antibodies with the role of B cells in control of primary influenza virus infection. Mice deficient in IgM (IgM(-/-)), but capable of producing other Igisotypes, exhibited increased pulmonary virus titers compared to wild-type mice. However, IgM(-/-) mice were less susceptible compared to B cell-deficient micro MT) mice. CD4(+) T cells from spleen and lung draining lymph nodes of infected micro MT mice showed reduced proliferation upon virus re-stimulation in vitro. Furthermore, numbers of IFN-gamma-producing CD4(+) effector T cells were reduced in the alveolar lavage (BAL) of micro MT mice but not IgM(-/-) mice. In contrast, total number of virus-specific CTL was almost comparable in BAL of micro MT and wild-type mice. Pulmonary recruitment of inflammatory macrophages and neutrophils occurred normally in both micro MT and IgM(-/-) mice. Interestingly, virus-specific IgG2a and IgG2b antibody responses were affected locally in the BAL and in the serum of IgM(-/-) mice, while IgG1 responses remained largely normal. Taken together, our data suggest a role for B cells to promote effector T cell responses and a role of both IgM and IgG antibodies in the defense against acute influenza virus infection.     

Properties of murine (CD8+)CD27- T cells

In humans, loss of CD27 expression is associated with the stable acquisition of effector functions by CD8+ T cells. We found that murine (CD8+)CD27- T cells were confined to the primed CD62L(dull/-)CD44(bright)CCR7- T cell population. (CD8+)CD27- T cells were absent from lymph nodes but could be found in blood, spleen and in non-lymphoid organs such as lung and liver. Late after primary influenza virus infection, low percentages of antigen-specific CD27- cells emerged in the lung and spleen. After recovery from secondary influenza virus infection, high percentages of influenza-specific CD27- T cells were found in the lung and the loss of CD27 on lung CD8+ T cells coincided with high granzyme B expression. After murine cytomegalovirus infection, loss of CD27 expression on virus-specific CD8+ T cell populations was sustained and especially marked in liver and lung. We suggest that in mice, CD27 is lost from CD8+ T cells only after repetitive antigenic stimulation. Moreover, the high expression of both granzyme B and perforin in the CD27- T cells suggests that the lack of CD27 on murine CD8+ T cells can be used to identify memory T cells with expression of cytotoxic effector molecules.     

Characterization of age-related changes in natural killer cells during primary influenza infection in mice

The current investigation examined the importance of natural killer (NK) cells during the innate immune response to primary influenza infection in young and aged mice. Young (6-8 weeks) and aged (22 months) C57BL/6 mice were infected intranasally with influenza A virus, and NK cell-mediated cytotoxicity was determined in lung and spleen during the first 4 days of infection. Aged mice demonstrated both a decrease in influenza-inducible NK activity and a reduction in the percentage and number of NK1.1+ cells in response to primary influenza infection, relative to young mice. In order to further establish a role for NK cells in controlling influenza infection, young mice were depleted of NK cells in vivo by injecting rabbit anit-NK1.1 antibody 2 days and 1 day prior to influenza infection. Young mice depleted of NK cells exhibited increased weight loss and lung virus titers during the course of infection, compared to young mice infected with influenza virus. These data indicate that NK cell function is impaired in response to primary influenza infection in aged mice. More importantly, these results underscore the essential role of NK cells in controlling virus titers in lung during the early course of influenza infection, regardless of age.     

Cells Mediating Delayed-Type Hypersensitivity in the Lungs of Mice Infected with an Influenza A Virus

Effector cells that demonstrate delayed-type hypersensitivity (DTH) on transfer with antigen to naive mice can he recovered from the lungs of mice inoculated intranasally 6 days earlier with a lethal dose (usually 5 × 10⁴EID₅₀) of influenza A virus. The activity recovered was proportional to the dose of virus instilled intranasally and the extent of lung consolidation observed. Active cells could also be recovered from the draining lymph nodes and from the peripheral blood. The effector cells were identified as T lymphocytes of Ly I phenotype and required I-region sharing between donor and recipient for activity to be elicited. They were cross-reactive within the A group of influenza viruses. Two experiments are reported in which immune cell preparations that expressed DTH activity but had very little cytotoxic T cell activity were transferred to mice inoculated 1 or 2 days earlier with a lethal dose of virus. The mice were not protected from death, and in both experiments, the recipient mice died more rapidly than the controls. These results contrast with earlier results in which cell preparations with high cytotoxic T-cell activity were shown to protect recipient infected mice from death.     

Lethal synergism induced in mice by influenza type A virus and type Ia group B streptococci.

Intranasal inoculation of CD-1 or BALB/c mice with low doses of influenza A/PR8/34 (HON1) virus followed 48 h later by intranasal inoculation of low doses of type Ia group B streptococci effected a lethal synergism. At a constant input dose of virus, a direct relationship between input dose of bacteria and percent mortality was observed; the converse was also true. An inverse relationship between input dose of group B streptococci, but not input dose of virus, and mean time to death was observed in CD-1 but not in BALB/c mice. The kinetics of influenza A/PR8/34 virus and group B streptococcal replication in singly and dually infected BALB/c mice was determined by assaying samples from the lungs, liver, spleen, and blood for viable group B streptococci and infectious influenza A/PR8/34 virus. No significant difference in virus replication in the lung was observed between singly and dually infected mice. Extrapulmonary dissemination of virus was not observed. Concurrent virus infection effected a 10,000- to 100,000-fold increase in the levels of type Ia group B streptococci in the lung. Potentiation of group B streptococcal infection of the lung was not associated with bacteremia or infection of the liver or spleen, a finding contrary to previous observations of fulminant septicemia after intranasal inoculation of mice with input doses of group B streptococci less than one-tenth of the pulmonary levels observed in the present study.     

Evidence for two T-helper populations with distinct specificity in the humoral response to influenza A viruses.

Virus specificity of T-helper cells for the humoral antibody response to influenza A viruses was studied with a hapten-carrier secondary adoptive transfer system, using whole virus, or viral components inserted into liposomes as carrier with B cells primed to DNP human gamma globulin. Evidence was obtained for two distinct T-helper cell populations from mice primed by influenza infection: a T-helper cell cross-reactive for all type A influenza viruses and a second T-helper population specific for the variant haemagglutinin. In vivo the virus cross-reactive T helpers recognized whole virus, but did not recognize isolated surface glycoproteins or internal virus proteins.     

An improved protocol for measuring cytotoxic T cell activity in anatomic compartments with low cell numbers.

The study of target cell lysis and cytokine production are valuable tools to characterize antigen-specific T and NK cell function during virus infections. After localized infections in compartments such as the lung or the brain, however, cell numbers isolated from these organs are too low to perform standard assays with individual mice. Here, we report a few simple modifications of the classical 51Cr release assay allowing reduction of the number of required effector cells by a factor of 10 without loosing sensitivity or specificity. Using not more than 4x10(5) effector cells, we were able to study ex vivo virus-specific CTL or NK activity from the lungs of individual mice after infection with respiratory syncytial virus (RSV) and from the brains of mice infected with Borna disease virus (BDV). Flow cytometric analysis of interferon-gamma production by virus-specific T cells including appropriate controls was achieved with as few as 10(5) effector cells.     

Induction and characterization of delayed-type hypersensitivity to influenza virus in mice.

Mice infected with an aerosol of influenza virus or immunized with purified UV- inactivated whole virus or viral subunits developed delayed-type hypersensitivity (DTH) to influenza virus. The level of DTH was greatly enhanced when mice were injected intraperitoneally 2 days prior to immunization with 200 mg/kg of cyclophosphamide. The reaction peaked at 24 h after elicitation, had the classical DTH histology, and was transferable by immune cells but not by immune serum. DTH induced in cyclophosphamide-pretreated mice persisted for at least 40 days after sensitization. DTH induced by deoxycholate-treated subviral particles (DC particles) or the matrix protein of the influenza virus was type-specific, i.e. DTH induced by DC particles or matrix protein of type A virus cross-reacted only with type A virus and not with type B virus. DTH induced by purified hemagglutinin (HA) subunits, on the other hand, showed subtype specificty. Thus, DTH induced by HA of X 31 virus (H3) did not cross-react with PR 8 virus (H 0). However, DTH induced by HA of one subtype cross-reacted significantly with the variants of the same subtype. Thus, it appears that the effector T cells of DTH do not discriminate between the antigenic variants of influenza virus that are distinguishable by serology. DTH induced by purified, UV- inactivated whole virus showed extensive cross-reactivity. This nonspecific reaction was shown to be due to host-derived material, the lipid bilayer. - The theoretical and practical implications of these findings are discussed.     

APRIL affects antibody responses and early leukocyte infiltration, but not influenza A viral control

A proliferation inducing ligand (APRIL) is implicated in the regulation of class switch recombination to IgA in T-independent B cell responses. Since B cells play an important role in the immunity to influenza A virus and resistance against the virus is partly controlled by T-independent IgA B cell responses, we studied the role of APRIL during an influenza A infection in vivo. APRIL transgenic, wild-type and APRIL deficient mice were intranasally infected with a non-lethal dose of a mouse adapted strain of influenza A. Compared to wild-type mice, APRIL deficient mice showed a twofold reduction in the amount of macrophages in the lungs and a tendency towards decreased granulocyte influx in the early leukocyte recruitment phase. Although the T cell immune response against influenza was unaffected, APRIL Tg mice showed prolonged influenza-specific IgM production and differential class switching. Unexpectedly, the IgA B cell response was completely T helper cell dependent and also not affected by the absence or presence of APRIL. In addition, viral clearance and recovery from the infection was not influenced by APRIL. Combined these results indicate that APRIL affects specific aspects of the anti-influenza response, but plays a limited role in disease recovery.     

Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity

Mice previously infected with an aerosol of A/Rec 31 influenza virus were strongly protected against an aerosol challenge with A/Vic influenza as judged by lung virus titers recovered 2 days after the challenge infection. Such complete homotypic immunity was not achieved by priming with live Rec 31 virus injected i.v. or UV-inactivated Rec 31 virus administered s.c. together with Al(OH)₃ and saponin. The reason for the superior protective effect of the natural infection was investigated. The protection induced by respiratory infection with Rec 31 virus was specific for influenza A viruses. It was not correlated with specific serum hemagglutination inhibition antibody titer or cross-reactive cytotoxic T (T_c) cell reactivity. Moreover, the transfer of splenic and lymphoid T cell populations with strong secondary T_c activity did not significantly reduce lung virus titers in recipient mice 3 days after infection. The protection however occurred in parallel with the presence of cross-reactive IgA antibody in the lung washings. It thus appears that local secretory IgA plays a causal role in the prevention of cross-infection by influenza A virus. Serum antibody and T_c cells, on the other hand, may be crucial for recovery from such infection. All mice primed with live Rec 31 virus, administered i.v. or by aerosol and expressing equally high levels of T_c reactivity, survived a lethal challenge with A/PR8 virus. The same challenge, however, killed half of the mice immunized s.c. with inactivated Rec 31 virus which induced only a low level of T_c reactivity.     

Induction of cytolytic T- and B-cell responses against influenza virus infections.

Inoculation of mice with live influenza virus results in the induction of cytotoxic thymus-derived (T) lymphocytes and of bone marrow-derived (B) cells producing antiviral antibodies. An assay system was developed to evaluate both types of immune responses on a cellular basis within the same lymphocyte pool with no need to separate out the different effector cell classes. The test system represented a modification of the 51Cr-release assay. T-cell activity was measured exclusively in the absence of active complement using targets that were compatible for determinants encoded by the mouse major histocompatibility gene complex, H-2. H-2-different and even xenogeneic target cells were lysed in the presence of either non-inactivated fetal calf serum or normal rabbit serum as a complement source. Cytotoxicity was mediated in the latter case by direct interaction of B-cell-produced immunoglobulin directed to viral antigens expressed by the target cell and complement. Antibody-dependent cell-mediated cytotoxicity mechanisms did not contribute to cytotoxicity in the test system described. It was demontrated that the cytolytic B-cell responses of one particular strain of mice (BALB/c) against different influenza A viruses were restricted to the immunizing virus on the effector cell level. In another strain of mice (C3H), B cells revealed a broad cross-reactive response resembling that of killer T cells.     

Cellular Changes in Lungs of Mice Infected with Influenza Virus: Characterization of the Cytotoxic Responses

Transpleural lavage of lungs from uninfected C3H mice yielded an average of 300,000 leukocytes per mouse. This number increased eightfold within 6 days after intranasal inoculation with virulent influenza A/Hong Kong/68 (H3N2) virus. Macrophages and lymphocytes in approximately equal numbers comprised 90% or more of the leukocytes both before and during infection. B, T, and null lymphocytes comprised, respectively, 9, 21, and 18% of the leukocytes before infection and 7, 26, and 5% by day 6. In absolute numbers, macrophages and T lymphocytes provided the major increments during infection. Cytotoxic activity of mononuclear cells from lung lavages was compared in a chromium release assay using syngeneic L929 target cells with the activity of mediastinal lymph nodes, spleens, and peripheral blood of uninfected and infected C3H mice. Nonspecific cytotoxicity for target cells infected with H3hkNeq1 or B/Lee influenza virus was found with mononuclear cells from uninfected mice. This activity tended to be highest with lavage leukocytes and was associated with adherent cells, presumably macrophages. Increased virus-specific cytotoxicity was detected with lavage cells by day 6 and persisted through day 9, the period of maximal pneumonia. Similar cytotoxic activity also appeared in cells from the nodes and spleen at this same time but was not detected in peripheral blood cells. The virus-specific cytotoxicity of lavage cells was due largely to a nonadherent cell possessing Fc receptors and theta antigen but lacking C3 receptors; these properties are compatible with actively cytotoxic T lymphocytes. The cytological characteristics of the infiltrating leukocytes and the cytotoxicity data suggest that the local T cell response to influenza virus infection in the lung is a major contributor to the pneumonia observed in this mouse model.     

IL-18, but not IL-12, is required for optimal cytokine production by influenza virus-specific CD8+ T cells

The potent innate cytokines IL-12 and IL-18 are considered to be important antigen-independent mediators of IFN-gamma production by NK cells and T lymphocytes. The present analysis addresses the physiological role of IL-12 and IL-18 in the generation of virus-specific CD8+ T cells. Both wt C57BL/6J (B6) mice and mice with disrupted IL-12p40 (IL-12p40(-/-)) or IL-18 (IL-18(-/-)) genes were infected with an influenza A virus and the characteristics of the resultant epitope-specific CD8+ T cell responses were compared. While IL-12 appeared to have no notable effect on either virus growth or on CD8+ T cell response profiles, the absence of IL-18 was associated with delayed virus clearance from the lung and, despite normal numbers, a significantly reduced production of IFN-gamma, TNF-alpha, and IL-2 by epitope-specific CD8+ T cells. While this cytokine phenotype was broadly maintained in IL-12p40/IL-18 double-knockout mice, no evidence was seen for any additive effect. Together, our results suggest that IL-18, but not IL-12, induces optimal, antigen-specific production of key cytokines by CD8+ T cells for the efficient clearance of influenza virus from the lungs of infected mice.     

The Generation of 'Cytotoxic' Macrophages in Mice during Infection with Influenza A or Sendai Virus

Injection of infectious but not of non-infections influenza A virus or of infectious or non-infectious Sendai virus intraperitoneally into mice induces the generation of plastic-adherent cells that arc able to effect release of ⁵¹Cr from labelled virus-infected target cells but not from labelled, uninfcctcd cells. Their activity is greatly diminished by exposure to silica or carrageenan but not by anti-Thy I antibody and complement treatment. Similarly, the activity of the cell preparation cannot be explained by contamination with natural killer or 'K' cells. Thus, the effector cells were identified as macrophages and for convenience are called 'cytotoxic macrophages'. The maximum cytotoxic activity was recovered from the peritoneal cavity 5 days after virus injection and declined thereafter. Although the effector cells are cross-reactive in that cells activated by an influenza A strain virus lyse target cells infected with the same or other A strain viruses or with Sendai virus, there is preferential lysis of cells infected with the homologous virus. The action of the effector cells is not H-2-restrictcd. Preliminary experiments showed that similar effector cells can be recovered from the lungs of mice 5 days after intranasal inoculation of infectious influenza virus, so they may contribute to the host control of the disease.