Natural killer cells vs cytotoxic T cells in the peripheral blood of virus-infected mice

The cell-mediated immune response of mice against various enveloped RNA and DNA viruses expressed by immune lymphocytes from the spleen and the peripheral blood (PBL) were compared. PBL from mice of various strains infected with vaccinia virus, vesicular stomatitis virus (VSV), or lymphocytic choriomeningitis virus (LCMV) were tested on histocompatible or incompatible target cells infected with the homologous virus. PBL from immune mice showed clear H-2 restriction, but additionally, they expressed high natural killing (NK) activity on YAC-1 cells. The high NK-cytolytic activity of PBL on YAC-1 differed significantly from that expressed by splenic lymphocytes. In both lymphocyte populations lysis was detected as early as 1 day after infection; NK activity decreased in the spleen after day 4 post infection, whereas that of PBL persisted at high levels for up to 10 days after infection. Treatment of mice with anti-asialo GM1 in vivo abrogated NK activity in PBL effector cells tested in vitro. These results may explain some of the difficulties to observe MHC-restricted cytotoxic T cells in PBL from humans or primates during primary infections with virus.     

Interleukin-4 causes delayed virus clearance in influenza virus-infected mice.

Two different subsets of T cells, Th1 and Th2 cells, have been demonstrated to secrete different profiles of cytokines and to influence various infections in different ways. Whereas cytokines secreted by Th1 cells, particularly gamma interferon, promote the generation of cell-mediated immunity, Th2 cells and their cytokines (interleukin-4 [IL-4], IL-5, IL-10, and IL-13) have been shown to function in recovery from parasitic infections and in antibody responses. In this study, we analyzed the effects of the dominant Th2 cytokine, IL-4, on immunity to virus infection. We assessed the effects of IL-4 on both secondary immune responses by an adoptive transfer assay and primary immune responses by in vivo treatment of influenza virus-infected mice with IL-4. The results demonstrated that IL-4 can function to inhibit antiviral immunity at both stages. We found that IL-4 treatment of sensitized cells during secondary stimulation in vitro had little effect on their ability to lyse virus-infected target cells in a 51Cr release assay. Nevertheless, the clearance of influenza A/PR/8/34 (H1N1) virus from the lungs of infected BALB/c mice was significantly delayed after the transfer of virus-specific T cells secondarily stimulated in the presence of IL-4 in comparison to virus clearance in recipients of cells stimulated in the absence of IL-4. In contrast to the adoptive transfer results, the treatment of PR8 virus-infected mice with IL-4 during primary infection greatly suppressed the generation of cytotoxic T-cell precursors, as assessed by secondary stimulation in vitro. In addition, culture supernatants of secondarily stimulated spleen cells from IL-4-treated mice contained significantly less gamma interferon and more IL-4 than did spleen cells from controls. More importantly, the treatment of mice with IL-4 resulted in an extremely significant delay in virus clearance. Thus, IL-4 can inhibit both primary and secondary antiviral immune responses.     

Suppression of cell-mediated immunity by street rabies virus infection.

An attempt to define a severe suppression of cell-mediated immunity by street rabies virus infection was undertaken by using the mice lethally and peripherally infected with a street rabies virus (1088 strain). The cell-mediated cytotoxic (CMC) activity of the spleen cells from those mice once slightly increased until day 4 after infection but declined rapidly thereafter until their death on days 10 to 12 after infection. In parallel with a decrease of CMC response of the spleen cells from 1088-infected mice, proliferative response to Con A, IL-2 activity in the culture supernatants of Con A-induced proliferation, responsiveness to exogenously added IL-2 and to Con A to express IL-2R, of those cells became suppressed, and the marked decrease of the total number of spleen cells was observed. Selective depletion of CD4+ and CD8+ cells in the spleens, abnormalities of IL-1 and E-type prostaglandins (PGE2) production or production of inhibitory component able to block IL-2 activity by spleen cells were not observed and these factors did not appear to be associated with the suppression of proliferative response to Con A. However, an apparent association of CD8+ cells in the suppression of differentiation of pre-cytotoxic lymphocytes (CTL) into CTL was demonstrated in the co-culture experiments of the spleen cells from 1088-infected mice with spleen cells of mice infected with an attenuated rabies virus (ERA strain) which can induce higher levels of CMC response. There was no evidence of the productive replication of rabies virus in thymus and spleen of 1088-infected mice. The relationship of these observations to current theories on virus-induced immunosuppression was discussed.     

Cellular immunity in chronic Theiler's virus central nervous system infection.

After (IC) inoculation of the DA strain of TMEV, SJL/J mice develop chronic CNS infection with marked mononuclear cell infiltration of spinal cord leptomeninges and white matter and concomitant demyelination. In the present study the temporal course of cell-mediated and humoral immune responses to virus were measured in this infection. It was shown that chronic TMEV infection is associated with the development of immunologically specific spleen cell reactivity as judged by in vitro incorporation of 3H-TdR into DNA in response to inactivated TMEV antigen. Spleen cell reactivity is first detectable about 2 months after infection, persists for at least 1 year, and correlates with the temporal development of serum-neutralizing antibody. The late development of sensitized spleen cells is not the result of an immunosuppressive effect of this virus infection since infected mice exhibit normal spleen cell proliferative responses to T cell mitogens and produce normal antibody responses to a heterologous protein antigen, sheep red blood cells. In addition, anti-viral antibody inhibits virus-induced spleen cell reactivity. Finally, the antigen-reactive lymphocyte subpopulation within the spleen responsible for proliferation to TMEV antigen are T cells and not B cells.     

The effect of the bronchoalveolar fluid of mice infected with the influenza virus on hematopoietic bone marrow precursors]

The influence of bronchoalveolar washing fluid (BAWF), as well as BAWF cells, obtained from mice infected with influenza virus, on the formation of exogenic spleen colony-forming units (CFUs) of lethally irradiated syngeneic recipients was studied. BAWF and BAWF cells of intact syngeneic mice stimulated the growth of CFUs. BAWF of mice infected with nonpathogenic strain A/PR/8/34 lost its capacity for stimulating the growth of colonies, and BAWF cells greatly suppressed colony formation in the spleen of recipients. The participation of interferon, colony-stimulating factor and the virus itself in the process of the modulation of colony formation is discussed.     

Effect of vesicular stomatitis virus (VSV) infection on the development and regulation of T cell-mediated immune responses

Infection of mice with vesicular stomatitis virus (VSV) at the time of immunization failed to enhance T cell-mediated immune response to azobenzenearsonate-(ABA) conjugated spleen cells as measured by delayed-type hypersensitivity and by in vitro proliferation and in vitro generation of ABA-specific cytotoxic T cells. However, mice infected with VSV are incapable of responding to signals from suppressor T cells or their soluble factors. Further analysis revealed that VSV infection does not interfere with the induction of Ts-1 or Ts-2 cells. Because infection of Ts-1 or Ts-2 donors had no effect on the subsequent response seen in the recipients of antigen and suppressor T cells, the most likely candidate for the target of VSV infection is therefore the Ts-3 cell or another T cell interacting with Ts-3. This is supported by our observation that it is possible to bypass the VSV effect by providing the recipients of VSV with normal Lyt-2+-bearing T cells.     

Lymphotoxin-alpha-deficient mice make delayed,but effective,T and B cell responses to influenza

Lymphotoxin-alpha(-/-) (LTalpha(-/-)) mice are thought to be unable to generate effective T and B cell responses. This is attributed to the lack of lymph nodes and the disrupted splenic architecture of these mice. However, despite these defects we found that LTalpha(-/-) mice could survive infection with a virulent influenza A virus. LTalpha(-/-) mice and normal wild-type mice infected with influenza A generated similar numbers of influenza-specific CD8 T cells that were able to produce IFN-gamma and kill target cells presenting influenza peptides. Furthermore influenza-infected LTalpha(-/-) mice produced high titers of influenza-specific IgM, IgG, and IgA. However, both CD8 and B cell immune responses were delayed in LTalpha(-/-) mice by 2-3 days. The delayed cellular and humoral immune response was sufficient to mediate viral clearance in LTalpha(-/-) mice that were infected with relatively low doses of influenza virus. However, when LTalpha(-/-) mice were infected with larger doses of influenza, they succumbed to infection before the immune response was initiated. These results demonstrate that neither LTalpha nor constitutively organized lymphoid tissues, such as lymph nodes and spleen, are absolutely required for the generation of effective immunity against the respiratory virus influenza A. However, the presence of LTalpha and/or lymph nodes does accelerate the initiation of immune responses, which leads to protection from larger doses of virus.     

Role of immunity in age-related resistance to paralysis after murine leukemia virus infection.

Resistance to the paralytic effects of a wild mouse (Cas-Br-M) murine leukemia virus infection develops with age and is complete by 10 days of age in susceptible NFS mice. The possibility that cell-mediated immunity plays a significant role in this resistance was suggested by the observation that treatment of 10-day-old mice with antithymocyte serum rendered them susceptible to paralysis. By comparison, mice rendered incapable of generating a humoral immune response by treatment from birth to 1 month of age with anti-immunoglobulin M serum did not develop paralysis after challenge with virus at day 10. Transfer of unseparated and T-cell-enriched populations of Cas-Br-M murine leukemia virus-immune spleen cells protected neonatally infected NFS recipients from paralysis; transfer of Cas-Br-M murine leukemia virus-immune populations enriched for B cells delayed the onset but did not ultimately protect neonatally infected NFS mice from paralysis. Transfer of naive adult spleen cells had no protective effect in neonatally infected NFS mice. High-level virus replication occurred in the spleens and brains of all mice that developed paralysis regardless of treatment; low-level virus replication in spleen and barely detectable replication in brain occurred in mice that remained clinically normal. These studies suggest that the age-acquired resistance to the paralytic effect of Cas-Br-M murine leukemia virus infection is immunologically mediated and that T cells may play a major role.     

Virus-specific T-cell sensitization

Syngeneic, semiallogeneic, or allogeneic spleen lymphocytes were transferred intonu/nu BALB/c mice, which were infected with vaccinia virus. Specific Sensitization of transferred thymus-derived cells was determined in vivo by mean survival time and virus titer in the spleen six days after infection, and in vitro by cell-mediated cytolysis of vaccinia virus-infected syngeneic target cells. Virus-specific Sensitization took place only after transfer of syngeneic or semiallogeneic spleen lymphocytes; allogeneic lymphocytes had no influence on mean survival time or virus titer and showed no virus-specific cytolytic activity in vitro. Infection of mice with vaccinia virus-strain WR, Elstree, DIs, or DIs-infected syngeneic fibroblasts resulted in the generation of virus-specific effector cells, while injection of a high amount of inactivated virus particles caused no Sensitization. These results suggest H-2 homology for production of virus-specific effector cells. Propagation of virus is not necessary, since early surface antigens, combined with syngeneic H-2 antigens, suffice for Sensitization of cytolytic T lymphocytes.Abbreviations used in this paper are as follows CMC cell-mediated cytolysis - CTL cytolytic T lymphocyte - LCM lymphocytic choriomeningitis - MHC major histocompatibility complex - MST mean survival time - T cell thymus-derived cell - TCID₅₀ 50 percent tissue culture infective dose     

Evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice

Influenza virus-infected cells undergo apoptosis and become susceptible to phagocytosis by macrophages in vitro, and this leads to the propagation of the virus being inhibited. We previously showed that inhibitors of phagocytosis increased the rate of mortality among influenza virus-infected mice. However, the mode of the phagocytosis of influenza virus-infected cells in vivo has not been investigated. We, in this study, assessed this issue by histochemically analyzing bronchoalveolar lavage cells and lung tissue obtained from C57BL/6 mice infected with influenza A/WSN (H1N1) virus. Both neutrophils and macrophages accumulated in the lung soon after the viral challenge, and either type of cell was capable of phagocytosing influenza virus-infected, apoptotic cells. Changes in the level of phagocytosis and the amount of virus in lung tissue roughly correlated with each other. Furthermore, alveolar macrophages prepared from influenza virus-infected mice showed greater phagocytic activity than those from uninfected mice. The phagocytic activity of macrophages was stimulated in vitro by a heat-labile substance(s) released from influenza virus-infected cells undergoing apoptosis. These results suggested that the level of phagocytosis is augmented both quantitatively and qualitatively in the lung of influenza virus-infected animals so that infected cells are effectively eliminated. Finally, lack of TLR4 caused an increase in the rate of mortality among influenza virus-challenged mice and a decrease in the level of phagocytosis of apoptotic cells in the lung. TLR4 could thus play an important role in the host defense against influenza by positively regulating the phagocytic elimination of infected cells.     

Influenza virus-induced type I interferon leads to polyclonal B-cell activation but does not break down B-cell tolerance

The link between infection and autoimmunity is not yet well understood. This study was designed to evaluate if an acute viral infection known to induce type I interferon production, like influenza, can by itself be responsible for the breakdown of immune tolerance and for autoimmunity. We first tested the effects of influenza virus on B cells in vitro. We then infected different transgenic mice expressing human rheumatoid factors (RF) in the absence or in the constitutive presence of the autoantigen (human immunoglobulin G [IgG]) and young lupus-prone mice [(NZB x NZW)F(1)] with influenza virus and looked for B-cell activation. In vitro, the virus induces B-cell activation through type I interferon production by non-B cells but does not directly stimulate purified B cells. In vivo, both RF and non-RF B cells were activated in an autoantigen-independent manner. This activation was abortive since IgM and IgM-RF production levels were not increased in infected mice compared to uninfected controls, whether or not anti-influenza virus human IgG was detected and even after viral rechallenge. As in RF transgenic mice, acute viral infection of (NZB x NZW)F(1) mice induced only an abortive activation of B cells and no increase in autoantibody production compared to uninfected animals. Taken together, these experiments show that virus-induced acute type I interferon production is not able by itself to break down B-cell tolerance in both normal and autoimmune genetic backgrounds.     

Multifunctional CD4 cells expressing gamma interferon and perforin mediate protection against lethal influenza virus infection

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. IFN-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ(-/-) CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-γ-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme B (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 T cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection.     

Enhanced immune response late in primary cytomegalovirus infection of mice.

Murine cytomegalovirus (MCMV) infection has been previously shown to depress humoral and cell-mediated functions to non-MCMV antigens. In this report we show that in C3D2 mice undergoing nonlethal primary infection the depressed anti-sheep RBC plaque-forming cell (PFC) response is followed by an enhanced PFC response. Infected mice often generated twice the number of PFC per spleen than that of control mice. Total numbers of spleen cells as well as the recovery of virus from spleens of infected mice did not distinguish the depressed from the enhanced phase of the response. Investigation of the kinetics of the response revealed a defect in shutdown regulation. This enhanced PFC response during primary MCMV infection was not reflected in measurements of serum hemagglutinin. These findings suggest that MCMV induces an impairment of immunoregulation.     

In vivo administration of anti-asialo-GM1 antibody enhances splenic clearance of Listeria monocytogenes

Resistance of mice to infection by Listeria monocytogenes involves a biphasic response. The first phase consists of the first 48 h after infection, during which there is multiplication of Listeria in the liver and spleen of infected mice. In these nonimmune mice, macrophages and polymorphonuclear leukocytes are the effector cells involved in controlling multiplication. In the second phase, cell-mediated immunity develops, beginning on day 2, during which multiplication of Listeria is prevented by macrophages possessing increased microbicidal activity that is mediated through the action of lymphokines released by immunologically committed T lymphocytes. The purpose of the present study was to define a role for natural killer (NK) cells in natural resistance to Listeria during the first 48 h after infection, prior to the development of specific immunity. Splenic NK cell activity was enhanced following a sublethal intravenous injection of viable Listeria as early as 24 h after injection and remained elevated throughout the nonimmune phase of infection. Interestingly, treatment of mice with anti-asialo-GM1 significantly enhanced the ability of mice to clear Listeria from the spleen relative to infected controls possessing intact NK cell populations. This was evidenced by 23-fold fewer bacteria obtained from the spleens of anti-asialo-GM1-treated mice. In addition, Percoll-enriched NK cell populations obtained from 48-hour Listeria-infected mice do not exhibit in vitro listericidal activity. These observations suggest a regulatory role of NK cells in resistance against Listeria and preclude a role for NK cells in direct cytolysis. Perhaps these cells modulate the immune response to Listeria by down-regulating the activity of the immune cells crucial to listerial resistance.     

Pattern recognition molecule mindin promotes intranasal clearance of influenza viruses

The innate immune response is essential for host defense against microbial pathogen infections and is mediated by pattern recognition molecules recognizing pathogen-associated molecular patterns. Our previous work has demonstrated that the extracellular matrix protein mindin functions as a pattern recognition molecule for bacterial pathogens. In this study, we examined the role of mindin in influenza virus infection. We found that intranasal infection of mindin-deficient mice by influenza virus resulted in dramatically increased virus titers in the lung and intranasal cavity of mutant mice. In contrast, lungs from intratracheally infected mindin-deficient mice contained similar influenza virus titers. We showed that mindin interacted with influenza virus particles directly and that mindin-deficient macrophages exhibited impaired activation after influenza virus infection in vitro. Furthermore, intranasal administration of recombinant mindin significantly enhanced the clearance of influenza virus in wild-type mice. Together, these results demonstrate that mindin plays an essential role in the host innate immune response to influenza virus infection and suggest that mindin may be used as an immune-enhancing agent in influenza infection.     

Recovery from a viral respiratory tract infection. IV. Specificity of protection by cytotoxic T lymphocytes

Immune spleen cells enhanced for influenza-specific cytotoxic activity after exposure to virus-infected stimulator cells in vitro effect recovery when transferred to nude and immunocompetent mice with influenza pneumonia (5). This protective effect correlated with the virus-specific cytotoxic activity of the transferred lymphocytes and is removed by treatment with anti-0 serum and complement. The experiments presented here indicate that spleen cells taken directly from mice undergoing a primary or secondary infection are less protective than immune spleen cells that are restimulated in vitro before transfer. This decreased ability to clear pulmonary virus and effect survival correlated with their relatively lower levels of influenza-specific cytotoxicity. Protection did not correlate with the level of natural killer cell activity of transferred cells. The results also indicate the immune spleen cells that are protective are influenza A subtype cross-reactive and are H-2-restricted; H-2d immune spleen cells effected recovery of H-2d but not H-2k challenged mice.     

A mouse model for immunization with ex vivo virus-infected dendritic cells

Dendritic cells (DCs) have been demonstrated to be an important if not essential inducer of cellular immune responses. The ability to grow these cells in vitro may open up new avenues for protective immunizations. In this study we have analyzed the virus-specific memory response generated following immunization with ex vivo-infected bone marrow-derived dendritic cells. We demonstrate that mouse DCs are efficiently infected with influenza virus but do not release infectious progeny virus. Ex vivo-infected DCs secrete interleukin-12 (IL-12) and induce a potent T helper (Th)1-like immune response when injected into mice. This was demonstrated by the generation of cytotoxic T lymphocytes, the production of high levels of gamma-interferon, and undetectable levels of IL-4 upon in vitro restimulation of splenocytes from immunized animals. In addition, the virus-specific antibody response is primarily of the IgG2a isotype, consistent with the expansion of Th1 cells. Animals immunized with DCs infected with X-31 influenza virus and challenged with PR8 influenza virus cleared the infection faster than animals not vaccinated. Thus, infected DCs efficiently activate the cellular immune response and induce heterosubtypic immunity in mice.     

Regulation of granulomatous inflammation in murine schistosomiasis. IV. Antigen-induced suppressor T cells down-regulate proliferation and IL-2 production

In murine schistosomiasis mansoni the cell-mediated immune response to the deposited eggs is mediated by CD4+ delayed-type hypersensitivity effector T (TDH) cells that produce vigorous granulomatous responses in the liver and intestines of acutely infected animals. The response is significantly down-modulated in chronically infected mice by Ag-specific Ts cells. The present study was undertaken to establish an in vitro model by which TDH-Ts cell interactions could be analyzed. To this end, Ts cells were induced in vitro by preculture of chronic or acute infection spleen cells with soluble egg Ag (SEA) for 48 h. The induced cells suppressed the SEA-specific proliferation of acute infection spleen cells by 80 to 95%. The induced suppressor cells were Ag specific in both induction and elicitation of function, and were not cytotoxic to the acute infection splenic target cells. Suppression by the induced cells was manifested within the first 24 h of the SEA-induced response as IL-2 produced by acute infection spleen cells was suppressed 62%. Phenotypic analysis by flow cytometry of the induced suppressor cells showed that CD8+ cells from acute infection spleens and CD4+ and CD8+ cells from chronic infection spleens were effector Ts cells. Taken together, CD4+ and CD8+ SEA-specific Ts cells can be induced in vitro to effectively suppress the SEA-specific lymphoproliferation and IL-2 production of acute infection spleen cells. Establishment of this in vitro model will allow us to further analyze the mechanisms of Ts cell-mediated suppression of TDH cells.     

Macrophage immunity to influenza virus: in vitro and in vivo studies.

Using M-TUR, a macrophage-adapted avian influenza A virus (Hav1, Nav3), antiviral resistance of peritoneal macrophages obtained from specifically or nonspecifically immunized mice towards in vitro infection was assessed. M-TUR grew to high titers in macrophages from nonimmune mice thereby causing a marked cytopathic effect. In contrast, peritoneal macrophages from mice specifically immunized with TUR virus were not affected by infection with M-TUR in vitro. This antiviral immunity was specific: mice immunized with antigenetically unrelated influenza strains such as influenza A/Hong Kong/1/68 (H3, N2) or influenza B/Lee yielded susceptible macrophages. Specific macrophage immunity could be abrogated by trypsin treatment in vitro. Susceptible macrophages from nonimmune hosts became resistant following in vitro exposure to homologous anti-TUR sera. Peritoneal exudate cells from BCG-infected animals were less susceptible to in vitro challenge with M-TUR than control macrophages. In vivo treatment of mice with the unspecific immunostimulants BCG or Corynebacterium parvum did not protect the animals against lethal infection with a hepatotropic variant of TUR.