Specifically immune mouse T-cells can destroy H-2 compatible murine target cells infected with herpes simplex virus types 1 or 2.

Lymphocytes from the spleen (SL) or peritoneal cavities (PL) of HSV-1- or HSV-2-immunized C 3 H mice expressed pronounced cytotoxicity, as measured by 51Cr release assay, against L cells experimentally infected with HSF-types 1 or 2 but not against uninfected L cells. HSV-1 or HSV-2 immune lymphocytes induced substantially more 51Cr release when L-cell targets were infected with homotypic virus compared to those infected with heterotypic virus. Inasmuch as the cytotoxicity of specifically immune C 3 H SL for HSV-infected L cells was selectively eliminated by treatment with AKR anti-theta C 3 H serum plus complement, the effector cells in present system were theta-bearing T cells. Evidence has been provided which indicates that specifically immune T cells express cytotoxicity exclusively directed against different HSV-infected target cells (mouse embryo cells or mouse peritoneal macrophages) which share H-2 antigens with the effector cells.     

Establishment of an enzyme release assay for cytotoxic T lymphocyte activity.

In the present report, we describe the establishment of a cell line that can be used as the target for measuring the activity of cytotoxic T lymphocytes (CTL) by an enzyme release assay. We transfected P3/NS1-Ag4-1 (NS-1), a myeloma cell line derived from BALB/c mice with Escherichia coli beta-galactosidase (beta-Gal) gene, and isolated a stable transformant designated as NS-1/Z that expressed a high level of the enzyme activity intracellularly. The effector cells showing cytotoxicity against NS-1/Z were induced when the spleen cells of AKR or C3H mice were cultured with mitomycin C-treated BALB/c spleen cells for 4 days. When 2 x 10(4) NS-1/Z cells were incubated with varying numbers of effector cells, beta-Gal activity was released from the target cells depending on the number of effector cells and the time of incubation for up to 8 h. A highly sensitive enzyme assay was performed by using a fluorescent substrate, 4-methylumbelliferyl-beta-D-galactoside. The cytotoxicity was specific for H-2 haplotype of the stimulator cells, and was abolished by treating the effector cells with anti-Lyt 2 plus complement. The sensitivity of the enzyme release assay was comparable to that of 51Cr release assay. These results indicate that NS-1/Z can be used as a target cell line for the non-radioactive measurement of CTL activity.     

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.     

Cell-Mediated Cytotoxicity Toward Measles Virus-Infected Target Cells in Randomly Bred Syrian Hamsters

Cell-mediated cytotoxicity (CMC) toward measles virus-infected cells was studied by a ⁵¹Cr release assay with spleen cells from hamsters inoculated with measles virus (strain Lec) or control antigen and with spleen cells from normal hamsters. Spleen cells from measles virus-inoculated hamsters showed greater CMC toward infected than toward noninfected target cells (designated specific CMC). Specific CMC was maximal 7 days after virus inoculation and was declining by 9 to 10 days. Effector cells were present in a nonadherent cell population. Specific CMC was reduced after treatments of effector cells with antithymocyte serum plus complement. The decrease in cytotoxicity was greater toward infected target cells than toward noninfected target cells. Treatment of infected target cells with antimeasles serum did not increase specific CMC by effector cells from the majority of virus-inoculated hamsters. CMC toward infected target cells by normal spleen cells (natural killer cells) or spleen cells from hamsters inoculated with control antigen was approximately the same as, or more often less than, CMC toward noninfected target cells. Natural killer cells were present in a nonadherent cell population. Treatment of natural killer cells with antithymocyte serum plus complement caused a similar decrease in cytotoxicity toward both infected and noninfected target cells. This study demonstrated virus-specific cellular cytotoxicity of effector spleen cells from measles virus-inoculated hamsters. Although the data were compatible with T cells as the source of effector cells in the virus-specific CMC, definitive identification could not be made. Additional membrane markers for better characterization of hamster lymphocyte subpopulations are required.     

Cell-mediated immunity to tumor antigen in Marek''s disease: susceptibility of effector cells to antithymocyte serum and enhancement of cytotoxic activity by Vibrio cholerae neuraminidase.

Spleen cells from chickens inoculated 7 to 8 days previously with Marek's disease virus were cytotoxic for 51Cr-labeled cells of a Marek's disease lymphoblastoid cell line (MSB-1 line) in a 4-h in vitro cytotoxic assay. The cytotoxic activity of spleen cells was inhibited by pretreatment with antithymocyte serum and complement, but not with complement alone or in combination with anti-bursa cell serum or normal preimmune serum. The conclusion was that the effector cell in the above cytotoxic assay was a thymus-derived lymphocyte. Also, pretreatment of target cells with Vibrio cholerae neuraminidase enhanced in vitro cytotoxic activity of effector cells. Similar enzymatic treatment of effector cells had a negligible effect on cytotoxicity.     

Modulation of human natural killer cytotoxicity by influenza virus and its subunit protein.

The influence of intact influenza virus and purified detergent solubilized haemagglutinin (HA) subunits from these viruses on human natural killer (NK) cell activity was examined. Effector cells incubated with whole influenza virus for 18 hr initiated the production of alpha interferon which was associated with the enhancement of NK cell activity. In contrast, purified influenza virus HA suppressed NK activity in a dose-dependent manner, when added at the onset of the cytotoxicity assay, or when used to pre-treated effector cells prior to assay for cytotoxicity against K562 target cells. Effector cells exposed to influenza HA for 90 min, washed and re-incubated in fresh medium for up to 18 hr, failed to regain their cytotoxicity. Suppression of NK cell cytotoxicity could not be ascribed to direct toxicity of HA preparations or residual detergent and preservative in these preparations. The augmented cytotoxicity of activated human effector cells was also susceptible to suppression by virus HA, and pretreatment of human PBL effector cells with HA for 90 min, prior to exposure to human alpha interferon caused NK effector cells to become refractive to the enhancing effects of HIFN. That direct interaction between influenza virus HA and effector cells was a requirement for suppression of activity was shown in experiments using Bromelain-released influenza HA, which would not be expected to bind to cells and which failed to suppress NK cell activity.     

Immunization with soluble simian virus 40 large T antigen induces a specific response of CD3+ CD4- CD8+ cytotoxic T lymphocytes in mice.

C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.     

Cytotoxic T cell response against lymphoblasts infected with Moloney (Abelson) murine leukemia virus. Methodological aspects and H-2 requirements

A method for infection of lymphocytes with Moloney(Abelson) murine leukemia virus [M(A)-MuLV] is described. Only lymphoblasts obtained after stimulation of normal spleen cells by the B cell mitogen lipopolysaccharide (LPS) were satisfactory targets for virus-specific, secondary cytotoxic T lymphocytes (CTL), whereas spleen cells stimulated by the T cell mitogen concanavalin A were not. The secondary CTL response against M(A)-MuLV could be efficiently measured using M(A)-MulV-infected LPS blasts as stimulating cells for secondary in vitro restimulation and as target cells for virus-specific destruction. Cold target inhibition demonstrated virus specificity of CTL. The T cell character of the cytotoxic cells was demonstrated by their sensitivity to anti-Thy-1.2 treatment. Using syngeneic virus-infected LPS blasts as target and stimulator, CTL responses were measured with effector cells from C57BL mice of the H-2^b haplotype and of recombinant haplotypes sharing either K or D alleles with H-2^b. In analogy with previous studies on Moloney virus-specific CTL. it was observed that C57BL/6 (H-2^b) effector cells predominantly lysed D^b-compatible, virus-infected target cells; B10.A(5R), (K^bD^d) effector cells showed a poor CTL response against syngeneic, virus-infected target cells. The combined findings indicate the existence of an Ir gene in the H-2D region regulating the CTL response against Moloney leukemia virus.     

Secondary cytotoxic cell response to lymphocytic choriomeningitis virus II. Nature and specificity of effector cells.

The method described in the previous paper was used to induce secondary responses in spleen cells from CBA/H mice, pre-primed with lymphocytic choriomeningitis (LCM) virus by culturing them with LCM-infected peritoneal cells. The cytolytic effector cells thus generated have been characterized. Effector cells were sensitive to treatment with anti-theta ascitic fluid and complement. Separation procedures based on rosetting of certain categories of lymphocytes with sheep red cells through an Isopaque-Ficoll gradient indicated that effector cells lacked surface immunoglobulin and generally did not bear Fc receptors. Cytolytic activity was restricted by the H-2 gene complex. Killing had single-hit characteristics. All these results suggested that the cells from memory cultures mediating cytolysis were T cells. There was evidence for two T cell subsets, a major subpopulation directed against antigens on infected targets and a minor one directed against antigens on uninfected, H-2-compatible targets. Specificity was present at the infected cell:memory responder and killer:target levels between LCM virus (an arenavirus) and ectromelia virus (a poxvirus).     

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.     

Influenzal pneumonia: early appearance of cross-reactive T cells in lungs of mice primed with heterologous type A viruses.

Mice were first primed with a type A or a type B influenza virus and then challenged intranasally at least 1 month later with another type A virus. Potent cytotoxic T cell populations were found in lung, and effector T cell function was also demonstrated in blood, spleen and mediastinal lymph nodes. Lymphocytes isolated from all of these anatomical sites were active against target cells infected with the same, or with serologically different, type A influenza viruses. Also, prior exposure to another type A virus resulted in more rapid development of effector function than was seen in mice that had first been infected with B/Lee. Cytotoxic T cell populations generated in mice with influenza thus tend overall to be type-specific, and there is substantial localization of these effector lymphocytes in the pneumonic lung.     

The Recovery of Mice from Influenza Virus Infection: Adoptive Transfer of Immunity with Immune T Lymphocytes

Transfer of primary or secondary influenza-immune spleen cells to mice infected intranasally with influenza virus resulted in a significant clearance of virus from the lungs and the protection of the recipients from death. The antiviral activity was associated only with intact, viable cells and was not due to carryover of virus. The effector cell population responsible for the antiviral effect was shown to be T cells. Thus, the removal of adherent, phagocytic and Ig+ cells did not affect the antiviral activity, whereas it was destroyed with antitheta serum and complement. Antiviral activity was specific and was best expressed if the virus used to infect the recipients and to generate immune cells was the same strain. Further work will be necessary to define rigorously the role of different viral antigens in cell-mediated immune response to influenza virus infection.     

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.     

Lymphocyte-mediated cytotoxicity against allogeneic tumour cells. IV. Fine specificity mapping and characterization of concanavalin A-activated cytotoxic effector lymphocytes.

Concanavalin A (Con A)-activated cytotoxic lymphocytes have been investigated, mapping the genetic differences between the P815 target and the effector cells required for cell-mediated lympholysis to occur. The target antigens recognized during the effector phase and the phenotype of the killer cell population(s) were also determined. It was found that Con A could activate a population of primed cytotoxic lymphocytes capable of killing target cells that were identical at the major histocompatibility complex (MHC) but differed at other background genes. Thus, after in vivo priming with DBA/2, B10.D2 lymphocytes cultured with Con A were capable of killing the P815 target. Unprimed B10.D2 cells, however, would not. Studies on the involvement of the MHC indicated that differences in the H-2K through H-2S, as well as differences in H-2D and H-2L alone could cause lysis. This killing could not be accounted for by additional differences at Qa-2, a MHC-linked locus. However, the contribution of other similar non-MHC linked loci could not be excluded. Cold target competition experiments indicated that MHC encoded alloantigens were involved as recognition structures on the target cell surface. Antisera plus complement depletion of cytotoxic effector function demonstrated that the cytotoxic cells had the cell surface phenotypes Thy 1.2+, Lyt 2.2+ and natural killer (NK) 1.1-. We conclude that Con A polyclonally activates population(s) of T cells that express antigen-specific cytotoxicity through clonally distributed recognition receptors intrinsic to their membranes when lectin is omitted from the cytotoxic assay.     

Generation of virus specific cytotoxic T cells in vitro. III. Spleen cells stimulated by viral antigens generate alloreactive cytotoxic T cells.

Cytotoxic T lymphocytes (CTL) from DBA/2 strain mice primed with Sendai virus (SV) in vivo were activated by secondary stimulation of spleen cells with viral antigens in vitro and analyzed for their target antigen specificity. These effector cells lysed syngeneic Sendai virus infected target cells, marginally a variety of non-infected targets and had a strong cytotoxic effect on H-2b targets. Studies on the antigenic requirements revealed that all SV preparations which generated specific CTL also induced the alloreactive populations. Similar results were found in the response to Newcastle disease virus (NDV) and some influenza A viruses; all these viruses were mitogenic for lymphocytes. Experiments on the cellular requirements indicated that virus specific and alloreactive cells can be separated by their requirements for help and for restimulation. By competition experiments both activities could be attributed to clearly separable T cell subpopulations. The induction mechanism of alloreactive T cells by viral antigens is discussed.     

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.     

In vitro culture of coxsackievirus group B, type 3 immune spleen cells on infected endothelial cells and biological activity of the cultured cells in vivo.

Spleen cells from male BALB/c mice infected 7 days earlier by an intraperitoneal injection of 3 X 10(4) PFU of a myocarditic strain of coxsackievirus B-3 lysed virus-infected endothelial cells in a 51Cr release assay. Cytotoxic activity in the in vivo sensitized spleen cell population could be further increased by culturing the immune spleen cells from infected mice on virus-infected or uninfected endothelial cells for 6 to 7 days in vitro. Cytotoxicity of in vitro cultured spleen cells to infected targets was mediated by T lymphocytes since reactivity was abolished by treatment of the spleen cells with anti-thy 1.2 serum and complement. Reciprocal assays with BALB/c and C57BL cells indicated that maximum cytotoxicity occurred when spleen cells were sensitized on syngeneic endothelial cells. Other experiments showed that spleen cells sensitized to coxsackievirus B-3 or encephalomyocarditis virus were selectively cytolytic to targets infected with the homologous virus. Adoptive transfer of T cells cultured in vitro on infected endothelial cells retained their ability to induce myocarditis in T-lymphocyte-deficient mice.     

Cytotoxic T lymphocyte recognition sites on influenza virus hemagglutinin

When influenza virus infection occurs, part of the cytotoxic T lymphocyte responses induced are directed to the major surface molecule of the virus, the hemagglutinin. However, despite their potential use as a peptide vaccine, little information is available concerning the submolecular areas in the hemagglutinin that are responsible for its immunologic recognition by cytotoxic T lymphocytes. The primary goal of this study is to determine whether submolecular areas recognized by antibodies and helper T cells are also important in the virus-specific, T lymphocyte-mediated cytotoxic responses generated towards virus-infected cells. A panel of synthetic peptides representing areas of the hemagglutinin, homologous to those in influenza AX-31 virus which have previously been shown to bind anti-influenza virus antibodies and provoke proliferation of virus-primed T-helper lymphocytes, was tested in two different cytotoxicity assays. In the experiments presented here, it was found that when selected peptides were incubated with appropriate L929 target cells, lysis by virus-specific cytotoxic T cells was observed. In addition, AX-31-primed lymphocytes preincubated with these synthetic peptides (both individually and as an equimolar mixture) exhibited enhanced lysis of virus-infected syngeneic targets. The cytotoxic responses showed dose-response characteristics in all cases, and in each of the two assays used the same patterns of cytotoxic induction were observed. The recognition of peptides was MHC-restricted since virus-specific cytotoxic T cells from C3H/He mice (H-2k) lysed L929 (H-2k) target cells after incubation with peptides or viruses, but did not lyse P815 (H-2d) targets under the same conditions.     

Cell-mediated immunity to Sendai virus infection in mice.

The development of a cell-mediated immune response to Sendai virus infection in mice was examined by the use of a 51Cr release assay of cytotoxicity. A low level of "background cytotoxicity" to Sendai virus-infected L cells was found in the spleens of uninfected CBA mice. Spleen cells from Sendai-infected mice showed an elevated level of cytotoxicity against these target cells for a period of 5 weeks, commencing 4 days after infection of the mice. A more transient response was observed in the spleens of mice infected with a serologically distinct virus, the Kunz strain of influenza. This cross-reacting, cell-mediated immune response was intermediate between that observed in unsensitized and Sendai-sensitized spleen cells. The relevance of these cell-mediated immune responses to respiratory tract virus infections is discussed.     

Decreased cell-mediated cytotoxicity against virus-infected cells in systemic lupus erythematosus.

Cell-mediated cytotoxicity, directed against virus-infected tissue culture cells, was studied with peripheral blood mononuclear cells from 11 patients with systemic lupus erythematosus (SLE) and 12 matched, normal subjects in a 51Cr release assay. Baseline (preimmunization) levels of cytotoxicity against target cells infected with influenza A/Victoria, influenza B/Hong Kong, Newcastle disease virus, and herpes simplex virus were significantly decreased in patients with SLE compared to normal subjects (P less than 0.001), although serum antibody levels to the respective viruses were similar in both groups. After intramuscular administration of inactivated influenza A/Victoria vaccine, SLE patients failed to generate elevated levels of cytotoxicity against A/Victoria-infected cells, in contrast to normal subjects. SLE patients responded with levels of serum hemagglutination-inhibition antibody which were similar to those of normal subjects. Thus, SLE patients manifest decreased cell-mediated cytotoxicity against virus-infected target cells, although humoral antibody responses appeared to be intact. Studies of SLE patients with influenza may help to define the role of cell-mediated immunity in the pathogenesis of certain viral infections.