Stimulating effect of aminoethylisothiuronium on the immune response and interferonogenesis in irradiated mice

Aminoethylisothiuronium (AET) stimulated the formation of antibodies against sheep erythrocytes, not against E. coli, in X-irradiated (4 Gy) mice. The serum containing AET-induced interferon had the same effect. AET also promoted the rejection of the allogenic skin graft in mice irradiated with the same dose. In addition, AET and cystaphos stimulated the induction of interferon by the Newcastle disease virus in mice exposed to doses of 4, 5 or 6 Gy.     

Cellular immunologic reactions in mice tolerant to the transplantation antigens after immunization with the cross-reacting microbial antigens]

In combined administration of cyclophosphamide and lymphocytes of mice (CC57BR XX C3H) F1 to mice CC57BR there was observed a tolerance to alloantigens of mice C3H. Immunization of the tolerant mice with the vaccines of streptococcus, group A, and Candida albicans, containing antigens similar to the transplantation ones, led, to the partial destruction of the tolerance. This was expressed in the reduction in the CC57BR mice of the survival of skin allotransplants of mice C3H and the appearance in the lymphoid organs of lymphocytes with the cytotoxic activity against the allogenic target cells. In case of the tolerance destruction the amount of the recipient's lymphocytes forming rosettes with the erythrocytes of mice C3H remained unchanged, but the stem cell count fell in the spleen and the lymph nodes. The total amount of the T- and B-lymphocytes in the lymphoid organs was unchanged in destruction of the tolerance.     

Alterations of interferon production in a mouse model of thermal injury

The effect of thermal injury on the response of interferon (IFN) production in vivo and in vitro after stimulation with eight representative inducers was investigated in a mouse model. The response of mice to immune IFN (IFN-gamma) inducers, staphylococcal enterotoxin A, concanavalin A, and a specific antigen for BCG-sensitized lymphocytes (purified protein derivative) was impaired after a 30% total body surface area third-degree burn. Suppression of IFN-gamma production was observed at day 2 and persisted until day 7 after burn. Decreased IFN-gamma production correlated closely with the percentage of total body surface area burned. When virus type IFN (IFN-alpha/beta) inducers, Newcastle disease virus, polyriboinosinic-polyribocytidylic acid, 10-carboxymethyl-9-acridanone, and E. coli endotoxin, were administered to mice, no change in IFN response was observed after thermal injury. Similar results were obtained when spleen cells obtained from thermally injured mice were stimulated with IFN-gamma inducers in vitro. These studies suggest that although the capacity for IFN-alpha/beta production remains intact in thermally injured mice, IFN-gamma production may be selectively decreased in burned animals and in their spleen cells.     

Role of T cells in virus control and disease after infection with pneumonia virus of mice

Infection of mice with pneumonia virus of mice (PVM) is used as a natural host experimental model for studying the pathogenesis of infection with the closely related human respiratory syncytial virus. We analyzed the contribution of T cells to virus control and pathology after PVM infection. Control of a sublethal infection with PVM strain 15 in C57BL/6 mice was accompanied by a 100-fold increase in pulmonary cytotoxic T lymphocytes, 20% of which were specific for PVM. T-cell-deficient mice failed to eliminate PVM and became virus carriers in the absence of the clinical or histopathological signs of pneumonia that occurred after infection of control mice. Mice with limited T-cell numbers did not achieve virus control without weight loss, indicating that T-cell-mediated virus control was closely linked to immunopathology. Both CD4 and CD8 T cells independently contributed to virus elimination and disease. Virus control and disease were similar in the absence of perforin, gamma interferon, or tumor necrosis factor alpha. Interestingly, disease and mortality after lethal high-dose PVM infection were independent of T cells. These data illustrate a key role for T cells in control of PVM infection and demonstrate that both T-cell-dependent and -independent pathways contribute to disease in a viral dose-dependent fashion.     

Cytotoxic T cell activity is strain-specific in outbred mice infected with lymphocytic choriomeningitis virus.

The cytotoxic T cell response in outbred mice infected with lymphocytic choriomeningitis virus (LCMV) is strain specific. The same is true for adoptive transfer of fatal LCM disease. The response of individuals within an outbred strain is completely cross-reactive, as shown by using immune lymphocytes and virus-infected macrophage targets from individual mice. Reciprocal exclusion of cytotoxic T cell activity between inbred and outbred mouse strains is the rule, the exception being one strain (H) known to have some C57BL ancestry. Immune T cells from one of 7 H mice specifically lysed LCMV-infected C57BL macrophages. Experiments with inbred mice have shown that only one allele need be shared at either the H-2K or H-2D locus for cytotoxic T cell activity to be manifest. Adoptive transfer protocols may thus be considered in outbred situations, providing that T cells are effective before allograft rejection occurs. Also, the LCMV cytotoxic T cell assay may be useful for determining the degree of H-2 variability in wild mouse populations, as novel H-2 types can be detected and mice need not be congenic.     

Effect of Antilymphocytic Serum on Circulating Interferon in Mice as a Function of the Inducer

MOST investigators concerned with interferon synthesis in vivo have used the experimental procedure described by Baron and Buckler¹, in which circulating interferon is induced by intravenous administration of viruses. When interpreting results, however, it is difficult to know which cells are responsible for circulating interferon synthesis in the animal. Using a radiobiological approach, we have shown that after an intravenous injection of virus, interferon released into the blood stream of mice originates in cell populations of varying radiosensitivities, depending on the virus inoculated². Myxo-virus-induced circulating interferon production is characterized by high radiosensitivity, for serum interferon titres are decreased by more than 90% in C3H/He mice after one total body X-irradiation of 250 r. Moreover, the species specificity of interferon has enabled us to show that circulating interferon induced by Newcastle disease virus (NDV) is of donor type in xenogeneic radiochimaeras, from which we concluded that cells responsible for interferon synthesis with this virus originate from haemopoietic stem cells^3,4. Both granulocytes and lymphocytes fulfil the criteria of very radiosensitive elements derived from haemopoietic stem cells^5,6. We wish to report that myxovirus-induced circulating interferon production is selectively depressed after administration of antilymphocyte serum (ALS).     

Effect of retinoic acid on indices of humoral immunity

The influence of all-trans- and 13-cys-methylretinoate on antibacterial and antiviral immunity was studied in experiments on noninbred C57Bl/6 and (CBA X C57Bl/6) F1 mice. All-trans-methylretinoate was shown to stimulate the production of antibodies to E. coli antigens, with the effect being dose-dependent. At the same time both compounds inhibited the production of inhibitors, interferon and antibodies to influenza virus.     

Therapeutic implications of NK cell regulation of allogeneic CD8 T cell-mediated immune responses stimulated through the direct pathway of antigen presentation in transplantation

Natural killer (NK) cells are a population of innate type I lymphoid cells essential for early anti-viral responses and are known to modulate the course of humoral and cellular-mediated T cell responses. We assessed the role of NK cells in allogeneic CD8 T cell-mediated responses in an immunocompetent mouse model across an MHC class I histocompatibility barrier to determine its impact in therapeutic clinical interventions with polyclonal or monoclonal antibodies (mAbs) targeting lymphoid cells in transplantation. The administration of an NK cell depleting antibody to either CD8 T cell replete or CD8 T cell-depleted naïve C57BL/6 immunocompetent mice accelerated graft rejection. This accelerated rejection response was associated with an in vivo increased cytotoxic activity of CD8 T cells against bm1 allogeneic hematopoietic cells and bm1 skin allografts. These findings show that NK cells were implicated in the control host anti-donor cytotoxic responses, likely by competing for common cell growth factors in both CD8 T cell replete and CD8 T cell-depleted mice, the latter reconstituting in response to lymphopenia. Our data calls for precaution in solid organ transplantation under tolerogenic protocols involving extensive depletion of lymphocytes. These pharmacological biologics with depleting properties over NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic alloresponses refractory to current immunosuppression.     

Modulation by gamma interferon of antiviral cell-mediated immune responses in vivo.

Mice were infected with lymphocytic choriomeningitis virus and injected once 24 h later with a monoclonal antibody directed against gamma interferon. In comparison with controls, the increase of numbers of CD8+ T cells and the generation of virus-specific cytotoxic T lymphocytes in spleens and virus clearance from organs were diminished, as was the ability of spleen cells to transmit adoptive immunity to infected recipients. The same treatment slightly but consistently lessened rather than augmented the virus titers early in infection, which was also observed in thymusless nu/nu mice. Injection into infected mice of the lymphokine itself in quantities probably higher than are produced endogenously resulted in lower virus titers in spleens but higher titers in livers. The adoptive immunity in infected mice achieved by infusion of immune spleen cells was not altered by treating the recipients with gamma interferon monoclonal antibody. Such treatment did not measurably affect the production of antiviral serum antibodies. We conclude that in lymphocytic choriomeningitis virus-infected mice, gamma interferon is needed for the generation of antivirally active CD8+ T lymphocytes, and furthermore that in this experimental model, direct antiviral effects of the lymphokine elude detection.     

Different effects of the immunostimulatory drug Stimforte on infections of hepatitis C virus and herpes simplex virus type 1

Stimforte, an immune response-stimulating preparation, is active with respect to hepatitis C virus (HCV) and herpes simplex virus type I (HSV-1). The effects of Stimforte in animals infected with either HCV or HSV-1 are fundamentally different. In mice with acute herpes virus infection, Stimforte administration leads to a higher activity of natural killer cells and cytotoxic lymphocytes, and the amount of interferon (IFN) λ grows. In mice infected with HCV, Stimforte administration results in a significant increase in IFN-β but not IFN-λ in blood and affected organs. Stimforte has been found to affect directly HCV reproduction that causes the infected cell death, but it does not affect HSV-1 reproduction in the Vero cells (V).     

Intensification of cellular immune response by immunization of mice with the cells preincubated in interferon]

Mice F1(CBA X C57BL/6) were immunized intraperitoneally with a single injection of L-1210 cells preincubated in interferon or control "false" preparation. CBA mice were injected with MX-11 cells: similarly treated according to the same scheme. Injection of interferon-treated cells was accompanied by the enhancement of the cellular immune response. The greatest cytotoxic activity was possessed by the lymphocytes of mice to which M-11 cells were administered together with interferon in which the cells were formerly incubated.     

Alternative mechanisms of respiratory syncytial virus clearance in perforin knockout mice lead to enhanced disease

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/granzyme-mediated 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.     

IFN-gamma- and cell-to-cell contact-dependent cytotoxicity of allograft-induced macrophages against syngeneic tumor cells and cell lines: an application of allografting to cancer treatment.

In allogeneic tumor or skin transplantation, the rejection process that destroys the allogeneic cells leaves syngeneic cells intact by discrimination between self and nonself. Here, we examined whether the cells infiltrating into the allografts could be cytotoxic against syngeneic immortal cells in vitro and in vivo. The leukocytes (i.e., macrophages (Mphi; 55-65% of bulk infiltrates), granulocytes (20-25%), and lymphocytes (15-20%)) infiltrating into allografts, but not into autografts, in C57BL/6 mice were cytotoxic against syngeneic tumor cells and cell lines, whereas the cytotoxic activity was hardly induced in allografted, IFN-gamma-/- C57BL/6 mice. Among the leukocytes, Mphi were the major population of cytotoxic cells; and the cytotoxic activity appeared to be cell-to-cell contact dependent. When syngeneic tumor cells were s.c. injected into normal C57BL/6 mice simultaneously with the Mphi-rich population or allogeneic, but not syngeneic, fibroblastic cells, tumor growth was suppressed in a cell number-dependent manner, and tumor cells were rejected either with a Mphi:tumor ratio of about 30 or with an allograft:tumor ratio of approximately 200. In the case of IFN-gamma-/- C57BL/6 mice, however, the s.c. injection of the allograft simultaneously with tumor cells had no effect on the tumor growth. These results suggest that allograft or allograft-induced Mphi may be applicable for use in cancer treatment and that IFN-gamma induction by the allograft may be crucial for the treatment.     

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.     

Characterization of Virus- and Endotoxin-induced Interferons Obtained from the Serum and Urine of Rabbits

Interferons induced in the rabbit by Newcastle disease virus or by endotoxin have been further characterized as to their physicochemical stability and molecular size by Sephadex G-100 gel filtration. Endotoxin-induced interferon obtained from serum was more labile than virus-induced interferon. Both endotoxin and virus induced interferons obtained from serum contained two peaks: a minor high molecular weight (>100,000) peak and a major lower molecular weight peak. The molecular weight of the major peak induced by endotoxin was 54,000, and that induced by Newcastle disease virus was 46,000. The gel filtration pattern of interferon recovered from the urine of animals inoculated with virus reflected faithfully the pattern found in serum except that there was proportionately less of the high molecular weight peak. However, the urine interferon from endotoxin-inoculated animals contained only one broad peak with a molecular weight of 35,000. This was not the peak fraction present in the serum of such animals. It is postulated that this may represent the basic unit of endotoxin-induced interferon, and that the serum components are either polymers or conjugates of this basic unit.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Mechanisms of leukemogenesis. I. Generation of autoreactive lymphocytes in response to a murine leukemia virus.

Examined in this paper is the capacity of 334C murine leukemia virus (MuLV) to stimulate the generation of virus-specific cytotoxic effector cells in mice of the C57BL/6 strain that are relatively resistant to Friend, Moloney, and Rauscher (FMR) MuLV-induced leukemia, and in BALB/c mice that are relatively susceptible to leukemia induced by FMR MuLV. Generation of cytotoxicity requires in vivo administration of the virus followed by in vitro culture of lymphoid cells from virus-injected animals. Lymphoid cells from MuLV-resistant C57BL/6 donors develop high levels of specific cytotoxicity after secondary in vitro stimulation with syngeneic MuLV-induced tumor cells. Cells derived from these same donors, cultured in the absence of MuLV-induced tumor cells, fail to exhibit cytotoxicity. Secondary in vitro stimulation of lymphocytes from MuLV-susceptible BALB/c animals results not only in generation of cytotoxic reactivity against syngeneic MuLV-induced tumor cells but also induces apparently autoreactive effector cells capable of lysing other H-2d tumor cells as well as normal peritoneal cells bearing H-2d antigens. Moreover, generation of cytotoxicity by BALB/c lymphocytes occurs whether or not MuLV-induced tumor cells are included in the secondary culture system.     

Effect of interferon on the course of experimental E coli-induced pyelonephritis

The effect of the type I interferon on the development and process of experimental pyelonephritis caused by E. coli was studied on mice weighing 12 to 14 g. Interferon was administered intraperitoneally in a dose of 1000 units on days 3 and 7 of the disease. It was shown that the administration of the type I interferon to the mice with experimental pyelonephritis promoted rapid elimination of bacteria from the kidneys, prevented their penetration to the contralateral (intact) kidney, prevented marked macro- and microscopic damages in the kidneys, lowered the intensity of the inflammatory reaction, and increased the phagocytic activity of neutrophils and the number of the E-rosette-forming lymphocytes in the thymus. The data provided experimental grounding for clinical trials of interferon preparations in treatment of bacterial pyelonephritis.     

Suppression of interferon synthesis during the "graft-versus-host" reaction in F1(CBAXC57BL/6) mice]

The development of the graft-versus-host reaction (GVHR) in the F1(1CBA X C57BL/6 hybrid mice after the transplantation of spleen cells from the C57BL/6 parent donor resulted in a strong inhibition of the serum interferon production induced by the intraperitoneal injection of the Newcastle disease virus. In vitro with the mouse bone marrow cells during the development of the GVHR the interferon response was first reduced and then disappeared completely. The described phenomenon could therefore serve as an index of the development of the GVHR.     

ICOS-Expressing Lymphocytes Promote Resolution of CD8-Mediated Lung Injury in a Mouse Model of Lung Rejection

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8⁺ T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8⁺ T cell–mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG^-/- mice, we found that CD4⁺ T cells and ICOS^+/+ T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4⁺Foxp3 ⁺ ICOS⁺ T cells were enriched in the lungs of animals surviving lung injury and ICOS^+/+ Tregs promoted survival in animals that received ICOS^-/- T cells. Direct comparison of ICOS^-/- Tregs to ICOS^+/+ Tregs found defects in vitro but no differences in the ability of ICOS^-/- Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.