Modulation of Immune System Function by Measles Virus Infection: Role of Soluble Factor and Direct Infection

Measles virus infection can result in a variety of immunologic defects. We have begun studies to determine the basis for the lack of immune responsiveness to antigen and mitogen following infection. Here we present data showing that Epstein-Barr virus-transformed B-cell lines infected with measles virus produce a soluble factor that can inhibit antigen-specific T-cell proliferation and inhibit the proliferation of uninfected B cells. The soluble factor was neither interleukin-10, transforming growth factor β, nor alpha/beta interferon. B cells infected with measles virus or treated with the soluble factor were unable to present antigen to T cells in a manner that supported antigen-specific proliferation. This could represent one mechanism of how measles virus limits T-cell expansion. However, we found that once CD4⁺ or CD8⁺ T cells were activated, their cytolytic activity was intact whether infected with measles virus or treated with soluble factor. Thus, while slow to be generated these cytoxic cells could participate in viral clearance.     

乙型肝炎病毒特异性细胞毒性T淋巴细胞在病毒清除过程中的作用

在乙型肝炎病毒(HBV)感染过程中,适应性免疫与病毒的致病和清除密切相关。一般认为,体液免疫产生的抗体可以清除外周循环的病毒颗粒,从而阻止病毒在宿主体内的传播,细胞免疫主要清除被感染细胞中的病毒。HBV特异性的细胞毒性T淋巴细胞(CTL)在抑制HBV复制过程中发挥着重要的作用。CTL在肝内主要通过分泌γ干扰素抑制病毒,同时,当CTL识别HBV抗原后,HBV特异性CTL募集抗原非特异性炎症细胞对肝组织浸润,造成肝细胞的损伤。对CTL抗病毒作用进行深入研究,将为乙型肝炎的治疗开辟新的途径。     

Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Selected immunological reactions to measles virus in immunosuppressed guinea pigs

Cyclophosphamide and hydrocortisone treatment of measles virus infected guinea pigs resulted in suppression of cele- and antibody mediated immune reactions although a high dose of measles virus (10(5.5) TCID50) was used for infection: MIT was delayed and depressed; HI serum antibody response was almost completely inhibited.     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

Acute and chronic infection of human lymphoblastoid cell lines with measles virus.

Several human continuous lymphoblastoid cell lines (LCL) having T or B characteristics were infected with low and high passage strains of measles virus. All of the cell lines were susceptible to one or the other or to both strains of measles virus with the production of typical syncytial giant cells and released cell-free infectious virus into the supernatant medium. There was no consistent pattern of susceptibility of LCL with either T or B characteristics to infection by measles virus. Viral induced cytolysis of the lymphoblastoid cells in many of the lines was marked, but in the LCL that could be maintained over longer periods of time, a state of chronic, less cytolytic and persistent infection could be established. The infection was characterized by the production of moderate amounts of cell-free infectious virus for up to 4 1/2 months after initial infection with little change in the number of viable cells in culture. Long-term low multiplicity of infection (MOI) experiments demonstrated that the cell-free infectious virus was being produced only by a small number of cells, but the majority of cells in culture contained measles antigen that was in a cell-restricted, noninfectious, or defective form. Electron microscopic examination of the chronically infected cells demonstrated that many of them contained aggregates of hollow tubular intranuclear nucleocapsids whose "stripped" appearance was in marked contrast to the larger granular intracytoplasmic nucleocapsids found during earlier stages of infection. It is theorized that the persistent infection of LCL may serve as a model in understanding the immune mechanisms which permit latent and chronic measles infection in man.     

Antibody-mediated modulation of the immune response

We have studied the ability of monoclonal IgM and IgG antibodies to enhance or suppress immune responses and attempted to dissect the underlying mechanisms. Both IgM and IgG1 antibodies increased the rate of clearance of antigen from the circulation. Monoclonal IgM antibody to SRBC was found to specifically increase antibody responses, enhancement being insensitive to low doses of irradiation (150 R). IgM antibody specifically depressed the delayed hypersensitivity response to SRBC in vivo. Following administration of IgM in vivo, in vitro responses to SRBC were also enhanced. This in vitro enhancement appeared to depend on both T cells and B cells. In contrast, monoclonal IgG1 antibody to SRBC specifically depressed antibody responses in vivo. Such depressed antibody responses were also seen in vitro following IgG1 in vivo and did not appear to be due to the induction of suppressor T cells.     

Separation of immunomodulatory effects of mannan from Candida albicans into stimulatory and suppressive components

Mannan extracted from Candida albicans was studied for its immunomodulatory activity on in vivo antibody responses to type III pneumococcal polysaccharide (SSS-III), a helper-T-cell-independent antigen, and to sheep erythrocytes (SRBC), a helper-T-cell-dependent antigen. In some studies, the antibody response to SSS-III was converted to a helper-T-cell-dependent response by attaching it to a carrier (horse erythrocytes, HRBC); this complex then was used to immunize mice primed with a subimmunogenic dose of HRBC. Mannan enhanced the antibody response to both SSS-III and SRBC when administered at the same time or 1 or 2 days after immunogen. However, when both mannan and SSS-III were coated onto HRBC for immunization, either enhancement or suppression was noted; the effect depended upon the amount of mannan used. Larger amounts stimulated, whereas smaller amounts suppressed, the antibody response to SSS-III. The enhancing and suppressive components of mannan could be separated by molecular size or charge by chromatography on Sepharose 4B or on DEAE-Sephadex A-50 columns, indicating that mannan extracts contain individual components having opposing immunomodulatory properties. These components can be separated on the basis of molecular size and charge.     

The role of antigen in the activation of regulatory T cells by immune B cells

The transfer of B cells from mice immunized with Type III pneumococcal polysaccharide (SSS-III) results in the activation of suppressor and amplifier T cells that control the magnitude of the antibody response in recipient mice, immunized subsequently with SSS-III. Prior treatment of transferred B cells with an excess of enzyme (polysaccharide depolymerase) capable of hydrolyzing SSS-III, does not alter the capacity of these cells to activate regulatory T cells. These findings indicate that the activation of regulatory T cells by immune B cells is not mediated by residual antigen on the surface of transferred cells.     

Measles virus-specific CD4 T-cell activity does not correlate with protection against lung infection or viral clearance

Acute measles in children can be prevented by immunization with the live attenuated measles vaccine virus. Although immunization is able to induce CD4 and CD8 T cells as well as neutralizing antibodies, only the latter have been correlated with protective immunity. CD8 T cells, however, have been documented to be important in viral clearance in the respiratory tract, whereas CD4 T cells have been shown to be protective in a mouse encephalitis model. In order to investigate the CD4 T-cell response in infection of the respiratory tract, we have defined a T-cell epitope in the hemagglutinin (H) protein for immunization and developed a monoclonal antibody for depletion of CD4 T cells in the cotton rat model. Although the kinetics of CD4 T-cell development correlated with clearance of virus, the depletion of CD4 T cells during the primary infection did not influence viral titers in lung tissue. Immunization with the H epitope induced a CD4 T-cell response but did not protect against infection. Immunization in the presence of maternal antibodies resulted in the development of a CD4 T-cell response which (in the absence of neutralizing antibodies) did not protect against infection. In summary, CD4 T cells do not seem to protect against infection after immunization and do not participate in clearance of virus infection from lung tissue during measles virus infection. We speculate that the major role of CD4 T cells is to control and clear virus infection from other affected organs like the brain.     

Cell-associated IgM, but not IgD or I-A/E, is important in the activation of suppressor T cells by antigen-primed B cells

The ability of transferred antigen-primed immune B cells to induce T cell-mediated suppression of the antibody response to Type III pneumococcal polysaccharide (SSS-III) could be blocked or eliminated by prior treatment of B cells with F(ab')2 anti-Ig or anti-IgM antibodies; however, F(ab')2 anti-IgD antibodies, or M5/114 (monoclonal anti-I-A/E antibody), had no effect on activation of suppression by SSS-III-primed B cells. Thus, cell-associated IgM antibody plays an important role in the activation of suppressor T cells during the antibody response to SSS-III.     

Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.

Measles virus infection of the central nervous system in the murine model of experimental measles virus-induced encephalitis is successfully controlled by virus-specific T-helper lymphocytes. T cells from BALB/c mice that are resistant to measles virus encephalitis proliferate well against measles virus in vitro, and bulk cultures recognize viral nucleocapsid and hemagglutinin as well as fusion proteins. The measles virus-specific T cells secrete large amounts of interleukin 2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) but no IL-4, IL-6, or IL-10, and hence the cytokine pattern is consistent with that of subtype 1 T-helper lymphocytes. In contrast, cells obtained from measles virus-infected susceptible C3H mice recognize measles virus proteins only weakly and secrete little IFN-gamma and TNF-alpha. Treatment of infected mice with anti-TNF-alpha antibodies has no effect on survival or virus clearance from the brain. Upon neutralization of IFN-gamma in vivo, the phenotype of measles virus-specific T-helper cells isolatable from BALB/c mice is reversed from subtype 1 to subtype 2-like. Anti-IFN-gamma antibody-treated BALB/c mice are susceptible to measles virus encephalitis, and viral clearance from the central nervous system is impaired. These results indicate that IFN-gamma plays a significant role in the control of measles virus infection of the central nervous system.     

Production of soluble suppressor factor by spleen cells from mice immunized with type III pneumococcal polysaccharide

Supernatant fluid (SF) derived from spleen cell cultures, obtained from mice 16 hr after immunization with 0.5 microgram of Type III pneumococcal polysaccharide (SSS-III), suppressed the antibody response when SF was given (i.v.) 3 hr before immunization with SSS-III. Such suppression was antigen specific and could be reproduced by SF derived from cultures of T cells from mice immunized with SSS-III (0.5 microgram) or by SF derived from cultures of spleen cells from mice primed with a subimmunogenic dose of SSS-III (0.005 microgram). Adsorption of SF with SSS-III covalently bound to a Sepharose 4B column did not alter the ability of SF to suppress the SSS-III-specific antibody response. However, adsorption of SF with Ig+ (B) cells from mice immunized with 0.5 microgram SSS-III completely removed the suppressive activity. Significant (p less than 0.05) suppression of the antibody response was observed only when SF was administered (i.v.) 24 hr before to 24 hr after immunization with 0.5 microgram of SSS-III. These results suggest that suppressor T cells generated in response to SSS-III function by releasing a soluble factor(s) that binds to determinants on B cells rather than antigen; this soluble factor(s) acts directly on antigen-stimulated B cells or inhibits the induction of amplifier T cells.     

Augmentation of suppressed antibody responses in mice during experimental Chagas' disease by T helper cells activated in a time-dependent mode of immunization

Mice infected with the protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas' disease, develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC), hapten-conjugated SRBC (TNP-SRBC), and horse erythrocytes (TNP-HRBC). Studies in vivo demonstrated that anti-SRBC responses were best enhanced when T. cruzi-infected mice were injected with primed T cells derived from normal or infected mice immunized four days previously. The presence of enhancing capacities for DPFC responses by T cells from T. cruzi-infected mice were also supported by experiments examining the hapten-carrier effect. Preimmunization of infected mice with SRBC or HRBC four days before injection of hapten-homologous (TNP-SRBC or TNP-HRBC) carrier resulted in markedly augmented anti-hapten antibody responses. These results show that functional help provided by T cells activated during priming and exposed to a challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in T. cruzi-infected mice.     

Augmentation of Suppressed Antibody Responses in Mice During Experimental Chagas'Disease by T Helper Cells Activated in a Time-Dependent Mode of Immunization

ABSTRACT. Mice infected with the protozoan parasite Trypanosoma cruzi , the causative agent of human Chagas'disease, develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC), hapten-conjugated SRBC (TNP-SRBC), and horse erythrocytes (TNP-HRBC). Studies in vivo demonstrated that anti-SRBC responses were best enhanced when T. cruz -infected mice were injected with primed T cells derived from normal or infected mice immunized four days previously. The presence of enhancing capacities for DPFC responses by T cells from T. cruzi -infected mice were also supported by experiments examining the hapten-carrier effect. Preimmunization of infected mice with SRBC or HRBC four days before injection of hapten-homologous (TNP-SRBC or TNP-HRBC) carrier resulted in markedly augmented anti-hapten antibody responses. These results show that functional help provided by T cells activated during priming and exposed to a challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in T. cruzi -infected mice.     

The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System

Background

The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus.

Methodology/Principal Findings

Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell–deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus–neutralizing antibody reach infected cells in the cerebellum of B cell–deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies.

Conclusions/Significance

The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus–specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues.     

Non-invasive Imaging of Sendai Virus Infection in Pharmacologically Immunocompromised Mice: NK and T Cells,but not Neutrophils,Promote Viral Clearance after Therapy with Cyclophosphamide and Dexamethasone

In immunocompromised patients, parainfluenza virus (PIV) infections have an increased potential to spread to the lower respiratory tract (LRT), resulting in increased morbidity and mortality. Understanding the immunologic defects that facilitate viral spread to the LRT will help in developing better management protocols. In this study, we immunosuppressed mice with dexamethasone and/or cyclophosphamide then monitored the spread of viral infection into the LRT by using a noninvasive bioluminescence imaging system and a reporter Sendai virus (murine PIV type 1). Our results show that immunosuppression led to delayed viral clearance and increased viral loads in the lungs. After cessation of cyclophosphamide treatment, viral clearance occurred before the generation of Sendai-specific antibody responses and coincided with rebounds in neutrophils, T lymphocytes, and natural killer (NK) cells. Neutrophil suppression using anti-Ly6G antibody had no effect on infection clearance, NK-cell suppression using anti-NK antibody delayed clearance, and T-cell suppression using anti-CD3 antibody resulted in no clearance (chronic infection). Therapeutic use of hematopoietic growth factors G-CSF and GM-CSF had no effect on clearance of infection. In contrast, treatment with Sendai virus—specific polysera or a monoclonal antibody limited viral spread into the lungs and accelerated clearance. Overall, noninvasive bioluminescence was shown to be a useful tool to study respiratory viral progression, revealing roles for NK and T cells, but not neutrophils, in Sendai virus clearance after treatment with dexamethasone and cyclophosphamide. Virus-specific antibodies appear to have therapeutic potential.     

Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2

Antibody production to sheep erythrocytes (SRBC) or hapten-conjugated SRBC (TNP-SRBC) was studied in mice with chronic Trypanosoma cruzi infections. Studies in vivo demonstrated that both IgM and IgG anti-SRBC responses were suppressed during chronic infection. Secondary IgG responses were suppressed regardless of whether the primary immunization was given before or after infection. The ability of cells from infected mice to provide help for antibody production was examined in vitro. Anti-SRBC responses were restored to cultures of whole spleen cells from infected mice by the addition of interleukin 2 (IL 2)-rich supernatants, indicating that these cells were capable of antibody production when sufficient help was provided. T cells from SRBC-primed infected mice were unable to provide significant help to normal B cell/M phi cultures for in vitro anti-TNP or anti-SRBC responses. The percentages of Thy-1+, Lyt-1+, and Lyt-2+ spleen cells were not significantly different between normal and infected mice. Anti-TNP and anti-SRBC responses were restored to cultures that contained T cells from infected mice and normal B cell/M phi by the addition of IL 2-rich spleen cell supernatants. The suppression of in vitro antibody responses in mice with chronic T. cruzi infections was associated with a lack of T cell help, which was provided by exogenous spleen cell supernatant.     

Suppression of measles virus expression by noncytolytic antibody in an immortalized macrophage cell line.

Immune regulation of measles virus (MV) expression was studied in a persistently infected mouse macrophage cell line. Synthesis of both membrane-associated and internal MV antigens was suppressed when infected macrophages were treated with polyclonal rabbit anti-MV antibody that was specific for MV proteins. Persistently infected macrophages were treated for 3, 5, or 7 days with increasing doses of anti-MV antibody. All MV proteins were down-regulated 2 days after treatment was terminated. One week after treatment was terminated, down-regulation was still evident but to a lesser degree. MV protein synthesis was suppressed whether or not complement components were inactivated by heating all serum supplements and antibodies. However, when complement was active, cell lysis accounted for some of the reduced MV protein synthesis. When lytic destruction of infected cells by antibody and complement was prevented by inactivation of complement, antibody alone reduced the cellular synthesis of viral proteins by noncytolytic mechanisms. The absence of cell death in the absence of complement was confirmed by the lack of 51Cr release from labeled cells, the lack of reduction in cell number, and the lack of a decrease in total protein synthesis when radiolabeled infected cells were treated with antibody. It is noteworthy that low doses of antibody were optimal for suppression in the longer-term experiments and did not cause lysis, even in the presence of active complement. Since infected macrophages disseminate virus in measles infection, noncytolytic regulation of these cells by antibody may supplement viral clearance by cytolytic T cells and other immune mechanisms.     

Antigen-Specific Expansion of Cytotoxic T Lymphocytes in Acute Measles Virus Infection

Skewing of the T-cell receptor repertoire of CD8⁺ T cells has been shown in some persistent infections with viruses, such as human immunodeficiency virus, simian immunodeficiency virus, and Epstein-Barr virus. We have demonstrated that similar distortions also occur in nonpersistent measles virus infection. In addition, two of four children immunized with live, attenuated measles virus showed larger and more persistent CD8⁺ T-cell expansions than their naturally infected counterparts. The expanded lymphocyte populations were monoclonal or oligoclonal and lysed target cells infected with recombinant vaccinia virus expressing measles virus protein. These results demonstrate that the expansions of CD8⁺ T lymphocytes are antigen driven.