Immunosuppression during viral oncogenesis. V. Resistance to virus-induced immunosuppressive factor

Rabbits given malignant rabbit fibroma virus (MV) develop severe immunologic dysfunction during the course of infection. Splenic T lymphocytes from these rabbits elaborate a soluble non-specific immunosuppressive factor (virus-induced suppressor factor (VISF]. As malignant rabbit fibroma virus infection progresses, normal immunologic responsiveness returns. This recovery is multi-factorial and involves production by T lymphocytes of a soluble factor capable of antagonizing the activity of VISF. This soluble anti-suppressor factor (ASF) is not a generalized immunologic potentiator. Its sole apparent effect on immune function appears to be to antagonize the activity of VISF. The protective effects of ASF are evident only when suppressor factors and ASF are simultaneously present in culture. Pre-treatment of target cells with ASF-containing culture supernatants does not render them insensitive to the immunosuppressive effects of subsequent treatment with VISF. In addition, ASF appears to be directly responsible for antagonizing VISF activity. That is, ASF does not appear to initiate an anti-suppressive cascade by activating a population of cells that in turn generate secondary protective factors. ASF-producing cells do not bind Vicia villosa lectin, as do contra-suppressor cells described by others. In almost all of these features, the system we describe herein differs from systems in which other investigators have described factors that antagonize the effects of suppressor factors.     

Immunosuppression in viral oncogenesis. III. Effects of virus infection on interleukin 1 and interleukin 2 generation and responsiveness

Malignant rabbit fibroma virus (MV) is an oncogenic immunosuppressive leporipoxvirus. We studied the effects of MV infection and MV-associated tumor-induced suppressor factor (TISF) on the production of and responsiveness to interleukins 1 and 2. Adherent cells from MV tumor-bearing rabbits elaborate adequate amounts of IL 1 in response to E. coli endotoxin. Neither live virus nor TISF alters the production or the responsiveness to IL 1. However, when we examined spleen cells from rabbits 7 days after MV inoculation, we noted that their ability to produce and respond to IL 2 is deficient. Despite their relatively poor capacity to produce IL 2, these spleen cells express receptor for IL 2 in normal amounts, as measured by the monoclonal antibody 7D4. TISF derived from T lymphocytes from MV tumor-bearing rabbits is by itself capable of inhibiting partially normal secretion of IL 2 and also the response of the cloned murine T cell line HT-2 to added IL 2. Full expression of the immunosuppressive capacity of spleen cells from MV tumor-bearing rabbits requires cell-cell contact, however, and cannot be replaced by either live virus or spleen cell supernatants. Such spleen cells inhibit normal mitogen responsiveness, a defect not remedied by adding exogenous IL 2. Immunologic dysfunction induced by MV infection is transient, and by 11 days after virus inoculation, actively mediated recovery from immunosuppression is observed. We found that spleen cells from rabbits studied 11 days postinoculation secreted IL 2 normally. Thus, immunologic dysfunction secondary to infection with malignant rabbit fibroma virus reflects deficiencies in both elaboration of and response to IL 2, and return of immune function later in the course of the infection is associated with return of the ability of lymphocytes to secrete IL 2.     

Generation by lipopolysaccharide of a late-acting soluble suppressor of antibody synthesis.

Polyclonal stimulation of normal mouse spleen cells by lipopolysaccharide (LPS) from Salmonella typhimurium resulted in the generation of a factor which was capable of suppressing the humoral immune response in vitro. LPS effectively induced the release of the inhibitory material into the supernatants of these cultures within 24 hr. The suppressive mediator, which was similar in properties to the antigen-generated, transiently-acting soluble suppressor (TASS) reported earlier, partially abrogated (by 30–80%) the anti-sheep erythrocyte plaque-forming cell response when added to test cultures ~20 hr prior to assay for direct hemolytic plaques. Although LPS, in submitogenic doses, also was effective in depressing the in vitro hemolysin response, the inhibitory activity of residual mitogen present in the test supernatants, and that of the LPS-induced factor, were shown to be different. By use of antisera and complement treatment to selectively deplete spleen cell populations of T or B lymphocytes, it was demonstrated that B cells were essential for production of the suppressive mediator.     

Enterically induced immunologic tolerance. I. Induction of suppressor T lymphoyctes by intragastric administration of soluble proteins.

Specific immune unresponsiveness was induced in inbred mice (BDF1) by the administration of soluble ovalbumin (OVA) by gastric intubation. Anti-hapten (DNP) responses likewise were specifically diminished when animals were fed autologous carrier (OVA or keyhole limpet hemocyanin). Adoptive transfer of spleen cells demonstrated that the tolerant state could be maintained in irradiated recipient mice, and specific anergy could be transferred to normal recipient animals. Adoptive suppression was mediated by T lymphocytes, as demonstrated by nylon wool fractionation and susceptibility of the cells to anti-Thy 1.2 and complement. Transferred B cells had neither suppressive nor augmentative effects. Enteric administration of OVA also specifically diminished antigen-induced DNA synthesis of primed lymph node T cells, although suppressor cells were not identified in the lymph nodes per se.     

Lymphocyte function in experimental African trypanosomiasis. IV. Immunosuppression and suppressor cells in the athymic nu/nu mouse

The role of T cells in the development and expression of antigen-nonspecific immunosuppression in experimental African trypanosomiasis was addressed. Nude ($\text{nu}\text{nu}$) C57BL/ 6 NIH mice and their thymus-bearing ($\text{nu}\text{+}$) littermates were infected with Trypanosoma rhodesiense and examined for suppression of splenic B-cell responses in vitro to the mitogen LPS. All animals developed splenic unresponsiveness to LPS. Further, both nu/nu and nu/ + infected mice displayed suppressor cell activity in their spleen cell populations upon transfer to normal uninfected mouse spleen cell cultures. On the basis of these findings we suggest that both the generalized immunosuppression and the development of suppressor cell activity in the spleens of mice infected with T. rhodesiense are T-independent processes.     

Immunologic dysfunction during viral oncogenesis. I. Nonspecific immunosuppression caused by malignant rabbit fibroma virus

Malignant rabbit fibroma virus (MV) is a potent oncogenic poxvirus that produces a rapidly progressive syndrome of disseminated myxosarcoma, immunosuppression, and fatal gram-negative infection. MV is probably a recombinant between Shope fibroma virus (SFV) and rabbit myxoma virus, and is capable of preventing or aborting the in vitro proliferative responses of rabbit lymphocytes to B and T lymphocyte mitogens. Proliferative responses to sheep erythrocytes (SRBC) are similarly affected, although MV does not alter ongoing antibody responses to SRBC. Splenic lymphocytes from MV tumor-bearing rabbits suppress antibody and proliferative responses to SRBC when added to lymphocytes from SRBC-primed rabbits. Finally, lysates of cultured splenic lymphocytes from rabbits given MV suppress both proliferative and antibody-forming responses to SRBC. When MV is removed from these lysates by UV inactivation or by centrifugation, the suppressive activity remains. We therefore conclude that MV induces immunologic unresponsiveness in rabbits by at least two mechanisms. First, a direct suppressive effect of added virus on in vitro lymphocyte proliferation is seen. There is no effect in this situation if an antibody response is already in progress. Second, spleen cells exposed to MV in vivo produce one or more soluble factors capable of suppressing both proliferative and antibody responses of normal lymphocytes.     

Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoacetic acid.

Phosphonoacetic acid specifically inhibited human cytomegalovirus DNA synthesis in virus-infected human fibroblasts as detected by virus-specific nucleic acid hybridization. Inhibition was reversible; viral DNA synthesis resumed upon the removal of the drug. The compound partially inhibited DNA synthesis of host cells in the log phase of growth but had little effect on confluent cells. Studies of partially purified enzymes indicated that phosphonoacetic acid specifically inhibited virus-induced DNA polymerase and had only a slight effect on normal host cell enzymes. The drug was shown to interact directly with virus-induced enzyme but not with the template-primers.     

Immunologic dysfunction during viral oncogenesis: II. Inhibition of cellular immunity to viral antigens by malignant rabbit fibroma virus

The ability of two related viruses—Shope fibroma virus (SFV) and malignant rabbit fibroma virus (MV)—to induce virus-specific immune responses in lymphocytes of recipient animals was studied. SFV produces a benign local tumor which regresses in 12–14 days. Using an assay for virus-induced lymphocyte blastogenesis lymphocytes reactive to SFV were detected, both in rabbits bearing SFV-induced tumors and in rabbits whose SFV-induced tumor had regressed. These virus-reactive cells were detected in peripheral blood and spleen, and in lymph nodes draining the primary tumor. In contrast, MV produces a disseminated tumor and eventual death. MV does not induce detectable blastogenic responses in lymphocyte populations. SFV and MV are antigenically cross reactive: rabbits immune to SFV do not develop MV-induced tumors, and antisera to each virus neutralize both equally. Lymphocytes from SFV-infected rabbits proliferate in vitro in response to MV that has been inactivated by ultraviolet light (uv/MV) but not to infectious MV. In contrast, lymphocytes from rabbits infected with MV do not respond to uv-inactivated MV or to SFV. Thus, infectious MV inhibits the development of normal blastogenic responses in vivo and prevents the expression of those responses in lymphocytes from MV-resistant, SFV-immune rabbits in vitro. The relevance of this impairment to the differences in the clinical courses of SFV- and MV-induced tumors is discussed.     

Somatic cell hybridization and problems of viral oncogenesis

In Vitro Cellular &; Developmental Biology - Plant -     

Cell-specific proteins regulate viral RNA translation and virus-induced disease.

Translation initiation of the picornavirus genome is regulated by an internal ribosome entry site (IRES). The IRES of a neurovirulent picornavirus, the GDVII strain of Theiler's murine encephalomyelitis virus, requires polypyrimidine tract-binding protein (PTB) for its function. Although neural cells are deficient in PTB, they express a neural-specific homologue of PTB (nPTB). We now show that nPTB and PTB bind similarly to multiple sites in the GDVII IRES, rendering it competent for efficient translation initiation. Mutation of a PTB or nPTB site results in a more prominent decrease in nPTB than PTB binding, a decrease in activity of nPTB compared with PTB in promoting translation initiation, and attenuation of the neurovirulence of the virus without a marked effect on virus growth in non-neural cells. The addition of a second-site mutation in the mutant IRES generates a new PTB (nPTB) binding site, and restores nPTB binding, translation initiation and neurovirulence. We conclude that the tissue-specific expression and differential RNA-binding properties of PTB and nPTB are important determinants of cell-specific translational control and viral neurovirulence.     

Role of suppressor T cells in herpes simplex virus-induced immune deviation.

Herpes simplex virus type 1 inoculated into the anterior chamber of the mouse eye induces suppression of anti-herpes simplex virus T-cell-mediated delayed hypersensitivity. This suppression is virus-specific, and mediated by splenic T lymphocytes, and it can be adoptively transferred to naive recipients.     

Programmed cell death of T lymphocytes during acute viral infection: a mechanism for virus-induced immune deficiency.

Acute viral infections induce immune deficiencies, as shown by unresponsiveness to mitogens and unrelated antigens. T lymphocytes isolated from mice acutely infected with lymphocytic choriomeningitis virus (LCMV) were found in this study to undergo activation-induced apoptosis upon signalling through the T-cell receptor (TcR)-CD3 complex. Kinetic studies demonstrated that this sensitivity to apoptosis directly correlated with the induction of immune deficiency, as measured by impaired proliferation in response to anti-CD3 antibody or to concanavalin A. Cell cycling in interleukin-2 (IL-2) alone stimulated proliferation of LCMV-induced T cells without inducing apoptosis, but preculturing of T cells from acutely infected mice in IL-2 accelerated apoptosis upon subsequent TcR-CD3 cross-linking. T lymphocytes isolated from mice after the acute infection were less responsive to IL-2, but those T cells, presumably memory T cells, responding to IL-2 were primed in each case to die a rapid apoptotic death upon TcR-CD3 cross-linking. These results indicate that virus infection-induced unresponsiveness to T-cell mitogens is due to apoptosis of the activated lymphocytes and suggest that the sensitization of memory cells by IL-2 induced during infection will cause them to die upon antigen recognition, thereby impairing specific responses to nonviral antigens.     

Fractionation and immunologic assessment of KCl-extracted cardiac antigens in coxsackievirus B3 virus-induced myocarditis.

Hypertonic salt extracts prepared from the heart tissues of adolescent CD-1 mice were fractionated on Sephadex G-100 columns. Two separate fractions were obtained. Fraction I, containing the antigenic immunoreactive activity, was able to inhibit the migration of CVB3-PPD immune mouse peritoneal exudate cells (IMPEC) as well as PEC from mice infected with CVB3 virus alone. Fraction II did not have antigenic activity as assessed by the agarose droplet cell migration inhibition assay. As controls, Fraction I prepared from the livers of spleens of CVB3-infected CD-1 mice was unable to inhibit the migration of CVB3 IMPEC. Unimmunized or "normal" mouse peritoneal exudate cells (NMPEC) were not inhibited by Fraction I. Antibodies prepared against Fractions I and II were unable to neutralize CVB3m virus in the plaque reduction test, and polyacrylamide gel analysis revealed multiple bands in 10% SDS gels.