Steven Specter

Dissociation between the adjuvant vs mitogenic activity of a synthetic muramyl dipeptide for murine splenocytes.

Summary A relatively low-molecular-weight synthetic muramyl dipeptide was found to stimulate antibody formation by normal mouse splenocytes immunized with sheep red blood cells in vitro. This material also resulted in a marked stimulation of “background” antibody-forming cells to the erythrocytes in cultures incubated without antigen. In contrast, the muramyl dipeptide, regardless of concentration, did not induce a marked blastogenic response in vitro, as occurs with adjuvants such as bacterial lipo-polysaccharides. These results suggest that the nonspecific immunostimulatory properties of a low-molecular-weight substance such as the muramyl dipeptide tested, which is commonly found in bacterial cell walls, may not be dependent upon mitogenicity.

Tumor-associated immunosuppressive factors.

Impairment of immune function in tumor-bearing animals is now a generally accepted phenomenon.’-3 It is not yet known, however, whether immunologic dysfunction in malignancy is a necessary component of the neoplastic process or merely an inconsequential sequela. Extensive studies during the past few decades, both clinical and experimental, have provided a broad base of information indicating that many malignancies are accompanied by impairment of either antibody formation, cell-mediated immune responses, or both. It seems plausible that such immunologic deficiencies may be directly related to the emergence and proliferation of transformed tumor cells. This seems likely since it is now evident that tumor cells possess on their surface neoant igen~.~-~ These either may be induced by neoplastic transformation by a tumorigenic agent such as a virus or chemical carcinogen or are histocompatibility antigens usually not present on differentiated adult cells, at least in detectable amounts. It seems reasonable that a fully functioning immune-response mechanism should be capable of dealing with antigenically distinct tumor cells. Indeed, most neoplastic cells in the body are probably rapidly destroyed when recognized as “foreign” by the immune system. In those instances where the immune mechanism is impaired, either by irradiation, immunosuppressive drugs, or other agents, or when individuals have an “innate” immune deficiency, malignancies appear to arise at a much higher rate than in otherwise “normal” individuals. Such observations have been interpreted by many to indicate that an “immunologic surveillance” mechanism normally functions to keep in check malignant Other interpretations are possible, however, including the possibility that immunosuppressed individuals lack a functional immune defense mechanism against latent oncogenic viruses. In this regard, extensive studies in this laboratory over the past decade have been concerned with the nature and mechanism of immunosuppression induced in susceptible strains of mice by oncomaviruses.10-14 Infection of mice with a murine leukemia virus resulted in marked impairment of immune responsiveness to antigens such as sheep erythrocytes, bacterial extracts, and allografts. During such studies it was noted that although a tumor virus per se could be shown to cause immunosuppression directly, virus-associated factors, including soluble extracts from the spleen of virus-infected mice, as well as ascitic fluid, also resulted in immunosuppression, both in vivo and in vitro. In in vifro systems, splenocytes from leukemia virus-infected mice, as well as soluble splenic extracts, markedly impaired the immune competence of normal spleen cells. Although the leukemia virus appeared to be the most likely mediator of such immunosuppression, it was also possible that substances associated with the host response to the Virus infection could be involved. Thus, attempts were made to determine if “virus-free” transplantable tumor cells also

Restoration of leukemia virus-suppressed immunocytes in vitro by peritoneal exudate cells.

Summary Depressed antibody responsiveness to sheep erythrocytes in mice infected with Friend leukemia virus continued in vitro when spleen cell cultures from infected animals were cultured in the presence of antigen. Addition of PE cells from normal donor mice to the immunologically depressed splenocyte cultures resulted in a marked restoration of antibody responsiveness. Restoration of the immune response was PE cell dose-dependent; a ratio of 1 PE cell per 10 splenocytes resulted in the largest numbers of PFCs. These results suggest that impaired antibody responsiveness by spleen cell cultures from FLV-infected mice may be due, in part, to effects on antigen-processing macrophages, since restoration of immune responsiveness occurs by PE cell supplements.

Suppressed Development of Mammary Tumorigenesis in R III Mice Treated Neonatally with BCG

Summary The effects of Bacillus Calmette Guerin on the development of spontaneous virus induced mammary adenocarcinomas in the mammary tumor prone R III strain of mice was investigated. A single injection of 1 × 106 infectious units of living BCG administered to mice between 3 to 4 weeks of age was found to significantly inhibit the rate of development of mammary adenocarcinoma as compared to control mice receiving a placebo injection of medium only. The incidence of spontaneous mammary tumors was essentially identical in both groups (96 vs 98%). However, mammary tumors developed at a consistently later time in the BCG treated animals as compared to the appearance of tumors in the control mice. Mice injected with BCG during the 2nd and 3rd month of life showed a moderately slower rate of development of tumors as compared to animals injected during the 3rd and 4th weeks of life. The possible role of BCG in nonspecific stimulation of resistance to the tumor virus mediated by immunologic factors seems likely.

DISCUSSION PAPER: IMMUNOSUPPRESSION INDUCED IN VIVO AND IN VITRO BY DIFFERENT CLASSES OF PLASMACYTOMAS

Murine plasma cell tumors were initially induced by plastic implants or by injection of mineral oil into BALB/c mice.’ These tumors have been reported to have pathological effects similar to those observed in human multiple myeloma, including severe depression of the immunoresponsiveness of the The nature and mechanism of immunosuppression induced by plasmacytomas are controversial, and varying results have been obtained in different laboratories. Zolla and coworkers reported that without exception plasmacytomas were immunosuppressive for humoral immune response^.^-^ They were not able, however, to demonstrate suppression of cellular immunity.R Conversely, Fenton and Havas have demonstrated only very limited suppression by plasmacytomas. Furthermore, other investigators studying plasmacytoma-induced immunosuppression have reported depressed cellular immunity in mice bearing plasma cell tumors.l0 Initially the immunologic dysfunction in plasmacytoma-bearing mice was attributed mainly to a feedback inhibition, possibly due to excess globulins secreted by the tumor cells.’? 3 More recent studies, however, propose two other possible mechanisms for immunosuppression. Heller and coworkers suggested that viral-associated RNA, isolated from tumor cells, was responsible for immunosuppression,1lp 1 2 whereas Tanapatchaiyapong and Zolla suggested that a soluble factor from the tumor cells mediated immunodepression since normal spleen cell implants, in cell-impermeable Millipore chambers, placed into the peritoneal cavity of plasmacytoma-bearing mice, were severely depressed in their ability to form antibody to sheep red blood c e k 7 They concluded that since normal plasma cells may synthesize “chalones,” which have been implicated as regulators of immune responses, plasmacytoma cells may secrete excess chalones, thus inhibiting immunoresponsiveness. Such chalones, if they exist, do not appear to be immunoglobulins (Ig) , since a non-Ig producing plasmacytoma still caused immunosuppression, thus ruling out feedback inhibition. It is possible that “host factors” other than those directly related to the tumor may influence immune responses. Therefore, an entirely in vitro system that would avoid possible influence of these host factors, using either splenocytes from plasmacytoma-bearing mice or normal spleen cells incubated with cells from tumor-bearing animals, would be a valuable alternative for studying plasmacytoma-induced immunosuppression. Thus, in the present study spleen cells from mice bearing one of four different classes of plasmacytomas were examined for antibody production to sheep erythrocytes, both in vivo and in vitro. Also, spleen and tumor cells from plasmacytoma-bearing mice were tested in vitro for their ability to influence the immunoresponsiveness of normal splenocytes.

INTERACTION OF VIRUSES WITH LYMPHOID CELLS

Publisher Summary This chapter discusses the interaction of viruses with lymphoid cells. Many tumor viruses, especially the leukemogenic oncornaviruses, are markedly immunosuppressive, especially during the initial stages of the tumorigenic process. It is suggested that immunosuppression associated with tumor virus infection may be a necessary prerequisite for development and appearance of a virus-induced malignancy. Various studies have shown that the murine leukemia viruses can interact with antibody-forming cells and their precursors. Infection with MuLV may also affect T cell immunity and macrophages. Infection of tumor virus susceptible mouse strains with a leukemia virus may result in rapid depression of immune responsiveness to a wide variety of antigens, including both T dependent and independent. Immunosuppression often develops prior to overt symptoms of the disease, and the level of suppression is directly related to virus dose and the time interval between virus injection and challenge immunization. Studies with intact animals have shown that leukemia viruses appear to preferentially affect B lymphocytes that are the precursors of antibody-forming immunocytes.

FACTORS FROM LYMPHOID CELL TUMOR AFFECTING IMMUNE RESPONSES

Friend leukemia virus induces erythroblastic leukemia in genetically susceptible BALB/c mice. FLV-containing leukemic cells markedly depressed the humoral immune response to SRBC in the appropriate mouse strain. Both immunosuppression and leukemogenesis were readily transmitted by cell-free virus-containing homogenates of the FLV leukemic splenocytes into normal BALB/c mice. In the present study it was found that both Friend leukemic splenocytes as well as virus containing extracts from the leukemic cells were neutralized by heating and by specific antisera. Suppressive activity passed through a 0.45 mu filter but not a 300,000 MW filter and could be pelleted at 100,000 x g. They were also highly resistant to inactivation by irradiation. Mice given leukemic splenocytes after irradiation with up to 32.000 rads still developed leukemia. Addition of either normal or irradiated FLV-leukemic cells to normal spleen cell cultures in vitro markedly suppressed antibody formation. At least 32,000 rads were required to significantly impair the immuno-suppressive activity of the FLV-leukemic cells. Thus, virus per se appears to be directly responsible for suppression of antibody formation to FLV.

Modulation of the immune response in vitro and in vivo by splenocytes from tumor-bearing mice.

Immunosuppression in mice infected with either a leukemia virus (Friend virus) or bearing a rapidly growing transplantable tumor (either a plasmacytoma or mastocytoma) was studied at the level of individual immunocytes to sheep erythrocytes both in vivo and in vitro. Immunization of mice with progressing tumors showed markedly depressed hemolytic antibody plaque responses in the spleen. Furthermore, spleen cell cultures derived from immunodepressed mice with the tumors revealed the continued impairment of antibody formation in vitro. Relatively small numbers of splenocytes from the tumor-bearing mice suppressed larger numbers of normal spleen cells from control mice. Immunosuppression in all three tumor systems could be related to subcellular factors in that cell-free extracts of the spleens and tumor bearing mice or even ascites fluid or plasma could suppress the normal antibody responsiveness of normal spleen cell cultures. The virus per se or a virus associated factor seemed important in the leukemia virus model but non-virus tumor associated or related substances seemed to be involved in the immunosuppression induced by the plasmacytoma or mastocytoma. Such results support the view that tumor related subcellular factors may be important mediators of immunologic impairment of a hosts immune defense mechanism to a neoplasm.