John R. Kateley

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

Immunosuppression in Vitro Induced by Leukemia Virus-infected Splenocytes

Summary Immunization of dispersed spleen cells from normal mice in vitro with SRBC was suppressed by simultaneous incubation of the spleen cell cultures with spleno-cytes from mice previously infected with FLV. Cell-free virus preparations alone did not suppress the antibody response. In contrast, relatively small numbers of splenocytes from infected mice, even when present at a ratio of 1-500 normal spleen cells, significantly suppressed the in vitro immune response to SRBC. Viable leukemic splenocytes were necessary for immunosuppression although the leukemic cells did not have to be in direct contact with the normal spleen cells. Specific anti-FLV serum, when added to the leukemic splenocytes or to normal spleen cells separated from infected cells by cell-impermeable membranes, prevented im-munodepression.

Modulation of antibody synthesis by cholera exotoxin: influence on helper thymocytes.

Abstract This report demonstrates that a major biological influence of cholera exotoxin (CT) on antibody formation to sheep erythrocytes is mediated by the toxins influence on the helper function of thymic lymphocytes. Hemolytic plaque-forming cell (PFC) responses for lethally X-irradiated mice repopulated with isologous thymus-marrow cell suspensions were stimulated three- to fivefold when 1.0 μg CT was administered at cell transfer. For mice given marrow cells only, CT did not elevate the PFC response; however, CT stimulated as many PFC in spleens of mice given marrow and 1 × 10 7 thymus cells as mice given marrow and 4 × 10 7 thymus cells. In other transfer experiments, depressed PFC responses were observed when irradiated mice were given marrow and thymus cells from donor mice inoculated with CT 24 hr prior to cell preparation and infusion, or given marrow cells from normal mice and thymus cells from CT-treated mice. In contrast, mice given marrow from CT-treated mice and thymus cells from normal mice responded as well as mice repopulated with normal thymus-marrow cell suspensions.

Elevated cyclic amp levels in mouse lymphoid tissue after stimulation by cholera enterotoxin in vitro.

Summary Addition of CT to suspensions of thymus, lymph node, spleen, or bone marrow cells in vitro resulted in a marked accumulation of cAMP with peak levels occurring 4-5 hr after incubation of cells with CT. Thymus cells showed the largest increase in cAMP, approximately 40-fold at 10 ng/ml CT. Bone marrow cells accumulated the least cAMP (1.5x), while intermediate levels were observed for spleen and lymph node cells (10-12x). Antiserum to CT prevented stimulation of increased cAMP levels. Repopulation studies using X-irradiated mice also showed that thymus-derived spleen cells accumulated more cAMP/107 cells than spleen cells from recipients given spleen or marrow cells. Spleen cells from athymic (nu/nu) mice also responded much less than did spleen cells from normal mice. Thymocytes appeared to bind CT to a greater degree than bone marrow cells. Spleen and lymph node cell suspensions also contained CT-binding cells and the number of CT-binding cells in these peripheral lym-phoid tissues appeared approximately equal to the summation of the numbers observed in thymocyte and bone marrow cell suspensions. Stimulation of cAMP in lymphoid cells, especially thymocytes, by CT provides a pharmacological tool to investigate the mechanism and role of this nucleotide in the early events of antibody formation.

Enhanced splenic atpase activity in immunized mice.

Summary The ATPase activity in spleens of mice immunized with sheep erythrocytes was examined. A maximal increase in ATPase activity was found at the time when peak numbers of specific antibody forming cells were evident in the mouse spleen, 4–5 days after immunization. A relationship was observed between antigen dose and the peak response of both antibody PFCs and ATPase activity. The Mg2+-ATPase activity was specifically enhanced, while no increase in Na+K+-ATPase activity was demonstrable. These results suggest that a relationship exists between Mg2+-ATPase activity and antibody formation in mouse spleens.

Mastocytoma-lnduced Suppression of in Vitro Antibody Formation

Summary Mastocytoma cells in vitro suppressed the immune response of normal syngeneic spleen cells to sheep erythrocytes. The immune response of dispersed spleen cells from normal mice in vitro was inhibited by simultaneous incubation with relatively small numbers of mastocytoma cells or a cell-free extract prepared from these cells. Inhibition occurred when diluted cell-free extracts from the mastocytoma cells or as few as 104 or 105 tumor cells were incubated with 5 million normal splenocytes. The supernatant factor from mastocytoma cells was labile to heating at 56° for 30 min. Detailed examination of the physicochemical properties of the masto-cytoma cell extract capable of suppressing immune responses to sheep red cells should be of value.

STIMULATION OF cAMP LEVELS AND MODULATION OF ANTIBODY FORMATION IN MICE IMMUNIZED WITH CHOLERA TOXIN

Injection of mice with 1.0 mu g of a purified exotoxin derived from Vitro cholerae together with a challenge injection of sheep erythrocytes (SRBC)P OR E. coli LPS markedly influenced the immune response to these antigens. Simultaneous injection of the toxin with antigen resulted in a delayed appearance of antibody-forming cells during the first few days after immunization, followed by a marked enhancement of the peak numbers of antibody-forming cells. In the case of the immune response to SRBC, both 19S and 7S plaque-forming cells (PFC) were enhanced on the peak day of response after simulataneous immunization of toxin-injected mice. The secondary immune response to SRBC was also similarly affected when cholera toxin was given along with a second injection of erythrocytes: i.e. a delay in appearance of the first antibody-forming cells followed by a marked enhancement of the peak 19S and 7S PFC response. Injection of cholera toxin 103 days prior to SRBC or LPS was immunosuppressive. The effect of cholera toxin on the level of splenic cyclic AMP appeared related to the effects on antibody formation.