Ds Nelson

Effects of CKS-17, a synthetic retroviral envelope peptide, on cell-mediated immunity in vivo: Immunosuppression, immunogenicity, and relation to immunosuppressive tumor products

SummaryCKS-17 is a heptadecapeptide corresponding to a region highly conserved in retroviral transmembrane proteins such as p15E. Because a relationship had previously been determined between p15E and immunosuppressive tumor cell products, we examined the effect of CKS-17, control peptides and conjugates thereof on the expression of cell-mediated immunity (delayed-type hypersensitivity, DTH) in mice. Conjugates of CKS-17 inhibited DTH reactions to sheep erythrocytes in the feet of mice. The degree of inhibition was dose-dependent. Unconjugated CKS-17 had almost no effect, and control peptide conjugates had no inhibitory effect. Immunization of mice with CKS-17 conjugates, but not with control conjugates, rendered them resistant to the depression of DTH reactions, not only by CKS-17 conjugates, but also by products of cultured tumor cells. CKS-17 conjugates, but not control conjugates, also depressed the cellular inflammatory reactions induced in mouse footpads by concanavalin A (ConA) and immunized mice against the depression of ConA reactions by products of cultured tumor cells. Injections of globulin from sera of mice immunized with CKS-17 conjugates conferred upon normal recipients resistance to the depression of footpad reactions to ConA by products of cultured tumor cells. Globulin from sera of normal mice or control immunized mice did not confer such resistance. Thus conjugates of a synthetic peptide not only mimic the immunosuppressive effects of tumor products in vivo, but can also immunize mice against those effects.

Evasion of host defences by tumours

Almost from the earliest days of immunology there have been hopes that the immune system could be harnessed to control the emergence, growth and spread of malignant cells. In 1929 these hopes were, for a time, dashed, when it was shown that resistance to transplanted tumours, as then studied, was largely or entirely due to immunity to foreign tissue (1). With the ready availability of inbred animals in the 1950s it became possible to demonstrate the true tumour-specific immunogenicity of some experimental tumours (2-5). Since then hopes for practical applications of tumour immunology have waxed and waned, and we are now in a period of guarded optimism.

Thy and Ly markers on lymphocytes initiating tumor rejection.

Abstract Nonadherent spleen cells from mice specifically immune to the syngeneic methylcholanthrene-induced fibrosarcoma H-7 caused suppression of tumor growth in footpads of nonimmune mice. The cells were Thy-1 positive, largely Ly-1 positive, but Ly-2 and Ly-3 negative, and resembled T cells causing delayed-type hypersensitivity reactions rather than cytolytic T cells. It is suggested that some tumors fail to elicit the production of rapidly cytolytic T cells but can be rejected by a mechanism akin to that of delayed-type hypersensitivity, mediated by a different class of T cells working in collaboration with macrophages.

Inhibition of interleukin-2 production by tumor cell products and by CKS-17, a synthetic retroviral envelope peptide

SummaryTumor cells of all types and species tested have been found to produce, in culture, substances that depress the expression of cell-mediated immunity, in the form of delayed-type hypersensitivity reactions in mouse feet. The factors responsible appear related immunologically to the retroviral envelope protein p15E. We have measured the effects of tumor products and conjugates of a p15E-related peptide, CKS-17, on interleukin-2 (IL-2) production by cultured, mitogen-stimulated EL4 cells; in this system IL-2 production is independent of IL-1. Supernatants of cultures of mouse, human and guinea-pig tumor cells inhibited IL-2 production in a dose-dependent fashion. CKS-17 conjugates, but not control conjugates, also inhibited IL-2 production. Responses to IL-2 of the CTLL line used were less inhibited by tumor products and very slightly inhibited by CKS-17 conjugates. IL-2 receptor density, assayed by flow cytometry, was not inhibited. IL-2 production was inhibited whether the tumor products or CKS-17 conjugates were added early or late in the course of culture of stimulated EL4 cells. Inhibition by CKS-17 conjugates was selective in that IL-2 production was inhibited to a greater degree than general protein synthesis in EL4 cells, and general protein synthesis by fibroblasts was unaffected. Measurement of IL-2 mRNA suggested that inhibition of IL-2 production was mediated post-transcriptionally. Fractionation of six different tumor supernatants on Sephacryl S-300 revealed a single peak of activity with an apparent molecular mass of 18 kDa. Antibodies to CKS-17 conjugates neutralized the inhibitory effect of native tumor products on IL-2 production. Inhibition of IL-2 production, by factors related to p15E, provides a strategically effective means of subversion of host defenses by tumors, and abrogation of this inhibition by means of antibodies might promote host resistance to tumor growth.

Macrophages and resistance to tumors

SummaryDelayed-type hypersensitivity (DTH) reactions in mouse feet were depressed by irradiation and by treatment with carrageenan, niridazole, or reserpine. Specific resistance of immunized mice to footpad challenge with a syngeneic methylcholanthrene-induced fibrosarcoma was also depressed by irradiation, carrageenan, niridazole, and reserpine. Growth of the tumor in the feet of normal mice was unaffected by irradiation or niridazole. It could be inhibited or enhanced by carrageenan treatment, depending on the dose of tumor cells injected. Paradoxically, treatment with reserpine inhibited tumor growth in the feet of nonimmune mice. It is suggested that: (a) specific, acquired resistance to this tumor is strongly akin to DTH; (b) mice offer some natural resistance to this tumor; (c) the establishment of an isograft of this tumor may depend on the occurrence of some degree of inflammation.

Festschrift Inhibition of cell-mediated immunity by tumour cell products: Depression of interleukin-2 production and responses to interleukin-2 by mouse spleen cells

Supernatants from cultures of mouse and human tumour cells inhibited the production of interleukin‐2 (IL‐2) by stimulated mouse spleen cells. The tumour cells tested, all of which were active, included a mouse and a human melanoma, three methylcholanthrene‐induced fibrosarcomas of mice, and human HeLa cells. Supernatants from normal mouse and human fibroblasts were inactive. Inhibition was dose‐dependent. Spleen cells from aged mice were more susceptible to inhibition than spleen cells from young mice. When tumour cell culture supernatants were fractionated on Sephacryl S‐300, two peaks of activity were found, with apparent molecular weights of approximately 50 and 18 kD. Supernatants from tumour cell and fibroblast cultures caused variable, but generally weak, inhibition of responses of lymphoblasts to IL‐2. It is suggested that inhibition of IL‐2 production may be an important mode of action of tumour cell products that inhibit cell‐mediated immunity.

Depression of cell-mediated immunity by tumour cell products: induction of resistance by immunotherapeutically active extracts of bovine ocular squamous cell carcinoma

SummaryTumours produce substances that inhibit the expression of cell-mediated immunity, in the form of delayed-type hypersensitivity in mice. Phenol-saline extracts of bovine ocular squamous cell carcinoma (BOSCC) which have immunotherapeutic activity in cattle were able to immunize mice against this depressive effect. Such immunization was effective against products of BOSCC, a spontaneous rat tumour, three of four human tumour cell lines and (in other experiments) mouse tumours. Phenol-saline extracts of mouse tumour cell lines were immunogenic (protective against depression of delayed-type hypersensitivity) in mice. Fractions of BOSCC phenol-saline extracts which were immunotherapeutically active in cattle were generally also protective in mice. The protective activity was lost after treatment with proteinase K, and was present in the supernatant after precipitation with 55% ammonium sulphate. It was not affected by treatment with RNase or DNase or by heating to 50 °C for 2 h. It was present in gel filtration fractions with an apparent molecular weight of 10,000–37,000 daltons. The immunogenic factor in mice and the immunotherapeutic factor in cattle may be related to each other.

Stimulation of Proliferation in Mixed Cultures of Mouse Tumor Cells and Nonimmune Peritoneal Cells. I. Occurrence of Stimulation and Cyclical Variation in Tumor Cell Activity

Mixtures of mouse tumor cells and resident peritoneal cells (PC) from nonimmune mice frequently incorporated [methyl-3H]thymidine [( 3H]dThd) in culture to a far greater extent than did either cell population alone. With PC-to-tumor cell ratios of 10:1, stimulation indices of up to 21 were recorded. Syngeneic, semisyngeneic, and allogeneic mixtures were active. PC elicited by thioglycollate or proteose-peptone could participate in the reaction. PC from mice injected with Corynebacterium parvum also could do so, provided they were precultured for 48 hours. Normal mouse spleen cells and fibroblasts and human Chang cells were inactive. There was considerable variation between experiments in the extent of stimulation observed, even with the same tumor. When tumors were cloned in vitro, freshly isolated clones differed widely in the extent of stimulation. Individual clones studied over a period of time varied markedly and cyclically in the degree of stimulation they exhibited in mixed cultures. This variation appeared to be unrelated to the density of the cultures from which the clones were obtained, to the base-line incorporation of [3H]dThd by tumor cells alone, or to the frequency of passage in vitro. It is suggested that the cyclical variation may be related to the generation of variant cells, which could be important in tumor progression and metastasis.

Two anti-tumour cytotoxic cells in the peritoneal cavity of rats: natural occurrence, augmentation and partial characterization.

Mononuclear cells cytotoxic to methylcholanthrene-induced fibrosarcoma cells were detected in the peritoneal cavities of normal rats by means of 16 h 51Cr release and 48 h 125IUdR release assays. Carriage of a tumour which induces concomitant immunity, or intraperitoneal injections of Corynebacterium parvum or proteose-peptone, led to increases in mononuclear cell numbers. Injections of C. parvum and proteose-peptone led to increases in cytotoxicity in 51Cr release assays. Carriage of an immunogenic tumour led to an increase in cytotoxicity to homologous tumour cells in the 125IUdR release assay. Cells were separated by sedimentation velocity, metrizamide density gradients and adherence to glass or plastic. Small cells were more active than large cells in 51Cr release assays. Larger cells tended to be more active in 125IUdR release assays. Generally, low density cells were more active in both assays, except that high density cells induced by C. parvum were most active in 125IUdR release. Adherent cells from C. parvum-injected rats were more active than non-adherent cells in both assays. On the basis of differential stimulation and separation by sedimentation velocity it is suggested that two cell types, possibly NK cells and macrophages, are operative. 125IUdR-labelled fibrosarcoma cells were lysed after intraperitoneal injection into normal and concomitantly immune rats, more rapidly extensively in the latter case. These cytotoxic cells may be important in both natural and acquired resistance of rats to tumour growth.

Macrophages in immunobiology.

SummaryMacrophages are now known to be important not only in resistance to infection but also in immunoregulation, in resistance to tumours and as secretory cells. Among problems requiring further investigation are the ways in which they destroy intracellular organisms, the ways in which they recognize and destroy tumour cells and the extent of their heterogeneity.