Antigenic reactivation in vitro of the cytotoxic activity of lymphoid cells from mice bearing syngeneic tumors]

The cytotoxic activity of lymphoid cells from mice bearing syngeneic sarcomas is increased when these cells are preincubated on heavily irradiated cells of the same tumours. This secondary stimulation is highest at the time when the cytotoxicity measured directly is relatively low. In consequence, whatever the age of the tumour, the level of cytotoxicity reached (35-45 %) cannot be increased further in normal conditions.     

In vitro studies of splenocyte cytotoxicity against syngeneic mammary tumors of mice

Summary Spleen cells from BALB/c mice immunized with D7T4S (MTV-negative) or D14 (MTV-positive) mammary tumors exhibited marked cytotoxic activity for the corresponding tumor cells in a terminal ⁵¹ -Cr-labeling cytotoxicity assay. A pronounced, seemingly nonspecific cytotoxic effect was displayed by splenocytes derived from normal BALB/c and BALB/cfC3H mice subjected to various surgical procedures 10–14 days before testing. Possible mechanisms underlying this phenomenon are discussed.     

Suppression of in vitro lymphocyte stimulation in mice bearing primary Moloney sarcoma virus-induced tumors

A marked depression of phytohemagglutinin (PHA) reactivity was observed in spleen cell cultures of C57B1/6N mice bearing primary Moloney sarcoma virus (MSV)-induced tumors. This defect was most pronounced 14 days after virus inoculation (MSV 14) and was reversed after regression of the tumor. Spleen cells from mice with primary methylcholanthrene-induced sarcomas were similarly deficient while no such effect was observed during the first weeks after inoculation of Moloney leukemia virus. The responses of MSV 14 spleen cells to Concanavalin A (Con A) were as consistently depressed as those to PHA, but reactivity to bacterial lipopolysaccharide was affected to a lesser degree. Stimulation by pokeweed mitogen (PWM) was not significantly lower in tumor-bearing mice than in control animals. Passage of MSV 14 spleen cells over rayon adherence columns which removed about 75% of the initial cell population led to an almost complete restoration of their PHA and Con A responses on a per cell basis. This may indicate that within MSV 14 spleens, T lymphocytes reactive to PHA and Con A are diluted out by a majority of unreactive cells. However, the possibility also exists that column passage removes a suppressor cell that actively inhibits these responses.     

Opsonization of tumor-reactive T cells in SJL/J mice bearing syngeneic tumors

Summary The role of antigen-reactive cell opsonization (ARCO) in a syngeneic tumor system and its effect on tumor progression was investigated. Thus, anti-tumor reactive T cells were prepared in vivo by immunization of normal SJL/J mice with mitomycin C-inactivated tumor cells of the syngeneic transplantable reticulum cell sarcoma (RCS) line LA-6. Dividing cells were subsequently labeled by injecting iodo-2-deoxyuridine (¹²⁵IUdR) into the same animals 3 days later. Antigen-reactive cells (*ARC) present in the radiolabeled, nylon wool-fractionated spleen cell population taken from these mice on day 4 and injected IV into syngeneic SJL/J mice bearing LA-6 tumors were diverted to the liver and away from the spleen. The effect was maximal by 8 days following inoculation of tumor cells, and was specific inasmuch as ¹²⁵IUdR-labeled cells prepared by immunization with allogeneic spleen or tumor cells which were not opsonized in day-8 LA-6 tumor-bearing mice. Opsonization of *ARC in day-8 LA-6 tumor-bearing mice was completely abrogated by either prior injection of heat-aggregated immunoglobulin into the mice or preincubation of the *ARC in solubilized tumor antigen before injection into tumor-bearing mice, demonstrating the involvement of Fc receptors in the host and antigen-specific receptors on the *ARC, respectively, in the opsonizing process. When anti-LA-6 reactive T cells were incubated in serum from LA-6 tumor-bearing mice and then injected IV into normal syngeneic SJL/J mice, a similar liver diversion was observed. Serum from cyclophosphamide-pretreated mice injected with LA-6 or serum from mice given mitomycin C-inactivated LA-6 cells did not cause opsonization of tumor-reactive T cells, while a mixture of these two sera did have some *ARC opsonizing activity. Further experiments with SJL/J mice bearing spontaneous RCS tumor indicate that tumor-reactive T cells are also opsonized in these mice. The above studies and others suggested that ARCO may play an important role in vivo in the survival of tumors. Abbreviations used in this paper are: ARC, antigen-reactive cells; ARC, radiolabeled antigen-reactive cells; ARCO, antigen-reactive cell opsonization; LA-6 tumor line derived in our laboratory; L.I., localization index; PEG, polyethylene glycol; RCS, reticulum cell sarcoma; STA, soluble tumor antigen; TBS, tumor-bearer serum     

In vitro reactivity of splenic lymphocytes from normal and UV-irradiated mice against syngeneic UV-induced tumors.

Skin tumors induced in mice by chronic ultraviolet (UV) irradiation are highly antigenic and are frequently immunologically rejected upon transplantation to normal syngeneic recipients. In this study we characterized this immune response with an in vitro microcytotoxicity test. Cytotoxic activity was present in the spleen cells of mice given a single injection of syngeneic UV-induced fibrosarcoma cells. After removal of adherent spleen cells, the remaining splenic lymphocytes were specifically cytotoxic for the immunizing tumor and showed no cross-reactivity with other syngeneic UV-induced or methylcholanthrene-induced tumors of similar histologic type. The level of cell-mediated reactivity against UV-induced tumors was quite high compared to that obtained with syngeneic tumors induced by methylcholanthrene, and the cytotoxicity was attributable to a population of theta antigen-bearing lymphocytes. With this in vitro test, we compared the response of normal mice, which reject a syngeneic tumor challenge, with that of UV-irradiated mice, in which the syngeneic UV-induced tumors grow progressively. After tumor cell inoculation, lymphocytes form the unirradiated (regressor) mice showed a high degree of cytotoxicity that reached a maximum level 8 days after injection. In contrast, no reactivity could be detected in the spleens of tumor-challenged UV-irradiated (progressor) mice.     

Characterization of suppressor cells in mice bearing syngeneic mastocytoma.

Suppressor cells from syngeneic P815 mastocytoma-bearing DBA/2 mice that inhibit in vitro generation of specific anti-tumor cytotoxicity were characterized. Suppressive activity was almost completely eliminated by treating suppressive spleen cells with anti-theta serum and complement. Treatment with anti-mouse lg serum and complement or with carbonyl iron did not affect their suppressive activity. When suppressive thymocytes from P815 tumor-bearing DBA/2 mice were tested for their capacity to inhibit the generation of cytotoxicity against L1210 cells, a leukemia line in DBa/2 mice, they did not affect the activity, indicating that the supressor cells in the thymocytes of P815 tumor-bearing mice are specific to the tumor. When Ficoll-Hypaque density cell separation was carried out with cytotoxic spleen cells and suppressive spleen cells from 815 tumor-bearing mice, the dense fraction was enriched for kiler cells whereas the suppressive activitty was mainly recovered in the light fraction. Therefore, killer cells and suppressor cells in P815 tumor-bearing mice are thought to be distinct populations.     

Generation of cytotoxic lymphocytes in vitro: response of immune rat spleen cells to a syngeneic gross virus-induced lymphoma in mixed lymphocyte-tumor culture.

Spleen cells from W/Fu rats 4 to 6 weeks after immunization with syngeneic Gross virus-induced lymphoma (C58NT)D cells usually lack detectable activity in a short-term 51Cr release assay. The results presented here demonstrate that these spleen cells retain the capacity to generate significant proliferative and cytotoxic activity upon re-exposure to mitomycin C-treated (C58NT)D cells in vitro. Optimal conditions were defined in W/Fu rats for this secondary immune response in vitro to the (C58NT)D cells. The cytotoxic response was observed to be quantitative, reproducible, and specific. Optimal generation occurred 5 days after initiation of cultures with a 30:1 responding cell:stimulating cell ratio. In vitro generated cytotoxic cells inhibit tumor growth in vivo when administered as a mixture with tumor cells.     

Cell-mediated cytotoxic responses of spleen cells from mice bearing primary, chemically induced tumors.

Sarcomas were induced in C57BL/6 mice by using 3-methylcholanthrene. Spleen cells taken from these mice bearing primary, chemically induced tumors and from matched control mice were assessed for the capacity to generate cell-mediated cytotoxic cell activity after in vitro sensitization. Spleen cells from tumor-bearing mice generated strong cell-mediated cytotoxic responses against alloantigens and antigens on syngeneic cells.     

Cell-mediated immune response to syngeneic ultraviolet-induced tumors. II. The properties and antigenic specificities of cytotoxic T lymphocytes generated in vitro following removal from syngeneic tumor-immunized mice.

The immune responses of C3Hf mice to syngeneic fibrosarcomas induced with either ultraviolet light or methychlolanthrene (MCA) were measured in vitro by the ability of cytotoxic lymphocytes (CTL) from immunized animals to kill ⁵¹Cr-labeled tumor targets in a 6-hr assay. The CTL were generated by the in vitro culturing of draining popliteal lymph node (DLN) cells derived from animals that were footpad immunized 8 days previously. It was determined that CTL activity could be generated using DLN from both normal (uv tumorresistant) and uv-exposed (uv tumor-susceptible) C3H mice. The kinetics of CTL generation between these two groups, however, was different in that the lymphocytes from normal animals appeared to differentiate into CTL faster than the lymphocytes from the uv-irradiated mice. The in vitro generation of CTL activity was found to be extremely radiosensitive and was also inhibited by the presence of viable tumor cells within the cell culture. Once generated, it was observed that the CTL were extremely insensitive to the effects of gamma irradiation. It was also established that the CTL is a T lymphocyte that appears to be Ia^?. The CTL derived from mice immunized to syngeneic uv- or MCA-induced tumors were capable of expressing cross-reactive non-MHC-restricted killing of multiple tumor targets. Cold cell inhibition experiments confirmed the presence of cross-reactive determinants on various tumors and also established the presence within a single CTL preparation of effector cells with specificity for both the unique tumor specific transplantation antigens as well as the common (cross-reactive) tumor-associated antigens.     

Functional hypersplenism in mice induced by adoptive transfer of syngeneic spleen cells

Investigation of peripheral blood cell count alterations in cases with hypersplenism, and an understanding of the relationship between splenic function and hematopoietic cell production require suitable experimental animal models. Previously described methods are either traumatic or require surgical intervention. We suggest a relatively simple method for achievement of a state mimicking hypersplenism in mice by intraperitoneal inoculation of syngeneic spleen cells. Mice were inoculated intraperitoneally with 3 x 10(7) splenocytes suspended in 0.3 ml phosphate buffered saline (PBS). After 2 months, the inoculated animals showed a progressive decrease in the peripheral white blood cell (WBC) counts and hyperplastic bone marrow that persisted until the experimental end point (7 months). Five days after inoculation of splenocytes stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE), the majority of the stained cells was present in the peritoneal cavity (33%) and in the liver (13%), whereas the percentage of stained cells in the peripheral blood and the spleen cell suspension was negligible. The mitogen response of the peripheral blood mononuclear cells (PBMC) from treated mice to concanavalin A (Con A) remained unaltered. Splenocyte-inoculated mice that were further splenectomized did not show leukocytosis after splenectomy, as was observed in animals in which the spleen was removed without any pretreatment. The lack of any signs of discomfort in animals from the study group, in comparison with the visibly ill appearance and even death of mice in which hypersplenism was achieved by repeated injections of methylcellulose (MC), which served as controls, favors the convenience of the method.     

Differences in antibody production after adoptive transfers of syngeneic and allogeneic spleen cells to irradiated mice

Immunologically competent spleen cells from syngeneic or allogeneic donors were transferred to supralethally irradiated mice. At the same time, or 72 h after transfer, sheep red cells were administered intraperitoneally to the hosts. Five days after administration of the sheep red cells, antibody production was determined in host spleen fragments by the method of haemolysis in agarose. It was found that an incompatible allogeneic environment markedly stimulated proliferation of the donor cells and that proliferation of clones of cells reacting specifically to sheep red cells by antibody production was also stimulated.     

Measles virus-induced suppression of lymphocyte reactivity in vitro

Measles virus (Edmonston strain B), in various multiplicities of infection, was added to human lymphocytes which were cultured in medium containing fetal bovine serum. Live measles virus was found to cause an almost complete inhibition of [³H]-thymidine incorporation in lymphocytes cultured in the presence of phytohemagglutinin, pokeweed mitogen, tuberculin purified protein derivate (PPD), or allogeneic lymphocytes. Analysis of cell size in the lymphocyte cultures revealed that blast transformation was inhibited as well. Measles virus, inactivated by heat or ultraviolet irradiation, did not cause inhibition. The inhibitory effect of measles virus was only measurable in the initial stages of culture; when added later, i.e., 24 hr before measuring [³H]-thymidine incorporation, it had no effect. The diminished reactivity of measles virus-infected lymphocytes cannot be explained by cytopathologic effects or by altered kinetics of lymphocyte transformation. When lymphocytes were cultured at 39 °C the extent of virus-induced suppression was significantly reduced. Very small amounts of pooled normal human serum, as well as IgG, prepared from the serum of a patient with subacute sclerosing panencephalitis, were able to prevent the inhibitory effect of measles virus.     

In situ lymphoid cells of mouse mammary tumors. IV. Comparison of functional activity of lymphoid cells separated from mammary tumors to that of spleen and lymph node cells of tumor-sensitized mice.

Lymphoid cells isolated form several types of mouse mammary tumors are capable of stimulating tumor cell growth or survival in MCT assays. Lymph node and spleen cells of mice bearing such a tumor are specifically cytotoxic to the tumor cells. Surgical removal of the tumor is followed in 4 to 7 days by the appearance of stimulatory capacity in spleens and lymph nodes. By day 10, cytotoxic cells specific for the sensitizing tumor are again detected. These reach a peak on day 13. By day 17 no reactivity is detectable. The functional distribution of tumor-reactive lymphoid cells is different between tumor masses and peripheral lymphoid organs.     

Genetics of mouse mammary tumor virus-induced mammary tumors: linkage of tumor induction to the gag gene

Retroviruses are believed to induce tumors by acting as insertional mutagens that activate expression of cellular protooncogenes. Indeed, almost 90% of mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/He mice show upregulation of Int protooncogenes. We have analyzed three different MMTV variants [MMTV(C3H), MMTV(HeJ), and a genetically engineered MMTV hybrid provirus (HP)] for tumorigenicity in mice from two distinct genetic backgrounds. All three viruses were tumor causing in BALB/cJ mice. However, only MMTV(C3H), but not MMTV(HeJ) or HP, induced mammary tumors in C3H/He mice. All of the viruses were infectious on either background and up-regulated expression of Int genes in tumors they induced. Like HP, MMTV(HeJ) was found to be a genetic recombinant between endogenous Mtv1 provirus and exogenous MMTV(C3H). Sequence comparison of MMTV variants linked the tumorigenicity of MMTV(C3H) to the gag region of the retrovirus.