Induction of simian virus 40 transplantation immunity in hamsters by gel-purified SV40 large T antigen.

Simian virus 40 (SV40) large T antigen and p53 cellular protein were isolated from an SV40-transformed hamster cell line by immunoprecipitation with anti-T sera and purified by sodium dodecyl sulfate-gel electrophoresis. These two protein were tested in hamsters for the presence of SV40 transplantation rejection antigenic sites by in vivo transplantation rejection assay. The large T antigen immunized the hamsters against a challenge of SV40 tumor cells and the protected animals generated cytotoxic spleen cells. Hamsters immunized with the p53 cellular protein were not protected against SV40-induced tumor but there was some delay in the appearance of tumor.

Spleen cell populations in normal and tumor-bearing hamsters.

Spleen cell subpopulations from normal and tumor-bearing hamsters (TBH) were quantified using Petri dishes coated with specific antibodies, and by flow cytometric immunofluorescence analysis. The relative numbers of T cells (Thy + cells) decreased, by both methods, as a function of tumor growth, while the number of B cells (bearing surface Ig) increased. Cells without T or B markers (null cells) were more numerous in the spleen of TBH and a large number of them expressed the receptor for the Fc fragment of IgG. Splenic cells were also sorted according to their light-scattering properties, and electron microscopic analysis was performed on the sorted fractions. It showed the presence of secreting plasmocytes and activated macrophages in the spleen of TBH.

Variations in the levels of IgG1 and IgG2 subclasses in the sera of normal, immunized and tumor-bearing hamsters.

The concentration of IgG1 and IgG2 subclasses in the sera of hamsters bearing tumors of different origins were compared to that of normal serum and to that of sera of animals rendered resistant to tumor take by immunization with viable SV40-transformed cells. In the sera of hamsters bearing tumors induced by virus-transformed cells an augmentation of IgG2 and a diminution of IgG1 was observed during the development of the tumor compared to sera of normal hamsters. In the sera of animals bearing tumors induced by methylcholanthrene ( MCH2 ) or by spontaneously transformed cells ( EHB ) the level of IgG2 was almost normal but IgG1 was barely detectable, especially in MCH2 tumors. On the other hand, the sera of animals immunized with virus-transformed cells showed a slight increase in both IgGs, but only that of IgG2 was significant. Antibody activity was tested in the sera as well as in the IgG1 and IgG2 fractions of the sera of hamsters immunized or bearing tumors induced by SV40 transformed cells. Both sera and the subclasses showed antibody activity, the activity being more pronounced in the IgG2 fraction than in the IgG1 fraction.

Immunologic and enzymatic studies of two breeding lines of NZB mice that differ in chromosome breakage.

Two sublines of NZB/BI mice were developed by selective matings according to chromosome breakage frequencies. These sublines--HB, a line with high chromosome breakages, and LB, a line with low or normal breakage rates--were studied in regard to the age of the animals as it related to two different aspects. The first aspect was immunologic: A decreased response to T-cell mitogens was found in old NZB mice, but this response was more pronounced in HB mice. The response to the B-cell mitogen (lipopolysaccharide) was increased in both sublines as compared to that in BALB/c mice. The percentages of IgG-positive and theta-positive spleen cells were evaluated in both sublines: Some increase in IgG-positive cells was observed in the spleens of 2- to 8-month-old NZB mice and a slight decrease was seen after age 8 months. The percentage of theta-positive cells diminished according to the age of the mice, and the decrease occurred earlier in HB than in LB mice. The second aspect studied was enzymatic and concerned the levels of DNA alpha- and beta-polymerases and terminal DNA nucleotidyltransferase in the thymuses and spleens of these animals. The major finding noted was an augmentation of 100-200% in the terminal DNA nucleotidyltransferase levels in HB thymuses by comparison with LB thymuses. The levels of both polymerases were increased in spleen cells of HB mice as compared to those of LB mice.

In vivo studies of spleen lymphoid cells implicated in antitumor immunity in hamsters.

Hamsters were immunized with repeated and progressive doses of strongly antigenic SV40-transformed cells (ZDCl25) and these animals rejected a tumor graft. According to the in vivo Winn test, spleen cells from such immunized animals could be used to transfer the antitumor immunity and protect naive recipients. To characterize the immune effector cells, different techniques of fractionation were used. In all cases the best protection was obtained by whole cell populations, even after reconstitution. The "T-depleted' fraction was more active than the T cell fraction. As shown by the treatment with specific antisera, part of the active population bore surface IgG. The protective activity could be the result of cooperation between different cell populations, one of which, unknown at present, could be the promotor of the transferred immunity and of the tumor neutralization effect.

Nude Syrian hamsters: some immunological and histological characteristics.

Nude mutants appeared in our colony of Syrian hamsters. They were hairless and just had rudiments of thymus. Only 3.5% of splenic cells were killed by rabbit anti-hamster thymocytes serum + C' whereas 55.5% of these cells were lyzed by an anti-hamster IgG + C' and 60-70% fixed the fluorescent protein A, but could not respond either to B-cell mitogens or to rat cell mitogens. The natural cytotoxic activity of the spleen cells from nude hamsters was evaluated in comparison with the same activity expressed by spleen cells of golden Syrian hamsters.

Isolation of two immunoglobulin G subclasses, IgG2 and IgG1, from hamster serum using protein A-sepharose.

The isolation of hamster immunoglobulin classes and subclasses by affinity chromatography on protein A and selective elution was studied using 0.1 M phosphate buffer, pH 8. The IgG fraction was completely absorbed, while IgM did not bind. Sequential application of buffers of decreasing pH allowed the elution of pure IgG2 (eluted at pH 6) and IgG1 (eluted at pH 5). Both subclasses were fully recovered. IgG2 could be subfractionated into 2 peaks eluted respectively at pH 6.5 and 6. Immunodiffusion of the whole IgG2 fraction against anti-hamster immunoglobulin serum gave 2 precipitation lines. One of these lines was missing in the pH 6.5 fraction. Until now only 2 IgG subclasses have been described and these results suggest heterogeneity of hamster IgG2.

Evolution of DNA polymerase alpha, beta and terminal deoxynucleotidyl transferase in hamster lymphoid populations during the development of different types of tumors.

We followed the evolution of DNA polymerase alpha, beta and terminal deoxynucleotidyl transferase (TdT) in the thymus, spleen and bone marrow of tumor bearing hamsters. Tumors were induced by spontaneously transformed fibroblasts (EHB), by SV40 (ZD) or by 20-methylcholanthrene (MCH2) transformed fibroblasts. We have shown that, in the thymus, the TdT activity of the various tumor bearing hamsters is generally inferior to the TdT activity of the control. In the spleen of animals bearing viral or spontaneously induced (ZD or EHB) tumors, the TdT activities were higher than in the controls. Moreover, DNA polymerase alpha and DNA polymerase beta activities in the spleen of the EHB tumor bearing hamsters were higher than in the controls. This fact, however, was not observed in the two other kinds of tumor, possibly because EHB tumors were growing much faster and led to earlier changes in DNA polymerases activities, as well as in spleen size and cellular populations. Finally, in the bone marrow, TdT, polymerase alpha and beta of ZD and EHB tumor bearers reached much higher activities than in the controls; for the MCH2 tumor bearing hamsters, no difference with the controls could be observed.

[Natural cytotoxic activity in different experimental conditions in hamsters (author's transl)]. / Activité cytotoxique naturelle dans différentes conditions expérimentales chez le hamster.

Lymphoid cells from normal hamsters from our inbred Z strain were evaluated for their natural cytotoxic activity against YAC cells (mouse lymphoma) and ZDc125 and EHB cells (cultured syngeneic hamster fibroblasts) employing the 51Cr-release cytotoxic essay. Natural cytotoxic reactivity was high in spleen, intermediate in peritoneal cells and very low in the thymus. Natural cytotoxic response was present in newborn animals, reaches a maximal level within 4-8 months and declines very slowly thereafter. Treatment of hamsters with a single dose of cyclophosphamide (Cy) augmented the natural cytotoxic reactivity 2 to 3 days after injection; this activity was suppressed thereafter and reappeared 20-25 days after the Cy injection. Growth of transplantable ZDc125 or EHB tumour in syngeneic hamsters inhibited the natural cytotoxic activity as soon as the tumour was palpable. Preincubation of target cells in sera of tumour-bearing animals diminished the level of natural cytotoxic activity of spleen lymphoid cells. One can postulate that this could be due to the presence of antibodies or immune complexes in those sera.

Cyclophosphamide induction of splenic suppressor cells in hamsters.

Injection of hamsters with a single sublethal dose of cyclophosphamide induced splenic atrophy followed by considerable hypertrophy. During the splenomegaly phase, the in vitro reactivity of spleen cells to concanavalin A (ConA) or protein A (ProtA) was decreased. The spleens of 6-8-month old animals contained cells able to suppress the in vitro reactivity of normal lymphocytes to ConA or ProtA. These cells were absent in 2-month old animals, and some suppressor activity could be shown in 3-month old hamsters. Suppressor activity was abolished by removal of adherent cells and by silica treatment, but antisera against thymocytes or IgG were ineffective.

Chromosome damaging agent of low molecular weight in the serum of New Zealand black mice.

Two substrains of NZB mice were developed by selective matings according to chromosome breakage frequencies in direct bone marrow preparations: HB, a line with increased chromosome breakage, and LB, a line with low or normal breakage rates. The serum of NZB mice from the HB strain contains a chromosome breaking factor that produces chromosome abnormalities in human lymphocytes. This clastogenic substance could not be detected in serum of NZB line. Nor does it exist in serum of Balb, C3H, C57 B1, Swiss, or AKR mice, in which no chromosomal breakage was observed in bone marrow. The breakage factor has a molecular weight between 1000 and 10,000 daltons.

Circulating immune complexes in sera of hamsters bearing simian virus 40-induced tumours.

Reported previously in certain human carcinomas and rat and mouse experimental tumor systems, circulating immune complexes (IC) have now been detected in Syrian hamsters bearing tumors produced by injection with syngeneic TSV5Cl2 cells. IC were detected by the Raji cell radioimmune assay, adapted for use in hamster sera. A novel feature of this test is the use of a stable covalently linked hamster immunoglobulin G aggregate as the reaction standard. Stable over six months on storage at -70 degrees and showing no tendency to form precipitates on thawing or during test procedures, this preparation greatly facilitated quantitation of hamster IC by Raji cells. Seven of 19 sera of hamsters bearing SV40-induced tumors from 60 to 128 days had IC concentrations exceeding 40 microgram aggregated hamster immunoglobulin G equivalents per ml, as contrasted to IC levels of less than 25 microgram for 19 age-matched normal hamsters. There appeared to be no significant correlation between IC levels and tumor weight or duration of tumor within the hamster host. The results suggest a complex relationship between IC and a number of factors connected with tumor growth.

[Differences in the fixation of antibodies in presence or absence of complement on SV40 transformed cells (author's transl)]. / Comparaison de la fixation d'anticorps en présence ou en absence de complément sur des cellules transformées par SV 40.

The fixation of antibodies directed against viral induced antigens was observed on SV40 transformed hamster fibroblasts. These cells were incubated for various periods of time with sera of tumor bearing hamster in the presence or absence of fresh guinea pig complement. The fixation of antibodies was revealed by an anti hamster immunoglobulins immune serum labeled with 125 iodine. Autoradiographic observations were made by optic and electron microscopy. The fixation of antibodies is quite different in the presence or in the absence of complement. Cells incubated with antibodies alone show a regular label on the plasma membrane with no decrease in the total cell population as compared to the controls. When the complement is added, one fraction of the cells is heavily labeled detached and progressively destroyed. Some of the cells (2/10) still attached on the flask are heavily labeled on the plasma membrane, the major part being nearly negative.

Renal deposition of soluble immune complexes in hamsters bearing an SV40-induced tumour.

Histologic and immunofluorescence studies of the kidneys of hamster bearing a tumour induced by injection of SV40-transformed cells show a proliferative glomerulonephritis associated with the presence of immune complex deposits along the glomerular basement membrane and in the mesangium. The intensity of the deposit is dependent of the size of the tumour. The presence of SV40-induced antigens was identified in the complexes by a heterologous immune serum. Immunoglobulins eluted from kidneys were shown to react with SV40 antigens by immunoprecipitation with TSV5CL2 cell extracts and by fluorescence on CV1 infected cells.

Humoral and cellular immune response during the growth of an SV40 induced tumour in hamsters.

Hamster fibroblasts transformed in vivo by the SV40 virus (TSV5Cl2) induce tumours when injected into adult hamsters and antibodies present in sera of tumour-bearing animals are used to demonstrate the presence of the antigens specific for the viral transformation. These tumours are a very useful tool in studying the evolution of the immune response during the tumour growth. A systematic histological examination of the spleen, thymus and lymph nodes was undertaken and the results thus obtained were correlated with a parallel study of the cells of the peritumoral region, the thymus and the spleen by immunofluorescence using antisera of different specificities. We were able to show that the plasma cells which make up the early peritumoral reaction and the lymphoblasts found later in spleen and thymus both synthesize antibodies directed against virus induced antigens. Circulating antibody directed against the same antigens is first detected at/or about the time that the peritumoral plasma cell reaction disappears and increases progressively as tumour weight increases.