Tumor-associated antigens in isoantigenic variants of a 3-methylcholanthrene-induced sarcoma.

A sarcoma was induced in an (A.CA X A.BY)F1 mouse. Two isoantigenic variants were selected by loss of one H-2 antigen. The tumor-associated transplantation antigens (TATA) of these variants were compared as to their specificities in (A.CA X A.BY)F1 mice. Both transplantation and indirect membrane immunfluorescence tests revealed that TATA of both variants did not cross-react. Thus selecting angainst different H-2 antigens also selected different TATA. Karyotype studies suggested that both variants originated from a unique clone.

Synergistic and selective stimulation of gelatinase B production in macrophages by lipopolysaccharide, trans-retinoic acid and CGP 41251, a protein kinase C regulator.

The production of gelatinase B by macrophages is relevant in the immunological and migratory functions of macrophages. CGP 41251, an inhibitor of protein kinase C (PKC), was found to stimulate the expression of gelatinase B in macrophages, as shown by the study of two different monocytic/macrophagic cell lines, mouse RAW 264.7 and human THP-1 cells. When human monocytes and rat peritoneal macrophages were treated with CGP 41251, insignificant increases of 10 and 25% were obtained. This can possibly be due to the presence of contaminating cells in these two enriched populations, since the CGP 41251 treatment of non-macrophagic cell lines inhibited their PMA-induced gelatinase B production. Taken together, these results suggest that the stimulatory effect of CGP 41251 is specific to cells of the monocytic lineage. Using RAW 264.7 cells as a model, the effect of CGP 41251 is additive to that obtained using lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), as revealed by gelatin zymography and Northern blot analysis. The stimulatory effect of CGP 41251 on gelatinase B production in RAW 264.7 was: (a) inhibited by calphostin C (as is the LPS-induced response), indicating a PKC-dependence; (b) inhibited by dexamethasone (as opposed to the LPS-induced response); and (c) enhanced by addition of trans-retinoic acid (RA). In fact, RA can induce gelatinase B production, either alone or in synergy with LPS and/or CGP 41251, since the combination of the three agents gives the highest gelatinase B response, at both the protein and the mRNA levels. This represents an important observation considering the RA is now being tested as an anti-cancer agent and proposed for prevention studies.

Induction, by adriamycin and mitomycin C, of modifications in lipid composition, size distribution, membrane fluidity and permeability of cultured RDM4 lymphoma cells.

Adriamycin and mitomycin C were previously found to modulate the sensitivity of lymphoma cells to lysis by certain effectors of immunity and this modulation was dependent on drug concentration. In the present studies, RDM4 lymphoma cells were treated with different concentrations of the two drugs for 24 h in culture. These treatments resulted in changes in the lipid composition, membrane fluidity, cell size distribution, and permeability to 51CrO4, Trypan blue, Acridine orange and trimethylaminodiphenylhexatriene (TMA-DPH) of the cells. Changes in some of these parameters, as a function of drug concentration, resulted in dose-response curves which were bell-like shaped, hence paradoxical similarities between non-drug-treated cells and cells treated with higher drug concentrations were observed.

Modulation of CD4 expression on lymphoma cells transplanted to mice fed (n - 3) polyunsaturated fatty acids.

Groups of adult AKR mice were fed well defined fats controlled diet regimens. These consisted of either saturated (beef tallow: 'BT') or (n - 3) polyunsaturated (fish oil: 'FO') fatty acids supplementation to basal mix mouse food. In other groups, the basal mix was given without any fat supplement ('NF'). Six weeks or more after the initiation of these diet regimens, mice received intraperitoneal injection of histocompatible RDM-4 lymphoma cells. Ascites RDM-4 tumors were harvested approximately two weeks later, and some of their physicochemical properties were studied. It was repeatedly found that: (1) the tumor grew considerably faster in the FO-fed donor than in the BT- or NF-fed donors; (2) cell membrane fluidity, content of C20(n - 3) and of C22(n - 3) fatty acids were significantly higher in the FO groups than in both BT and NF groups, while the content of C20(n - 6) and 22:4(n - 6) fatty acids was concomitantly decreased; (3) expression of the CD4 cell surface marker was always significantly diminished in the FO groups, whereas other markers such as CD8, H2K, Thy-1 and LFA-1 were not affected. Similar results were obtained, whether fats constituted from 1% to 16% by weight of the food intake. Use of a recently selected line of the RDM-4 lymphoma, exhibiting higher CD4 marker expression, resulted in similar observations. On the other hand, CD4 expression on cells from lymphoid organs of healthy adult AKR mice was not detectably modulated by the dietary fats.

Influence of lipid diets on the number of metastases and ganglioside content of H59 variant tumors.

We investigated the influence of the fatty acid composition of the diet on the number of hepatic metastases and the ganglioside profile of the primary tumor and metastases. C57BL/6 female mice were fed different diets containing either no fats (TEK) or 8% of fish oil (POL), linseed oil (LIN), safflower oil (SAF) or beef tallow (BT) and were injected subcutaneously in the dorsum with H59 cells, a variant of the Lewis lung carcinoma (3LLc) that metastasizes preferentially to the liver. The omega3 polyunsaturated fatty acid (PUFA)-rich diets (LIN and POL) elicited more metastases than the omega6 PUFA-rich (SAF), fat-free (TEK), or saturated fats (BT) diets. However, dietary fat did not influence the ganglioside composition of either the primary tumors or the metastases, at least in the glucidic part. However, comparison of diets with low (TEK, SAF, and BT) and high (LIN and POL) number of metastases showed that the levels of G3 (which could be a second band of GM2) were greater in metastases of the latter group. This study showed that the H59 hepatic metastases contained more GM2 than the s.c. tumors, irrespective of diet or the number of metastases produced. The small differences in the ganglioside profiles observed in this study could have resulted from the limitations of the HPTLC method. A detailed analysis of the lipid chains, as well as glycolipids other than gangliosides, could give more information on changes resulting from different lipid diets.

Differential variations of sensitivities of TNP-modified lymphoma cells to lysis by cytotoxic lymphocytes and by antibodies.

Cultured RDM4 cells were modified with 1 mM trinitrobenzene sulphonate (TNBS) and assayed for lysis by either in vitro generated cytotoxic T-lymphocytes (CTL) or complement-mediated humoral immunity. It was observed that cells harvested from the exponentially growing phase culture (as assessed by an important incorporation of thymidine) were more sensitive to CTL, but less sensitive to humoral immunity, than cells harvested from resting phase culture. Radiolabelled [14C]TNBS permitted to show that exponentially growing cells bound about 5 times less TNBS than cells at the resting phase. It is therefore concluded that the efficiency of the CTL on TNP-modified cells is not proportional to the density of TNP-groups on the targets, and that CTL on the one hand, and complement-mediated humoral immunity on the other hand, must have very different cell surface requirements to be able to lyze the targets.

Enhancement of antigen-specific interleukin 2 production by adding liposomes to rabies antigens for priming.

Antigen-specific IL-2 production was assessed, using splenocytes from rabies immune mice incubated for 24 h with rabies virus antigen. The antigenic material used for in vivo priming was either purified glycoprotein from rabies virus, or the inactivated virus. The time between priming, harvesting and restimulation of the splenocytes was 7 days. It was found that when antigenically inert liposomes were injected, together with antigenic material, to the prospective splenocyte donor mice, IL-2 production was enhanced. This augmentation was observed particularly when priming was performed with the inactivated rabies virus.

Tac expression induced by Epstein-Barr virus is restricted on non-transformable B lymphocytes.

During the course of a comparative study dealing with the immortalization of lymphocytes from a large number of normal healthy donors, we found that B cells of two of these individuals could not be immortalized by Epstein-Barr virus (EBV) under the standard conditions. The expression of the Tac antigen on the membrane of EBV-infected B cells from these two donors was compared with that of B cells from EBV immortalization-susceptible ones. The method used was two-colour immunofluorescence cytofluorometer analysis. We found that the Tac antigen expression was significantly and repeatedly reduced in the case of the two immortalization-resistant donors. This difference might be related to a genetic control of the resistance to EBV-immortalization.

Susceptibility of RDM4 lymphoma cells to LAK-mediated lysis is decreased in tumor bearers fed fish oil high fat regimen.

RDM4 lymphoma cells were grown intraperitoneally in genetically compatible AKR mice fed either regular mouse chow, or diet supplemented with either saturated fat (hydrogenated beef tallow = HBT) or unsaturated fat (fish oil = FO). It was observed that the lymphoma cells number was significantly greater in FO-fed hosts and lower in HBT-fed hosts, than in the mice fed regular chow. The tumor bearers diet did not dramatically influence the rate of DNA synthesis of RDM4 cells, as measured by [3H]thymidine uptake in culture, a few hours after harvesting from the peritoneal cavity. It was repeatedly found that FO feeding of the tumor bearers elicited an increased resistance of RDM4 cells to lysis by LAK effectors, as appraised in vitro by 51Cr release test and in vivo by the "Winn assay". Different FO percentage of the diet (16%, 8%, 4%) resulted in comparable reduction of susceptibility of RDM4 cells to lysis by LAK effectors. Lipid analysis showed that RDM4 cells grown in mice fed FO diet or HBT diet differed markedly in their fatty acid composition and that their resistance to lysis by LAK cells correlated with the quantity of oxidizable fatty acids especially of the n-6 type.

Peripheral blood lymphocytes resistant to Epstein-Barr virus immortalization manifest high natural killer (NK) type activity against NK-resistant target cells.

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. We found recently that PBL from two EBV-seropositive healthy adults were exceptionally resistant to immortalization by EBV. In contrast to PBL from other EBV-seropositive donors sensitive to immortalization by EBV (S-PBL), the "resistant" PBL (R-PBL) respond to EBV infection with an early interleukin-2 (IL-2) synthesis and high interferon gamma (IFN gamma) production. In order to determine whether these differences in cytokine responses between R-PBL and S-PBL could be associated with a detectable difference in lymphocyte cytotoxicity, we compared the natural killer (NK) activity of R-PBL and S-PBL effectors by using both NK-sensitive (i.e. K562) and NK-resistant (i.e. Raji) targets. We found that, while effectors from EBV-infected R-PBL and S-PBL cultures exhibited comparable NK activity against the K562 targets, they differed remarkably in their cytolytic activity against Raji cells. At days 3 and 5 of culture, effectors from EBV-infected R-PBL showed a significantly higher lytic activity against Raji targets, whereas S-PBL did not. Culture of EBV-infected R-PBL and S-PBL effectors in the presence of recombinant IL-2 (rIL-2) for 5 days resulted in increases of their lytic activity against Raji cells, whereas pretreatment of these effectors with recombinant IFN gamma (rIFN gamma) was found to increase only R-PBL cytotoxicity. These results suggest that the resistance of R-PBL to EBV immortalization could be associated with a lymphokine-mediated early cellular cytotoxic response of the NK/LAK (lymphokine-activated killer cell) type against EBV-infected cells.

Induction of natural killer cells and interferon during mouse hepatitis virus infection of resistant and susceptible inbred mouse strains.

Following infection by mouse hepatitis virus (JHM strain), an induction of natural killer (NK) cell activity was observed in C3H mice, which are considered to be sensitive to JHM virus infection. In contrast, mice of the resistant SJL strain did not show any increase of NK cell activity after JHM virus infection. However, infection of both SJL and C3H mice with mouse hepatitis virus type 3 (MHV3) resulted in an increase of NK level, comparable to that observed with the JHM virus infection in the C3H strain. No significant differences were observed in the NK cell activity of the peritoneal exudate or spleen cells of infected mice. Low levels of interferon were detected in serum or peritoneal exudate of C3H mice infected with JHM virus 18 or 24 hours before, but no detectable early interferon production was found. Also no interferon could be detected in the resistant SJL mice. After JHM virus infection, the number of peritoneal exudate cells (PEC) was increased significantly in C3H mice but not in SJL mice. Macrophages obtained from the C3H mice supported virus replication, whereas SJL macrophages did not. Our data suggest that NK cells do not play a role in the resistance of SJL mice against JHM virus infection but may participate in the defence mechanisms against this virus in C3H mice.

Cell-surface antigenic modifications with trinitrophenyl sulfonate versus trifluoromethyl-dinitrophenyl sulfonate.

The aim of the work was to justify the use of trifluoromethyl-dinitrobenzene sulfonate (CF3-DNBS) modification rather than trinitrobenzene-sulfonate (TNBS) modification, so as to be able to take advantage of the presence of fluorine atoms in the analogue, which allow the analysis of the hapten-carrier bonds by 19F-NMR nuclear magnetic resonance. Cell-surface antigenic modifications brought about by exposure to TNBS or CF3-DNBS were found to be immunologically cross-reactive, both in cell-mediated lymphocytotoxicity and in indirect immunofluorescence. The extent of haptenic derivatizations was found to be of the same order of magnitude, as appraised both by quantitative-absorption studies or by using radioactive hapten, provided that the less chemically reactive CF3-DNBS was used at the concentration of 10 mM and TNBS at the concentration of 1 mM. However, only TNBS-modified cells were sensitive to destruction by antibody-plus-complement-mediated cytotoxicity.

Interleukin 2 increases protection against experimental rabies.

Vaccination with either whole inactivated rabies virus or immunosome (rabies glycoprotein anchored on liposomes) induces a high level of interleukin 2 (IL 2) production after in vitro specific stimulation of splenocytes from primed mice (9). On the contrary, infection with a live rabies virus does not specifically induce the production of IL 2: splenocytes from ill mice previously infected with wild rabies virus cannot be specifically stimulated by rabies antigens, whereas they can be non-specifically stimulated by a mitogen (Concanavalin A (Con A]. When injected in mice, exogenous IL 2 (purified rat IL 2 or human recombinant IL 2) exhibits an adjuvant effect on rabies virus vaccine or subunit vaccine tested in a pre-exposure potency test (NIH test). When injected in hamsters, according to a post-exposure potency test (infection with a wild rabies virus followed by vaccination), IL 2 has no adjuvant effect on the rabies vaccine. Nevertheless, when injected alone, IL 2 protects thirty to fifty percent of the infected animals treated (1 hour, 3 and 7 days post-infection) with 10 international units of human recombinant IL 2.

Modification, by a poly I:C injection, of organ distribution of intravenously injected murine lymphoma cells and of blood coagulability.

51Cr-or 111In-labelled murine lymphoma cells were injected IV in control and poly I:C-treated mice. The organ distribution (lung, spleen, liver) of radioactivity was measured 2 h after injection. The results showed that if cell injection was performed 1 day after poly I:C treatment, the modifications of organ distribution did not fit with the expectations from a reinforcement of the NK function in vivo. In NK-suppressed mice, poly I:C affected the distribution of radioactivity in spleen and liver in the same manner as in normal mice, suggesting that the action does not entirely depend on the NK system. Additionally to that, poly I:C injections affected coagulability of the plasma from treated mice, by prolonging the coagulation time. It is concluded that poly I:C exerts a complex action on circulation and fixation of lymphoma cells.

Effect of 6(5H)-phenanthridinone, an inhibitor of poly(ADP-ribose) polymerase, on cultured tumor cells.

By catalyzing posttranslational modifications of nuclear proteins, poly(ADP-ribose) polymerase (PARP) controls their functions and therefore constitutes an enzyme of crucial importance in tumor development. In this study, we have investigated the action of 6(5H)-phenanthridinone, an isoquinoline derivative and one of the most potent PARP inhibitors described so far, on RDM4 murine lymphoma cells in culture. We also examined whether this compound could act synergistically with an antineoplastic drug in tumor-cell destruction. Our results demonstrate that a marked inhibition of PARP activity can be obtained in whole cells after a short incubation, and that this compound, when associated with an alkylating agent, dichloro-2,2' N-methyldiethylamine (chloromethine), leads to a marked drop in the RDM4 proliferation, indicative of a synergy between the two compounds.

Interactions between lymphoid cells and a thymic stromal cell line in vitro.

In the present study, we have optimalized the adherence assay to allow monitoring of the level of contact between thymocytes and a thymic epithelial cell line, E-5, in vitro. This type of interaction is not MHC-restricted, thus is unlikely to participate in the education of thymocytes to self. It was also shown that adherence does not vary from strain to strain, except for B6lpr/lpr immunodeficient mice which showed a markedly decreased adherence. This might be caused by the high level of L3T4-, Lyt-2- thymocytes in these mice (Davignon et al., 1985), since enriched double negative cells were shown not to adhere to E-5 cells. Preliminary characterization of adhering thymocytes suggests an heterogeneous mature phenotype. These cells appear around day 16 of fetal life and increase gradually until birth to remain constant throughout life. On the basis of contact duration, two populations of adhering thymocytes exist: one spontaneously detached after 1.5 hr, refractive to further adherence and the other which adheres for up to 14 hr. Contact between lymphoid and E-5 cells was shown to induce PHA responsiveness.

[Relationship between tumor antigens and histocompatibility antigens at the cell membrane]. / Relation entre antigènes tumoraux et antigènes d'histocompatibilité à la membrane cellulaire

The authors outline schematically the major histocompatibility complex as well as the relationship between tumor neo-antigen and pre-existing antigens at the cell surface. They note that these two antigenic systems are not independent and in particular, the major histocompatibility system (H-2) co-segregates with the tumour antigens. Considering the complexity of the H=2 system (a H-2 allele does not correspond to any single transplantation antigen but to a combination of several antigens units simultaneously present), the authors recall Boyse's hypothesis, revised by Haywood and Mc Khann, which propose that tumour antigens are a rearrangement of H-2 substructures. Moreover, it is possible that the relationship between H-2 and resistance to cancer may be attributed directly to the action of genes controlling the immune response (Ir region) which could also intervene in recognition of these tumour antigens. Finally, recent results obtained seem to show that some tumour antigens cross-react with H-2 fractions. This fact prevents the mice bearing these types of alleles from being immunized against the cross-reacting tumours. If this result could be transposed to the human situation, it would explain the frequency of certain types of tumours in defined H-LA groups and could allow the prediction of high-risk groups.

Lymphokine release as measurement of anti-mouse hepatitis virus type 3 (MHV3) cellular reactions in various mouse lines exhibiting differential susceptibilities to MHV3-induced paralysis.

We found that susceptibility to Murine Hepatitis Virus, type 3 (MHV3)-induced paralysis is controlled by genes of the H-2 complex. In this article, we compared MHV3 antigen specific cellular reactions, in congenic mice harbouring different H-2 genes (or gene). In a first set of experiments, paralysis susceptible (B10.A x A/J)F1, partly susceptible (B10.AQR x A/J)F1 and resistant (B10.Q x A/J)F1 hybrids were infected with live MHV3. Three weeks or more post-infection (p. i.), the spleens and peritoneal exudate (PE) cells from the mice were put into culture. Killed MHV3 was added to cultures, and antigen specific lymphokine production and utilization were measured: IL-1 production by PE cells after 24 hr in culture, IL-2 production by splenocytes after 24 hr in culture, IL-2 utilization (as appraised by splenocyte proliferation) after 96 hr in culture. No clearcut difference, resulting from genetic disparity, could be observed in the antigen-specific responses. In a second set of experiments, mice were primed with ultra-violet radiation killed MHV3. In that case, increases of IL-1 production by PE cells, of IL-2 production by splenocytes and splenocyte proliferation were always observed, compared to PE cells and splenocytes from non-primed (control) donor mice. However, in latter case, addition of MHV3 antigen to cultures did not result in augmentation of antigen specific IL-2 production and utilization. Here again, no genetic effect was observed. We conclude from these results that MHV3 infection elicited strong lymphokine responses, but that antigen-specific IL-1 and IL-2 production did not correlate with the susceptibility to MHV3-induced paralysis.