Anti-NeuGcGM3 antibodies, actively elicited by idiotypic vaccination in nonsmall cell lung cancer patients, induce tumor cell death by an oncosis-like mechanism.

1E10 is a murine anti-idiotypic mAb specific for an idiotypic mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In melanoma, breast, and lung cancer patients, this anti-idiotypic Ab was able to induce a specific Ab response against N-glycosylated gangliosides, attractive targets for cancer immunotherapy as these glycolipids are not naturally expressed in humans. A clinical study with nonsmall cell lung cancer patients showed encouraging clinical benefits. Immunological studies performed in 20 of these patients suggested a correlation between the induction of Abs against NeuGcGM3 and longer survival times. The induced anti-NeuGcGM3 Abs recognized and directly killed tumor cells expressing the Ag, by a mechanism independent of complement activation. In the present work, we show that this cytotoxicity differs from apoptosis because it is temperature independent, no chromatin condensation or caspase 3 induction are detected, and the DNA fragmentation induced has a different pattern than the one characteristic for apoptosis. It is a very quick process and involves cytosqeleton reorganization. The Abs induce cellular swelling and the formation of big membrane lesions that allow the leakage of cytoplasm and the loss of the cell membrane integrity. All of these characteristics resemble a process of oncotic necrosis. To our knowledge, this is the first report of the active induction in cancer patients of NeuGcGM3-specific Abs able to induce complement independent oncotic necrosis to tumor cells. These results contribute to reinforcing the therapeutic potential of anti-idiotypic vaccines and the importance of NeuGcGM3 ganglioside as antitumor target.

ERK5 knockdown generates mouse leukemia cells with low MHC class I levels that activate NK cells and block tumorigenesis.

Tumor cell-based vaccines are currently used in clinical trails, but they are in general poorly immunogenic because they are composed of cell extracts or apoptotic cells. Live tumor cells should be much better Ags provided that they are properly processed by the host immune system. We show herein that stable expression of a small hairpin RNA for ERK5 (shERK5) decreases ERK5 levels in human and mouse leukemic cells and leads to their elimination by NK cells in vivo. The shERK5 cells show down-regulation of MHC class I expression at the plasma membrane. Accordingly, ectopic activation of the ERK5 pathway induces MHC class I gene expression. Coinjection of shERK5-expressing cells into the peritoneum diminishes survival of engrafted wild-type tumor cells. Moreover, s.c. injection of shERK5-expressing cells strongly diminishes tumor development by wild-type cells. Our results show that shERK5 expression in leukemia cells effectively attenuates their tumor activity and allows their use as a tumor cell-based vaccine.

Combination of photodynamic therapy + immunotherapy + chemotherapy in murine leukiemia.

Photodynamic therapy (PDT) is a treatment for cancer based on the photosensitization of tumor cells by photosensitive drugs and their subsequent destruction on exposure to light of particular wavelength. The combination of drug uptake in malignant tissues and selective delivery of laser-generated light provides an effective therapy with efficient tumor citotoxicity and minimal normal tissue damage. Since immune response of the host is important in the control of tumor growth and spreading, PDT is able to increase the antitumor immunity. In our laboratory we examined the antitumor effect of combination of PDT, with photoactivated M-THPC (meta-tetrahydroxyphenylchlorin, FOSCAN, Temoporphirin), adoptive immunotherapy, with immune lymphocytes, and chemotherapy on advanced murine tumors. Mice bearing L1210 tumor were treated at day +4 with Navelbine (NVB 1mg/Kg), at day +5,+6 with PDT (0.3mg/Kg of mTHPC and 100mW/cm(2) x 200'' of exposure of laser light), and at day + 7 with immune lymphocytes(IL), collected from mice pretreated with PDT(2x10(7) cells). The results show that the combination NVB + PDT + IL demonstrates a significant synergistic antitumor effect while the chemotherapy treatment with low dose of the drug is uneffective. The same positive results were obtained with the combination of Cisplatin (CDDP 0.5mg/Kg), PDT and IL, while the CDDP treatment alone is completely uneffective. In conclusion, these results suggest that it is possible to completely cure animals bearing advanced tumors, with a combined therapy, PDT + adoptive immunotherapy + low dose chemotherapy.

Transplantation of leukemic bone marrow treated with cytotoxic antileukemic antibodies and complement.

The ability of antiserum against murine L1210 leukemia to remove residual leukemia cells from murine bone marrow was investigated. Leukemic marrow was treated in vitro with antiserum and complement and used to hematologically reconstitute mice that had been irradiated with doses lethal to bone marrow. Following infusion of treated leukemic marrow, normal marrow returned without evidence of leukemia. More than 90 percent of the animals have survived for 11 months without untoward effects, suggesting that the technique may be of use in the treatment of acute leukemia in humans.

Rice protein prolamin promotes anti-leukemia immunity and inhibits leukemia growth in vivo.

Prolamin is a heat-stable storage protein of rice (Oryza sativa). This study aimed to examine the effect of prolamin on anti-tumor immune response in vitro and leukemia growth in vivo. The prolamin-enriched rice fractions were prepared to stimulate peripheral blood mononuclear cells (MNC) from mice spleen. The MNC-conditioned medium (MNC-CM) was collected to treat leukemia L1210 cells. Human MNC-CM was prepared to treat Jurkat acute T cell leukemia cells. Purified prolamin was orally administered to syngeneic L1210-bearing DBA/2 mice to assess weights of tumor, liver and spleen, liver histopathology, peripheral blood neutrophil count and cytokine levels. Prolamin-prepared MNC-CM inhibited the viability of murine leukemia L1210 cells and human leukemia Jurkat cells, indicating an immunomodulatory effect. In syngeneic L1210-bearing DBA/2 mice, oral administration of purified prolamin dose-dependently decreased the tumor weight and attenuated the leukemia-induced reduction of liver and spleen weights. Prolamin inhibited the increase of peripheral blood leukocyte count. The levels of tumor necrosis factor-α and interferon-γ in MNC-CM and mice serum were significantly increased by prolamin treatment. No significant change in body weight, serum alanine aminotransferase and creatinine levels was noted by prolamin treatment. Rice prolamin could effectively promote anti-tumor immunity and inhibit leukemia growth without significant toxicity.

Injury of neoplastic cells by murine macrophages leads to inhibition of mitochondrial respiration.

Cytotoxic activated macrophages (CM) inhibited the growth of neoplastic L1210 cells in vitro but L1210 cell death was minimal to nonexistent. L1210 cells injured by CM were separated from macrophages and studied in an isolated system. CM-injured L1210 cells had an absolute requirement for glucose or another glycolyzable hexose (mannose or fructose) for at least 40 h after removal from macrophages. If the culture medium lacked sufficient concentration of one of these sugars, CM-injured L1210 cells died within 4 h. Uninjured L1210 cells cultured alone or with peptone-stimulated macrophages had no such requirement and maintained complete viability in hexoseless medium. The hexose requirement of CM-injured L1210 cells could not be fulfilled by other naturally occurring monosaccharides, glucose or mannose derivatives, or substrates that can be oxidized by mitochondria. The concentration requirements for glucose, mannose, and fructose by CM-injured L1210 cells correlated with the concentrations required to support maximal glycolysis of these sugars by other murine ascites cells. A concentration of 2-deoxy-D-glucose which completely inhibited L1210 cell glycolysis also complete prevented the ability of glucose or mannose to maintain viability of CM-injured L1210 cells. Interaction with CM led to inhibition of L1210 cell mitochondrial oxidative phosphorylation. This was supported by the findings that: (a) CM-injured L1210 cells had no Pasteur effect; their rate of aerobic glycolysis was the same as the rate of anaerobic glycolysis of uninjured L1210 cells, (b) Endogenous respiration of CM-injured L1210 cells was 15% of normal. Maximal inhibition of uninjured L1210 cell respiration by a specific mitochondrial poison (oligomycin) was nearly the same (13% of normal). It followed that CM-injured L1210 cells required hexose for chemical energy production via the glycolytic pathway. CM-induced mitochondrial injury occurred in five other neoplastic cell lines tested.

Tumor-infiltrating dendritic cell subsets of progressive or regressive tumors induce suppressive or protective immune responses.

Tumor-infiltrating dendritic cells (TID) have an ambivalent role in regulation of tumor regression or growth. However, their precise natures and molecular mechanisms have not been elucidated. In this study, we studied TIDs recruited in progressive P815 and regressive P198 tumors of the same origin. Our data showed that P815 tumors contained CD4+ 8+ and CD4- 8- TID815 subsets, whereas P198 tumors contained CD4+ 8+ and CD4+ 8- TID198 subsets. They similarly stimulate allogeneic T cell proliferation and have nitric oxide-mediated cytotoxicity to tumor cells with an exception of CD4- 8- TID815 with less efficiency. The newly identified fourth CD4+ 8+ TID815 or TID198 subset and the CD4+ 8- TID198 all express high levels of IFN-gamma and interleukin (IL)-6, whereas CD4- 8- TID815 secrete a marked level of transforming growth factor-beta. Vaccination of mice with P815 tumor lysate-pulsed CD4+ 8+ TID815 or TID198 and CD4+ 8- TID198 induced IFN-gamma-secreting Th1 and effective CTL responses leading to protective immunity against P815 tumor, whereas CD4- 8- TID815 stimulated IL-10-expressing Tr1 responses leading to immune suppression. Transfer of CD4+ Tr1 cells obtained from CD4- 8- TID815-immunized wild-type, but not IL-10(-/-) mice, into CD4+ 8+ TID815 immunized mice abolished otherwise inevitable development of antitumor immunity. Taken together, our findings provide an important insight into immunologic alterations in progressive and regressive tumors and an implication for dendritic cell-based approaches in the design of cancer vaccines.

The attacker: target-cell ratio and serum effects on in vitro cell-mediated immunity.

Sera collected from mice rejecting an allogeneic tumor and showing arming activity for normal splenocytes increased target-cell lysis by immune cells when low (less than or equal to 25:1) attacker:target-cell ratios (A:T) were used but inhibited cytotoxicity at higher (100:1) A:T. The importance of this methodologic variable in studies of serum activity on cellular cytotoxicity is emphasized and the possible mechanisms are discussed.

Increased immunogenicity of two lymphoma lines after drug treatment of athymic (nude) mice.

Highly immunogenic sublines of L1210 and LSTRA lymphomas were obtained from athymic (nude) mice treated with 4(5)-(3,3-dimethyl-1-triazeno)imidazole-5(4)carboxamide (DIC) in vivo. Conventional mice, compatible with the parent tumor, rejected the DIC-treated sublines and were relatively resistant to a subsequent challenge with the parent lines. The DIC-treated sublines were not rejected by athymic mice, which indicated that the transplantation resistance to these tumors in conventional mice was thymus-cell dependent. In addition, there was marginal or no increase of tumor-cell immunogenicity when the parent lines were passaged in nude mice without DIC treatment. This indicated that the DIC-dependent immunogenic changes in DIC-treated leukemic conventional mice could not be ascribed merely to protection by naturally occurring antigenic clones that resulted from DIC-induced immunodepression.

Distribution of Lyb-4.1 and other membrane antigens on murine lymphoid tumors.

The distribution of membrane antigens on 6 DBA/2-derived tumors (L1210, L5178Y, P815, ABLS 11, ABLS 12, and ABLS 13) was studied by direct cytotoxicity and quantitative absorption assays. Lyb-4.1 antigen was found solely on the L1210 tumor. Iad antigens were absent from all tumors, and H-2Kd and H-2Dd antigens were present on all tumors. Immunoglobulin was adsorbed to the ascites tumors and lost after 3 days or more in tissue culture. These studies were performed to characterize the distribution of DBA/2 membrane antigens on DBA/2-derived tumors as a base line for functional and chemical studies with these tumors and with their solubilized proteins.

Differential tumor immunogenicity of DBA/2 mouse lymphoma L1210 and its sublines. III. Control of host resistance to drug-resistant L1210 sublines by H-2-linked and non-H-2-linked genes.

The effects of the host's genetic composition on the host's resistance to the DBA/2 L1210 lymphoma and three drug-resistant L1210 sublines (which show an increased expression of tumor-associated antigens) were investigated. Histocompatible F1 mice between DBA/2 and other mouse strains were given injections ip of graded numbers of these tumor cells, and a marked difference in the resistance of the host to tumor growth was demonstrated. The resistant hybrid mice showed markedly higher resistance to the L1210 sublines than to the L1210 parent lymphoma cells. Experiments with F1 mice between DBA/2 (H-2d) and certain congeneic intra-H-2 recombinant strains showed that the H-2b/d heterozygosity at the H-2K+l-A regions and additional non-H-2 genetic factor(s) confer the resistance to the F1 mice. The resistance was abolished by treating host animals with injections of silica, an antimacrophage agent, or rabbit anti-mouse thymocyte serum. These results suggest that the resistance is immunologic and that the genetically controlled host resistance may be directed to surface changes related to the increased expression of tumor-associated antigens.

Humoral reponse to neuraminidase-treated tumor cells.

L1210 leukemia cells grew progressively and caused tumor deaths in all recipient mice. However, when these cells had been treated with Vibrio cholerae neuraminidase (VCN) prior to injection, tumor deaths did not occur. Both untreated and VCN-treated L1210 cells elicited a humoral response, as manifested by an increasing percent of cells in the spleen and peritoneal cavity, with various types of membrane-associated immunoglobulins. Progressive tumor growth was associated with a large percent of peritoneal exudate (PE) cells bearing membrane-associated IgG, a late increase in the percent of PE cells with IgG, and only a small percent of PE cells with IgM on their surfaces. Conversely, PE cells from mice given VCN-treated L1210 cells were characterized by a small percent with IgG, an early increase in percent of cells with IgG, and a large percent with membrane-associated IgM. An injection of VCN-treated L1210 cells into mice with progressively growing L1210 tumors caused frequent tumor remissions, with a corresponding alteration of the ongoing humoral responses. Both the degree of alteration and the number of cures depended on the tumor burden at the time VCN-treated tumor cells were injected. The humoral response in mice with tumor remission following immunization was comparable to the response detected after an injection of VCN-treated cells only.

Vesicular stomatitis virus-infected L1210 murine leukemia cells: increased immunogenicity and altered surface antigens.

Homogenates of L1210 cells infected in vitro with vesicular stomatitis virus (VSV) were immunogenic agains a tumor graft of 100 times the LD50 dose of L1210 cells" whereas those of uninfected cells were not. The immunogenicity of intact X-irradiated L1210 cells was distinguishable from that of VSV-infected cell homogenates on the basis of the susceptibility of immunogenicity to experimental procedures used in preparation of the immunogenic homogenates: Homogenization of intact X-irradiated cells or their infection with VSV prior to irradiation led to loss of immunogenicity. In addition, uninfected cell homogenates were not made immunogenic nor was the immunogenicity of VSV-infected cell homogenates eliminated by X-irradiation. At the time of tumor challenge, sera from mice that were effectively immunized with VSV-infected cell homogenate showed a high VSV-neutralizing titer but no complement-dependent cytotoxicity for L1210 cells. Quantitative absorption studies demonstrated that VSV infection led to a marked reduction in L1210 surface antigens recognized by cytotoxic alloantibody; spatial association between these antigens and VSV antigens was not demonstrable on VSV-infected cells. Antigens recognized by heterologous antiserum to L1210 cells were also reduced following VSV infection.

Modulation of cell-mediated alloimmunity by BCG. I. Antagonism and potentiation of immunosuppression caused by cytarabine.

The ability of iv administered BCG to antagonize immunosuppression caused by injection of the antimetabolite cytarabine (beta-cytosine arabinoside; ara-C) was investigated in C57BL/6 mice. Treatment with BCG 10 days before alloimmunization with killed L1210 tumor cells decreased spleen T-cell-mediated cytolysis against allogeneic P815Y tumor cells, as measured by a short-term 51Cr release assay, and potentiated immunosuppression due to ara-C. In contrast, spleen cell-mediated immunity (CMI) that was assayed by a 48-hour microcytotoxicity assay (MCA) was augmented by systemic BCG administered before alloimmunization. Pretreatment with BCG resulted in a complete and long-lasting protection against the immunosuppressive effects of ara-C on this CMI as measured by the MCA. Treatment with BCG after cytoreductive therapy resulted in a significant, although transient, reversal of immunosuppression. Depending on the type of response and thus the type of effector cell measured, BCG acts as a moderate immunosuppressive agent or a strong immunopotentiator of CMI.

Phosphatidylinositol-3-kinase regulates PKCtheta activity in cytotoxic T cells.

Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone. This suggests that diacylglycerol is neither sufficient nor required for PKCtheta activation. Furthermore, PKCtheta was only activated in a CTL clone stimulated with plate-bound anti-CD3 but not soluble anti-CD3. However, PMA or cross-linked anti-CD3 stimulated phosphorylation of PKCtheta as measured by a migratory shift, suggesting that phosphorylation was not sufficient for activity. Phosphatidylinositol 3-kinase activity was required for anti-CD3, but not PMA, stimulated phosphorylation and for immobilized anti-CD3-triggered PKCtheta activity. A substantial fraction of PKCtheta was constitutively membrane associated and PMA or CD3 stimulation did not significantly increase membrane association. Our data indicate that phosphorylation of PKCtheta is not a suitable surrogate measurement for PKCtheta activity and that additional, yet to be defined steps, are required for the regulation of PKCtheta enzymatic activity in CTL.

Differential expression of murine leukemia antigen on L1210 parental and drug-resistant sublines.

The different expression of surface antigens on L1210 leukemia DBA/2 and drug-resistant L1210 sublines was investigated. Indirect cytotoxic test, the anti-L1210/v alloantiserum reacted more strongly with subline cells than with parental cells. Absorption of the antiserum with Gross cellular surface antigen-positive AKR leukemia (AKSL-4) cells led to a much greater difference in this reactivity. Quantitative absorption experiments revealed that the drug-resistant sublines had 5 times higher absorption capacity than did the parental line. After complete absorption of antibodies against murine leukemia virus-related antigens, the anti-L1210/v alloantiserum still reacted with L1210 cells. This cytotoxicity could be removed after absorption with C3H mammary tumor (MAC-1) cells but not with normal C3H lymphocytes. These results provide evidence that the major cytotoxic activity of the antiserum against L1210 and L1210 subline cells was due to antibodies against murine mammary tumor virus-related antigen and that the drug-resistant sublines of leukemia L1210 have higher quantitative expression of mammary leukemia antigens.

Specific potentiation of L1210 vaccine by pyran copolymer.

Pyran copolymer (NSC 46015) was found to potentiate strongly the immune response of C57BL/6J X DBA/2 F1 mice to 10(4) live L1210 tumor cells following suboptimal vaccination with 10(7) radiation-inactivated L1210 cells. Optimal immunity to challenge was produced by concomitant i.p administration of pyran and L1210 vaccine, and activity was dependent upon both pyran and vaccine dosages. In addition, this immunopotentiation seemed to be related to the intrinsic viscosity of different pyran preparations tested, although all the pyran compounds had significant activity. Furthermore, the increased immunity of subsequent live tumor challenge appeared to be specific for the vaccinating cell type.

Acceleration of allogeneic antitumor immunity with immune RNA in vitro and T-cell suppression by L1210 tumor bearer spleen cells.

RNA fractions extracted from the spleens of immunized animals prepared against L1210 leukemia cells can transfer allospecific cell-mediated immunity both in vitro and in vivo. Both preculture of nonsensitized lymphocytes prior to the treatment with immune RNA and additional culture of lymphocytes after 1-hr treatment with immune RNA enhance the growth-inhibitory activity of immune RNA-treated lymphocytes. Preculture for 5 hr and additional culture for 24 hr are sufficient for maximal enhancement of the growth-inhibitory activity. There is no significant difference in inhibition between lymphocytes treated with immune RNA for 1 hr and 24 hr. The growth inhibition by lymphocytes is augmented proportionally to the increase in the dose of immune RNA in vitro but not in vivo. In mice given i.p. injections of immune RNA-treated syngeneic spleen cells (before or after inoculation with L1210 cells), a significant prolongation of the mean survival time [27.0 +/- 5.4 (S.D.) days, 0.05 < p < 0.1] was not achieved when compared with control mice (21.4 +/- 1.6 days). Evidence is presented that suppressor cells in the spleens of L1210-bearing mice are involved in preventing effector cell function in vivo. The present study revealed that 8 X 10(3) and 5 X 10(5) suppressor cells were sufficient to inhibit completely the activity of immune RNA-treated effector cells in vitro and in vivo, respectively. Suppressive activity was abolished by treatment with anti-Thy 1.2 serum and complement and partially abolished with irradiation (1500 R). The suppressor cells belonged to a radiation-sensitive T-cell population. The limited activity of immune RNA in vivo is probably due to the destruction of immune RNA by RNase present in the host plasma and tissues and the acquisition of suppressor cells which inhibits nonadherent peritoneal exudate cell activity in the animal.

Monoclonal antibodies to the L1210 murine leukemia cell line and to a drug-altered subline.

A panel of murine monoclonal antibodies (MAbs) was produced by fusing NS-0 myeloma cells with spleen cells of a BALB/c X DBA/2 F1 mouse hyperimmunized against a highly immunogenic subline of the L1210 leukemia obtained by in vivo treatment of the L1210 parental line with the antitumor drug 5-(3,3'-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC). Among the 52 MAbs produced 16 (anti-D) were specifically cytotoxic in a complement-dependent cytotoxicity assay for the drug-altered subline and the others (anti-L) also cross-reacted with the L1210 parental leukemia. Six anti-D and three anti-L MAbs were selected for detailed studies of tissue specificity. In quantitative absorption experiments the antigens defined by these antibodies could not be detected on cells from normal mouse tissues (lung, liver, kidney, heart, spleen, and thymus). The reactivities of both anti-D and anti-L MAbs against a panel of L1210/DTIC sublines obtained at different times were assayed. The results showed that the antigenic specificities defined by anti-L MAbs were expressed on almost every L1210/DTIC subline while the anti-D MAbs detected antigenic structures specific for the L1210/DTIC used for the immunization. None of the MAbs tested cross-reacted with the L5178Y lymphoma or with its DTIC-altered sublines. The failure of anti-D MAbs to cross-react with cells from other L1210/DTIC sublines supports the hypothesis that the immunological alterations induced by the DTIC treatment are the consequence of mutagenic activity of the drug. On the other hand the presence of anti-L antigens on the cells of every L1210 subline indicates that the DTIC alteration is not accompanied by a loss of the tumor-associated antigen from the L1210 leukemia.

Immunotherapeutic response of concanavalin A-bound L1210 vaccine enhanced by a streptococcal immunopotentiator, OK-432.

Immunotherapeutic response to concanavalin A (Con A)-bound L1210 murine leukemic vaccine and immunopotentiators was examined in histocompatible animals bearing a small burden L1210 leukemic cells. When combined with Con A-bound vaccine, a streptococcal immunopotentiator, OK-432 (NSC B116209), prepared from Streptococcus pyogenes, was potent in antitumor therapy and resulted in a number of cured animals. Administration of either Con-A-bound vaccine or OK-432 alone did not produce any beneficial effect on leukemic animals. The enhanced therapeutic response was dependent on the effectiveness of the dose and timing of the administration of OK-432 when given after vaccination. Combined modality of Con A-bound L1210 vaccine and OK-432 was not effective in animals bearing P388 murine leukemic cells, which indicates specificity of therapeutic response. In enhancing the therapeutic potency of Con A-bound leukemia vaccine, pyran copolymer (NSC 46015) was as effective as OK-432, whereas Bacillus Calmette-Guérin and Corynebacterium parvum were far less effective. When combined with OK-432, therapeutic response to Con A-bound L1210 vaccine was much greater than response to glutaraldehyde-, mitomycin C-, or Vibrio cholerae neuraminidase-treated L1210 vaccine.