Humoral response against murine lymphoma cells xenogenized by drug treatment in vivo

Treatment of murine lymphomas with triazene derivatives may lead to the appearance of novel drug-mediated tumor antigens, a phenomenon known as chemical xenogenization. Such antigens, which are capable of eliciting specific transplantation resistance in histocompatible mice, have been previously detected by in vivo and in vitro cell-mediated immune responses. In the present report we address the question of the humoral antibody response to a chemically xenogenized lymphoma. Histocompatible mice were given several injections of live cells of the xenogenized tumor. Ten days after each immunization, pooled sera from different animals were analyzed for Ab content by means of flow microfluorometry analysis and CEL-ISA assay. The results reveal that antibodies of both IgG and IgM classes capable of binding the xenogenized tumor can already be detected after one single sensitization. However, the Ab titer gradually increases through subsequent immunizations, reaching a peak level after 3-4 injections at a time when most of the humoral response is made up of antibodies of IgG class. The specificity of the anti-xenogenized tumor hyperimmune sera was subsequently investigated by its reaction with the parental, non-xenogenized line and with normal tissue cells of the same or allogeneic haplotypes. The data obtained point out that cross-reactivity with the parental line could be completely removed by absorption of the hyperimmune sera on parental cells, which removed most of the IgM antibodies. Moreover, the presence of an excess of anti-parental antibodies on the xenogenized tumor cells does not prevent the subsequent binding of the hyperimmune absorbed serum, thus indicating that the novel determinant(s) recognized on xenogenized cells are not spatially related to those shared with the original parental tumor. In addition, the hyperimmune absorbed serum does not cross-react with normal hemopoietic or lymphoid cells of the same (H-2d) or allogeneic H-2b and H-2k haplotypes. Furthermore, no alien histocompatibility antigens of H-2b or H-2k haplotypes could be detected on the xenogenized tumor cell surface. Taken together, these data provide evidence that chemical xenogenization of a murine lymphoma leads to the appearance of novel determinant(s) detectable by specific antibodies.     

Non-T-cell resistance against a mouse Moloney lymphoma.

We have previously shown that spleen cells from normal mice contain lymphocytes that kill certain in vitro-grown Moloney lymphoma lines in a 51Cr release test. The killing activity shows a marked dependence on the genotype of the donor mouse. In vivo rejection studies with a Moloney lymphoma line in various normal semisyngeneic F1 hybrids showed a clear positive correlation between in vitro natural cytotoxicity and in vivo rejection. Thus mice can be grouped as high- or low-reactive according to their in vitro cytolytic behavior as well as their in vivo rejection potential. A lymphoid cell without detectable T- or B-cell markers is responsible for the in vitro killing effect. The present study also shows that the in vivo growth inhibitory function is T-cell-independent. Lymphoid cells depleted of T- and B-cells and transferred together with Moloney lymphoma cells into an irradiated syngeneic recipent were highly efficient in delaying tumor growth. Furthermore, syngeneic or semisyngeneic thymecto-mized, irradiated, fetal-liver-reconstituted mice showed if anything an increased in vivo resistance to a Moloney lymphoma compared to control mice. In contrast, tumor cells histoincompatible with regard to the H-2 locus showed the expected preferential growth in the thymectomized animals. We thus conclude that the major in vivo resistance against transplantation of the syngeneic or semisyngeneic Moloney lymphoma used in this study is T-independent.     

Secondary cell-mediated cytotoxic response to challenge of rats with syngeneic Gross virus-induced lymphoma.

Secondary cell-mediated cytotoxicity generated in vivo against a syngeneic Gross virus-induced lymphoma [(C58NT)D] in WF rats was detected by the 4-hour 51Cr release assay. At 30 days or more following primary tumor cell inoculation, after the tumors had regressed, lymphoid cells had little or no detectable direct cytotoxic reactivity. At rechallenge with tumor cells, high levels of cytotoxicity were detected in the peritoneal exudate, peripheral blood, mesenteric lymph node, and spleen cells. The secondary cellular immune response after challenge developed earlier, reached higher levels, and lasted longer than the primary immune response. The secondary cytotoxic reactivity was shown to be immunologically specific by the use of various tumor cells both as target and inhibitor cells. Treatment of immune spleen cells with specific antiserum to rat T-cells and complement abolished their cytotoxic reactivity, whereas removal of complement receptor-bearing cells or phagocytic cells did not reduct the cytotoxicity. These data demonstrated that specific-memory T-cells persisted for long periods in the lymphoid organs of immune rats and could rapidly become cytotoxic from rechallenge with the tumor.     

Non-specific cytotoxic cells generated in vitro in response to a weakly immunogenic murine adenocarcinoma. In vivo correlations

Tumor 85 adenocarcinoma cells, which are very weakly immunogenic for C3H/HeN (C3H) mice, provide a model tumor system for studying the parameters of induction of a cell-mediated immune response which does not appear to be a traditional cytotoxic T-cell or NK-cell response. Mice pre-immunized with irradiated tumor 85 cells are protected about 50% of the time from a challenge of viable tumor 85 cells although it is never possible to protect 100% of the immunized mice. Optimal protection is observed in mice immunized with 10(6) irradiated tumor 85 cells 14 days prior to challenge. Protection is also observed if mice are immunized with Protection is also observed if mice are immunized with 3H or C3HfeB/HeN (C3Hf) tumors originally induced by the same carcinogen as that used to induce tumor 85. The injection of carrageenan 1 h before challenge completely reverses the protection observed in immunized mice and and tumors grow faster in carrageenan-treated immunized mice than in normal mice. Two populations of cells obtained from cultures of spleen cells stimulated by tumor 85 cells appear to prevent tumor growth in vivo. One population, which is cytotoxic for tumor 85 cells in vitro, is nylon-wool-adherent and expresses Thy 1.2. The second population, which is not observed to be cytotoxic in a 51Cr release assay, is phagocytic.     

Extremely potent,rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody

A recent study reported on an anti-CD19/anti-CD3 single-chain bispecific antibody (bscCD19xCD3) exhibiting high activity against human B lymphoma cell lines (L?ffler et al., Blood 2000;95:2098-103). In the present study, we have explored in detail the in vitro efficacy, T-cell donor variability, binding characteristics, specificity, kinetics and interleukin-2 (IL-2) dependence of bscCD19xCD3. We found that a majority of human donor T cells tested (n = 86) gave half-maximal B-lymphoma cell lysis (ED(50)) within a range of 10-50 pg/ml bscCD19xCD3, corresponding to sub-picomolar concentrations of the bispecific antibody. Under identical experimental conditions, the anti-CD20 monoclonal antibody rituximab had an at least 100,000-fold lower in vitro efficacy. The extreme potency of bscCD19xCD3 was in sharp contrast to the relatively low affinity of the anti-CD3 and anti-CD19 single-chain Fv portions in K(D) ranges of 10(-7) and 10(-9) M, respectively. Cell lysis by bscCD19xCD3 was predominantly mediated by the population of CD8/CD45RO-positive T cells. Both immortalized CD4- and CD8-positive human T-cell clones were highly active effector cells as well. Cell lysis by bscCD19xCD3 was rapid and specific. The respective parental monoclonal antibodies inhibited cell lysis and CD19-negative cells were not harmed by T cells in the presence of high amounts of bscCD19xCD3. The potent T-cell stimulus IL-2 could not markedly augment the activity of bscCD19xCD3-stimulated T cells. In conclusion, bscCD19xCD3 could redirect unstimulated cytotoxic T cells against CD19-positive cells in an unexpectedly potent, rapid and specific fashion.     

Secondary specific immune response in vitro to MSV tumor cells.

The interactions which occur between antigenic tumor cells and normal or immune lymphoid cells in a 3-day in vitro culture, have been studied with a murine sarcoma virus (MSV)-induced tumor. The 3H-thymidine incorporation of lymphoma cells growing in suspension, and the radioactive-chromium release of freshly sampled lymphoma cells regularly added to the culture, have been compared to determine the part played by immune lymphoid cells in cytolysis and cytostasis of the tumor-cell population. The cytolytic activity increases in the culture from day 0 to day 3. It is due, predominantly, to T-cells, and remains specific to antigens shared by MSV tumors and related lymphomas. This activity would be difficult to detect unless freshly sampled ascitic cells were used as targets, since the lymphoma cells spontaneously lose a part of their sensitivity to immune cytolysis during in vitro culture. The method used in the present experiments is a secondary chromium release test (SCRT), which measures the invitro secondary stimulation of cytotoxic T-lymphocytes (CTL) by tumor cells. In the absence of stimulatory cells, the CTL activity would have rapidly fallen in vitro. The cytostatic activity also increases during the 3 days in vitro, in parallel to the cytolytic activity: it is due to non-T-cells and remains mainly non-specific. The significance of these data for the interpretation of invitro demonstrated cell-mediated anti-tumor immune reactions is briefly discussed, as well as their relevance in the in vivo role of immune CTL.     

Inhibition of Moloney murine lymphoma and sarcoma growth in vivo by dietary retinoids

The effects of dietary retinoids on the growth of Moloney lymphoma (LSTRA) and sarcoma (MSC) in BALB/c mice were evaluated. Transplantable syngeneic Moloney lymphoma and sarcoma tumors are immunogenic. Preimmunization with LSTRA cells provides protection against subsequent challenge and sarcomas spontaneously regress following injection of an appropriate inoculum of MSC cells. In normal mice fed varying concentrations of all-trans-retinoic acid (RA) and given injections of 10(3) LSTRA cells, RA caused a dose-dependent increase in the number of survivors; 50% of the mice fed RA at 50 mg/kg of diet were long-term survivors. All animals died that were fed a control diet and challenged with 10(3) LSTRA cells. Athymic (nu/nu) mice fed RA were not protected against lymphoma growth, whereas euthymic (nu/+) mice were; therefore, the antitumor effect of RA was thymus dependent. Primary immunization with irradiated LSTRA in the presence of RA caused a significant increase in cell-mediated cytotoxicity by spleen cells at 4 days after immunization. However, challenge of animals preimmunized with LSTRA in the presence of dietary RA revealed a dose-dependent inhibition of memory. A significant reduction in MSC growth was also observed in normal mice fed 13-cis-retinoic acid (cRA). A comparison of the primary antilymphoma effect of dietary RA, cRA, N-(all-trans-retinoyl)-DL-leucine (RL), and N-(4-hydroxyphenyl)retinamide (4-HPR) revealed an efficacy hierarchy of RL greater than RA greater than cRA greater than 4-HPR with RL producing 70% long-term survivors at 115 days after challenge with 10(3) LSTRA cells. These studies indicate that retinoids can inhibit the growth of transplantable, retroviral-induced, immunogenic tumors by thymus-dependent mechanisms and that a newly synthesized retinoylamino acid (RL) is more potent than RA at inhibiting Moloney lymphoma growth.     

In vitro and in vivo enhancement of canine pulmonary alveolar macrophage cytotoxic activity against canine osteosarcoma cells

The combination of chemotherapy with immunotherapy may offer an advantage over either therapy alone and provide a greater potential for total tumor eradication. Monocyte/macrophage-mediated tumor cell killing is a major mechanism of the host's defense against primary and/or metastatic neoplasia. We evaluated the tumoricidal activity against canine osteosarcoma cells of canine pulmonary alveolar macrophages (PAM) exposed in vitro to two recombinant canine (rc) cytokines (rcTNF alpha and rcIFN gamma). We also evaluated the in vivo tumoricidal activity of PAM from dogs treated with the macrophage activator, liposome-encapsulated muramyl tripeptide-phosphatidyl-ethanolamine (L-MTP-PE) alone or in combination with doxorubicin (DOX). This study demonstrated that rcTNF alpha and rcIFN gamma significantly enhance in vitro canine PAM cytotoxicity against canine osteosarcoma cells, and that PAM from dogs treated with DOX + L-MTP-PE have enhanced cytotoxic activity against osteosarcoma cells when compared to dogs treated with DOX or L-MTP-PE alone. These findings support the rationale for combining a chemotherapy agent with an immunotherapy agent for the treatment of metastatic disease, and suggest a role for TNF alpha and IFN gamma as agents for stimulating the antitumor activity of macrophages.     

Interaction of interleukin-11 with cytotoxic therapies in vitro against CEM cells and in vivo against EMT-6 murine mammary carcinoma

Interleukin-II (rhIL-II) is a cytokine that has been shown to enhance the recovery of bone marrow and intestinal crypt cells after cytotoxic insult with radiation or anticancer drugs. The current study examined the effects of rhIL-II on the response of CEM human lymphoblastic leukemia cells and on the EMT-6 murine mammary carcinoma in vivo to cytotoxic anticancer therapies. Exposure of CEM cells to rhIL-II for 24 hr did not alter the cytotoxicity of melphalan or radiation, increased the cytotoxicity of CDDP (100 μM) and 4-hydroperoxycyclophosphamide (50 βM) and decreased the cytotoxicity of 5-fluorouracil and ara-C toward the cells. Treatment of mice bearing the EMT-6 tumor with rhIL-II twice daily for 4 days prior to and the day of cytotoxic therapy resulted in no significant change in the tumor cell killing or bone marrow CFU-GM killing by melphalan, cyclophosphamide, thiotepa, CDDP, radiation, 5-fluorouracil or ara-C. Administration of rhIL-II twice per day on days 7–18 to EMT-6 tumor bearing animals receiving high dose chemotherapy (melphalan, thiotepa or cyclophosphamide) as a single dose on day 7 followed by mobilized peripheral blood cells on day 8 and rhG-CSF on days 8–20, tended to prolong the tumor growth delay produced by the drugs. This rhIL-II treatment also resulted in a more rapid recovery of white blood cells and granulocytes in the animals. Furthermore, animals treated with rhIL-II had improved survival rates compared with animals receiving all other normal tissue support without rhIL-II. © 1996 Wiley-Liss, Inc.     

In vitro and in vivo antitumor activity of lymphokine-induced cytotoxic cells

The present study demonstrates that LICC possess both in vitro and in vivo antitumor activity. The LICC were generated by culturing normal spleen cells with syngeneic peritoneal cells and indomethacin, or with a conditioned medium containing IL 2 with or without a putative new lymphokine, the CCDF. The LICC thus generated selectively killed the lymphoid or solid tumor targets of different H-2 haplotypes and of different etiological origins. The precursors of LICC were probably NK-like cells. The effectors were neither classical NK nor classical cytotoxic T lymphocytes. The LICC were very effective in preventing growth of both lymphoid and solid tumors in vivo, and Thy I+ cells were essential for the anti-tumor effect. The ability to generate LICC was preserved in the tumor-bearing hosts until the terminal stage of tumor growth, when the generation of suppressor T-cells interfered with LICC induction. LICC seem to play an important role in defense against non-immunogenic tumors.     

Dihydroartemisinin is cytotoxic to papillomavirus-expressing epithelial cells in vitro and in vivo

Nearly all cervical cancers are etiologically attributable to human papillomavirus (HPV) infection and pharmaceutical treatments targeting HPV-infected cells would be of great medical benefit. Because many neoplastic cells (including cervical cancer cells) overexpress the transferrin receptor to increase their iron uptake, we hypothesized that iron-dependent, antimalarial drugs such as artemisinin might prove useful in treating HPV-infected or transformed cells. We tested three different artemisinin compounds and found that dihydroartemisinin (DHA) and artesunate displayed strong cytotoxic effects on HPV-immortalized and transformed cervical cells in vitro with little effect on normal cervical epithelial cells. DHA-induced cell death involved activation of the mitochondrial caspase pathway with resultant apoptosis. Apoptosis was p53 independent and was not the consequence of drug-induced reductions in viral oncogene expression. Due to its selective cytotoxicity, hydrophobicity, and known ability to penetrate epithelial surfaces, we postulated that DHA might be useful for the topical treatment of mucosal papillomavirus lesions. To test this hypothesis, we applied DHA to the oral mucosa of dogs that had been challenged with the canine oral papillomavirus. Although applied only intermittently, DHA strongly inhibited viral-induced tumor formation. Interestingly, the DHA-treated, tumor-negative dogs developed antibodies against the viral L1 capsid protein, suggesting that DHA had inhibited tumor growth but not early rounds of papillomavirus replication. These findings indicate that DHA and other artemisinin derivatives may be useful for the topical treatment of epithelial papillomavirus lesions, including those that have progressed to the neoplastic state.     

Studies on mouse Moloney virus induced tumours: I. The detection of p30 as a cytotoxic target on murine Moloney leukaemic spleen cells, and on an in vitro Moloney sarcoma line by antibody mediated cytotoxicity.

Antigenic determinants of p30, the most abundant internal virion protein of C type RNA viruses, were detected on the surface of spleen cells from mice bearing Moloney leukaemia and on an in vitro line of Moloney sarcoma, MSC. On both cell types, these determinants on the p30 molecules served as cytotoxic targets in a xenogenic complement dependent antibody mediated 51Cr release assay. Two antisera were used: a rat anti MLV -M induced lymphoma serum, and an antiserum raised in goats to either disrupted FeLV. The cytotoxic target antigens of these antisera were analysed by inhibition of cytotoxicity with viral and cellular proteins.     

Dynamic alterations in some surface properties of freshly explanted Moloney lymphoma cells.

When Moloney lymphoma (YAC) cells were freshly explanted from the tumor-bearing host into culture, two events occurred in the first 120 minutes: The cells lost their natural IgG coat, and their sensitivity to complement-dependent lysis (CdL) mediated by antibodies to Moloney lymphoma cells decreased or increased. An increasing sensitivity to CdL as a function of incubation time at 37 degrees C was likely to occur when the sensitivity to CdL was low at explantation. A decreasing sensitivity to CdL was probable in instances of a high sensitivity to CdL at explantation. Artifical coating of YAC cells with antibodies to Moloney lymphoma immediately after explantation moderated the alterations in their sensitivity to CdL. This occurred even though a functional antibody did not remain on the cells as evidenced from the gradual decreased sensitivity of these artifically coated cells to the addition of complement. Spent culture media in which freshly explanted cells grew for 60 or 120 minutes sometimes blocked CdL of YAC cells mediated by antibodies to Moloney lymphoma.     

Augmentation of cell-mediated cytotoxicity to a rat lymphoma. II. Characterization of the non-T cytotoxic cells stimulated in vivo by tumour cells as natural killer cells.

A variety of tumours injected into rats were found to rapidly stimulate cytotoxicity which was similar to naturally-occurring cytotoxicity of normal rats. Cytotoxic cells from the spleen and peritoneal cavity closely resembled NK cells in their lytic specificity and cell-surface characteristics. Thus, although cytotoxicity could be stimulated "non-specifically" with tumours which were resistant to lysis in vitro by NK cells, the cytotoxic cells exhibited patterns of specificity against a panel of target cells in direct lysis or competitive inhibition assays which were similar to those of NK cells from normal rats. These cells also closely resembled NK cells in being largely non-adherent, non-T cells, and in exhibiting a similar heterogeneity in the expression of Fc receptors. Thus, cytotoxicity which was augmented shortly after tumour inoculation appeared to be attributable to NK cells. However, whilst the majority of NK cells from normal or tumour-inoculated rats shared these properties, significant heterogeneity was observed. Minor populations of cytotoxic cells were adherent, were lysed by a heterologous anti-T-cell antiserum and complement and did not express an Fc receptor, although it was not determined whether the same subpopulation possessed all three characteristics.     

Cellular reactions against Burkitt lymphoma cells. 3. Effector cell activity of leukocytes stimulated in vitro with autochthonous cultured lymphoma cells

Peripheral blood leukocytes from four patients with Burkitt's lymphoma (BL) were studied for immunological reactions with lymphoblastoid culture line cells. In all four cases peripheral blood leukocytes could reproducibly be stimulated with autochthonous cultured cells derived from tumor biopsies. Stimulation could be accomplished despite the inability of the unstimulated lymphocytes to mediate a cytotoxic response. Leukocytes which have been stimulated with the autochthonous cells displayed strong colony inhibiting activity against the autochthonous BL-derived target cell, but considerably less activity against two non-BL-derived target cells. Autochthonous fibroblasts were ineffective as stimulator cells.     

Enhancement of paclitaxel activity against hormone-refractory prostate cancer cells in vitro and in vivo by quinacrine.

Cytoplasmic phospholipase A2 (PLA2) is known to be phosphorylated and activated by MAP kinase (Lin et al 1993, Cell 72: 269-278), an important downstream component of signal transduction, whereas paclitaxel has been shown to inhibit isoprenylation of ras proteins (Danesi et al 1995, Mol Pharmacol 47: 1106-1111). Given that quinacrine (Q), a PLA2 inhibitor, and paclitaxel (P) might act at different sites in the cell signalling pathway, our aim was to test whether they were synergistic in combination against prostate cancer cells. Cell viability of PC-3, PC-3M and DU145 cells in 96 - well plates was assessed 96 h after drugs were added concurrently. Using Chou analysis, we demonstrated synergy for the combination against all three cell lines. Further, synergy was present under both conservative (mutually non-exclusive) and non-conservative (mutually exclusive) models. Studies in the nude mouse xenograft model support the finding of synergy in vitro. In DU145-bearing mice, Q (50 mg kg(-1)) and P (0.5 mg kg(-1)) given daily for 12 consecutive days, either concurrently or sequentially, was more effective than either drug alone, at twice the dose intensity. In an enzyme-linked immunosorbent (ELISA) apoptosis assay, arachidonic acid was able to partially reverse Q- and P-induced apoptosis, suggesting PLA2 pathway involvement. Finally, the combination of lovastatin, another inhibitor of ras isoprenylation, and quinacrine had synergistic inhibitory effects on the growth of PC-3 cells in vitro, suggesting that the combination of these two classes of compounds might serve as an attractive therapeutic approach for prostate cancer.