A murine plasmacytoma MOPC 104E resistant to cyclophosphamide is resistant to immunotherapy

Summary A murine plasmacytoma MOPC 104E (MOPC) is highly sensitive to chemotherapeutic agents such as cyclophosphamide and mitomycin C as well as to immunotherapy (OK-432-combined adoptive immunotherapy using interleukin-2-cultured killer cells). In the present study, we prepared cyclophosphamide-resistant MOPC cells (MOPC-CPA/R) by serial in vivo passage of tumor cells following cyclophosphamide treatment. The in vivo sensitivity of MOPC-CPA/R to mitomycin C or to immunotherapy (OK-432-combined adoptive immunotherapy) was significantly decreased compared to the parent MOPC. In vitro experiments showed that MOPC-CPA/R were more resistant (five-fold) to lysis by cultured immune spleen cells than MOPC. Inhibition of the lytic activity of cultured immune spleen cells against MOPC was significantly increased (P <0.05) by the addition of unlabeled MOPC compared to unlabeled MOPC-CPA/R. These results suggest that MOPC-CPA/R express weaker antigenicity than MOPC. However, the transfer of immune spleen cells cultured with tumor extract derived from MOPC-CPA/R significantly prolonged the survival of MOPC-CPA/R-inoculated mice. Thus, by repeated cyclophosphamide treatment, tumor cells with low-antigenicity were selected. These tumor cells had lower sensitivity to another chemotherapeutic agent and immunotherapy. Such an immunological response may play an important role in cancer therapy.     

Inhibition of growth of MOPC 104E cells in immunosuppressed mice

In this report we provide evidence that suggests that MOPC 104E may come under regulation in highly immunosuppressed hosts depleted of T cells. Mice that are adult thymectomized, total body irradiated, and transplanted with bone marrow cells were able to resist the growth of MOPC 104E cells. Spleen cells from such animals had low NK activity and no cytotoxicity against MOPC 104E, and poor response to Con A, PHA, and LPS. The animals were deficient in Lyt-1+ and Lyt-2+ cells. The growth of MOPC 104E cells was measured by using the circulating level of MOPC 104E IgM in vivo in mice treated by different modalities. We observed that inhibition of tumor growth in vivo varied with the treatment of the host. Growth was inhibited in the host in the following order: ATXBM greater than XBM greater than NORMAL greater than ATx mice.     

Human autologous rosettes. I. Mechanism of binding of autologous erythrocytes by T cells.

A population of human peripheral blood lymphocytes is able to bind autologous erythrocytes in the presence of autologous serum. Erythrocyte binding is found to be more efficient at 4 °C after preincubation of lymphocytes in autologous serum for 30 min followed by overnight incubation. The overall cellular concentration and erythrocyte/lymphocyte ratio are also crucial in determining the weak erythrocyte binding to autologous lymphocytes. Membrane proteins are involved since the binding structures are sensitive to protease treatment. The 26% RFC obtained with an optimized assay are related to the T-cell lineage.     

Establishment of growth factor-dependent MOPC 104E cell line in vitro

The MOPC 104E cell line has been adapted to grow in vitro using a combination of feeder layer and growth factor(s). The growth of this myeloma cell line is dependent on the presence of growth factor(s). Growth-promoting activity generated from T-cell-mitogen-stimulated, Corynebacterium parvum-stimulated spleen cell culture supernatant, and peritoneal adherent cell culture supernatants gives dose-dependent proliferation. Generation of growth factors in the serum-free bovine serum albumin-substituted media and a rapid assay system based on [3H]thymidine uptake for the quantitation of growth promoting activity are described.     

Cytochemical characteristics of cells forming mast cell-lymphocyte rosettes

Cytochemical studies were performed to reveal activities of phosphatase, chloracetate esterase, neutral alpha-naphtyl acetate esterase and non-specific esterase, and their results enabled us to ascertain the nature of cells forming mastolymphocyte rosettes. Rosette-forming cells were shown to be mast cells to which non-mature cells of T-lymphocyte origin are joined.     

Re-expression of nonglycosylated surface IgA in trypsin-treated MOPC 315 plasmacytoma cells.

The importance of glycosylation for the re-expression of surface immunoglobulin in trypsin-treated MOPC 315 plasmacytoma cells was examined by using tunicamycin, an antibiotic that prevents glycosylation by inhibiting the formation of N-acetylglucosamine-lipid intermediates. Tunicamycin greatly inhibited the secretion of nonglycosylated MOPC 315 IgA in trypsin-treated cells. Two hours after trypsin treatment, there was an 80% inhibition of secretion as measured by immunoprecipitation assays of biosynthetically labeled immunoglobulin. However, tunicamycin had no effect on the time course of re-expression of surface IgA in these cells as measured by TNP-sheep erythrocyte rosette formation and [125I] TNP-albumin binding to the plasmacytoma cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I-labeled cell surface IgA re-expressed in the presence of tunicamycin revealed a protein with an apparent m.w. identical to nonglycosylated MOPC 315 alpha-chains, further suggesting that nonglycosylated surface IgA was being inserted into the plasma membrane. This protein did not bind to concanavalin A-Sepharose. These data suggest that in MOPC 315 plasmacytoma cells, glycosylation is necessary for immunoglobulin secretion but not for immunoglobulin expression at the cell surface.     

Tumor immunity directed against MOPC 104E: Effect of various therapeutic regimens

Summary Animals bearing the passable plasmacytoma MOPC 104E could be cured of palpable tumors (0.6–2.0×10⁸ cells) with single 10–250 mg/kg doses of cyclophosphamide or single localized x-ray doses greater than 1600 R. Residual tumor immunity of cured animals was determined by their ability to reject graded numbers of viable MOPC 104E cells 30 days following curative therapy. High doses of cyclophosphamide (250 mg/kg), although curative, left significantly less residual tumor immunity than either low dose cyclophosphamide (10 mg/kg) or localized irradiation. Animals cured of palpable tumors by high doses of cyclophosphamide nonetheless rejected greater numbers of cells in secondary challenge than did untreated control animals.This investigation received support from NIH Grants 13371, 17065, 05136, and 09082 from the National Cancer InstituteSubmitted in partial fulfilment of the degree Doctor of Philosophy in Radiation Biology     

Tissue localization of methotrexate-monoclonal-IgM immunoconjugates: Anti-SSEA-1 and MOPC 104E in mouse teratocarcinomas and normal tissues

Summary Methotrexate (MTX) was coupled to the tumor-targeting monoclonal IgM, anti-SSEA-1 and the nontargeting myeloma IgM, MOPC 104E. At 24-h intervals following injection, drug deposition in MH-15 teratocarcinomas and in several normal tissues was followed by immunoperoxidase microscopy using the M16 monoclonal antibody to MTX. MTX-anti-SSEA-1 was deposited on the surface and in the interior of living tumor cells 24 h after injection; at 48 h and after, only low-level binding to necrotic tissue was found. There was no significant gradation in staining from the outside to the interior of the tumors. In tumors, the control MOPC 104E immunoconjugate was detectable only in necrotic tissue. Binding to SSEA-1-expressing normal tissues was undetectable, except for pericryptal fibroblasts in the small intestine. No significant pathology was found in normal tissues that are SSEA-1 positive. High levels of the immunoconjugate were detected in the liver, where MTX was found predominantly in Kupffer cells and possibly in hepatocytes; again, no significant morphological changes were associated with this retention. Thus tumor-associated antigens can be suitable targets for antibody-drug conjugates even when present in normal tissues and in large quantities, provided that the antigens in normal tissues are inaccessible. Moreover, deposition in viable tumor tissue can be assessed using monoclonal antibodies to methotrexate.Supported by intramural research funds of the VAMC and by the Division of Urological Surgery, University of Pittsburgh     

Immunoglobulin M biosynthesis. Production of intermediates and excess of light chain in mouse myeloma MOPC 104E

1. Conditions have been established for the estimation of molecular weights of proteins by analytical gel filtration and sucrose-density-gradient centrifugation in 2.5m-potassium chloride-1m-sodium chloride; Halobacterium cutirubrum polynucleotide phosphorylase, DNA-dependent RNA polymerase and RNA-dependent RNA polymerase have been studied by these methods. 2. The RNA-dependent polymerase has also been studied by density-gradient centrifugation in the absence of salt. 3. All three proteins are of unusually low molecular weight compared with similar enzymes from non-halophilic bacteria.     

Formation of lipid-linked oligosaccharides by MOPC 315 plasmacytoma cells. Decreased synthesis by a nonsecretory variant

MOPC 315 is a BALB/c plasmacytoma which secretes a trinitrophenol-binding IgA lambda 2 paraprotein. We have investigated the incorporation of [3H]mannose into lipid-linked oligosaccharide precursors in wild-type MOPC 315/J and variant nonsecretory 315/P cells. In pulse labeling experiments, no differences could be detected in the ability of the two cell types to incorporate [3H]mannose into lipid-linked oligosaccharides containing 5 or less mannose residues. In contrast, quantitation of the incorporation of [3H]mannose into larger lipid-linked oligosaccharides and proteins revealed a 49 and 40% decrease, respectively, in the 315/P cells compared to wild-type cells. Further characterization of the lipid-linked structures documented a marked decrease in glucosylated oligosaccharides isolated from 315/P cells. When membranes from the two cell lines were analyzed for their ability to transfer [3H]glucose from UDP-[3H]glucose to [3H]glucosylphosphoryldolichol, an apparent deficiency was noted in the 315/P preparations. However, if assay conditions were adjusted to include AMP in the reaction mixtures, no differences in the in vitro synthesis of [3H]glucosylphosphoryldolichol or [3H]glucose-labeled oligosaccharide-lipid could be detected. In these reactions AMP was found to prevent hydrolysis of UDP-[3H]glucose by inhibiting nucleotide pyrophosphatase (EC 3.6.1.9), the specific activity of which was determined to be more than 100 times greater in variant 315/P compared to wild-type MOPC 315/J cells. This large difference in specific activity was not accompanied by similar differences in the activity of several other enzymes analyzed. A decrease in whole cell UDP-glucose pool size was not detected in 315/P cells. Therefore, if nucleotide pyrophosphatase is important for the control of substrates for glycosylation, it must regulate nucleotide sugar levels at a site other than the cytoplasm of cells, perhaps at the location of synthesis of the larger lipid-linked oligosaccharides.     

Proportion of lymphocytes forming E, EA, and EAC rosettes following treatment with human interferon preparations in vitro

The proportion of lymphocytes forming E, EA, and EAC rosettes after treatment with human interferon preparations in vitro was measured. While interferon increased the percentage of lymphocytes forming E rosettes, the percentage of cells forming EA rosettes was diminished. The proportion of lymphocytes forming EAC rosettes was not altered to any major extent by interferon treatment. The same effects were observed when fibroblast interferon, purified to homogeneity with regard to molecular weight, was used.     

Human autologous rosettes. IV. Their relation with interleukin 2 activity production and natural killer cells in cancer patients

Peripheral blood lymphocytes (PBL) of solid-tumor-bearing cancer patients produced a lower interleukin 2 (IL-2) activity after lectin stimulation than did those from normal subjects. Moreover natural killer (NK) cell activity and autologous rosette forming (ARF) cell rate are found significantly correlated with IL-2 production in these patients. No direct relation is observed between ARF cell ratio and NK cell activity in a given patient. A central role for IL-2 in cancer patient immune dysfunctions is suggested. Two lines of pathogenetic mechanisms are documented. First, PBL exhibited cellular function defects, namely, autologous receptor expression, IL-2 production, and NK activity. Second, these dysfunctions involved, at least partly, plasma factors. The possibility of specific deficiency, (e.g., thymic factors) is not documented. Conversely it is demonstrated that patient plasma contain immunosuppressive factor(s) that block(s) IL-2 production and ARF cell expression. Involvement of ARF cell receptor in T-cell activation is discussed.