Regulation of immunoglobulin biosynthesis in the murine plasmacytoma MOPC 315.

We have examined certain aspects of IgG biosynthesis by constructing hybrids between MPC11 (gamma2b, kappa) and MOPC 315 (alpha,lambda2) that have lost the ability to synthesize one or the other heavy chain. Cells express the three chains in a stable fashion, and both autologous (parental) and heterologous (nonparental) H and L chain pairs form and are secreted. The alpha H chain was found in polymeric form when associated with the heterologous kappa L chain. The lambda2 L chain covalently assembled to the heterologous gamma2b H chain. Surprisingly, autologous pairing was always favored over heterologous pairing in vivo by 5 to 10:1 in terms of rate of assembly. Similar ratios were maintained in the secreted protein. These results suggest that co-expression of particular H and L chain pairs is predetermined. Evolution presumably operates to improve antigen recognition as well as rate of assembly of active molecules.     

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.     

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.     

Sequential immunotherapy using interleukin-1 followed by interleukin-2 of ascitic MOPC104E-bearing mice

This study shows that intraperitoneal injection of interleukin-1 (IL-1), followed by interleukin-2 (IL-2), can effectively eradicate murine ascitic tumor cells. This antitumor effect of IL-1 and IL-2 was abolished when administration of IL-2 preceded that of IL-1. Solid tumors inoculated subcutaneously (s.c.) into the back of mice were also sensitive to this combined i.p. therapy, indicating a systemically-operating antitumor mechanism. Splenocytes from tumor-bearing mice treated with IL-1 followed by IL-2 showed a strong tumor-neutralizing activity. The population responsible proved to be Lyt2.2 (CD8)-positive cells.Abbreviations IL interleukin - LAK lymphokine activated killer - LU lytic unit - MST median survival time - SE sonicated tumor extract     

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     

Untranslated immunoglobulin kappa light chain mRNA in a lambda light chain-producing mouse myeloma, MOPC104E

Fourteen clones were isolated in culture from a mouse myeloma, MOPC104E. All clones had kappa and lambda types of light chain mRNAs in approximately equimolar quantity as assayed by hybridization with specific complementary DNA (cDNA). However, the myeloma produces and secretes only lambda-type light chain protein. Both kappa- and lambda-type mRNAs in these clones were indistinguishable from kappa- and lambda-type mRNAs of other myelomas with respect to (a) adsorption to oligo-(dT) cellulose, (b) molecular size (12.6 S), and (c) thermal stability of the hybrids formed with corresponding cDNA. The kappa chain mRNA of MOPC104E cells, however, was translated very inefficiently both in vivo and in vitro, whereas the lambda chain mRNA was translated efficiently. These results indicate that each cell of MOPC104E myeloma synthesizes a crippled kappa chain mRNA in addition to a normal lambda chain mRNA.     

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.     

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     

Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in alpha 1,2-linked mannose processing

Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.     

Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.     

The allogeneic effect on tumor growth. I. Inhibition of a murine plasmacytoma, MOPC 315, by the graft-vs-host reaction.

The allogeneic effect has been employed as a potent immunopotentiator in preventing the growth of a murine plasmacytoma and prolonging host survival. Parental BALB/c spleen cells were passively transferred to (BALB/c x A/H)F1 hybrid mice, who were then given a highly lethal dose of MOPC 315 plasmacytoma, a tumor of BALB/c origin. The resultant graft-vs-host reaction protected the recipient mice against growth of the tumor and significantly prolonged survival. This phenomenon was dependent upon the dose of BALB/c lymphoid cells employed, the route of administration, and the time interval between lymphoid cell transfer and tumor inoculation. A wide range of lymphoid cell doses and time intervals were effective, and repeated doses of allogeneic cells provided better protection than a single dose.     

Transplantation resistance to a murine plasmacytoma lacking MHC determinants.

A spontaneously arising murine plasmacytoma, HPC-202, derived from a BALB/c.H-2b congenic mouse that lacks any detectable H-2 determinants on its cell surface is described. However, the expression of H-2 determinants is inducible by interferon-gamma. The H-2 negative cell surface phenotype permits the HPC-202 tumor to escape H-2 allospecific cytotoxic cell lysis but not NK cell lysis, as well as to grow, to varying degrees, in some H-2 incompatible hosts. In those strains which exhibit a resistance to HPC-202 growth, resistance does not map to a single gene within the major histocompatibility complex of the mouse. Resistance is also radiosensitive and is therefore presumably due to a rapidly dividing cell population. The utility of this tumor as a model system to study both the non-H-2-restricted natural resistance to tumor growth, and the mechanism by which H-2 genes are regulated by cells is discussed.     

Passive immunity to murine plasmacytoma by rabbit antiidiotypic antibody to myeloma protein.

Antiidiotypic antisera to LPC-1 and MOPC-300 plasmacytoma globulins were produced in rabbits by immunization with the corresponding antigen and exhaustive immunoabsorption with normal BALB/c plasma and other myeloma globulins. IgG fractions of these antisera when given intraperitoneally on three consecutive days after tumor implantation, protected the animal specifically from the grafting of the corresponding plasmacytoma or induced regression of the tumors after their initial grafting and growth. In vitro cytotoxicity of the antisera to plasmacytoma cells was not demonstrated. Plasma of the antisera-treated normal BALB/c mice, though containing antiidiotypic antibody in high titers (320-640), was not cytotoxic to tumor cells. The inhibitory effect on tumor growth can be considered the result of passive immunization against plasmacytoma with xenogeneic humoral antibody.     

Therapeutic efficacy of sequential therapy with OK-432, cyclophosphamide,IL2-cultured lymphocytes andin vivo IL2 against advanced murine plasmacytoma

BALB/c mice inoculated IP with a syngeneic plasmacytoma MOPC104E were treated with a combination of a streptococcal preparation, OK-432 (1 KE, 0.1 mg/mouse), low-dose of cyclophosphamide (CPA, 1 mg/kg) and adoptive transfer of tumor-bearer-spleen cells (2 x 10(7) cells) cultured with IL2 and sonicated tumor extract (adoptive immunotherapy; AIT). The consecutive protocol of OK-432 (day 8, 9 post inoculation) - CPA (day 10) - AIT (day 11) was the most effective. Rate of complete remission was highest when recombinant (r-) IL2 was injected to the mice after AIT. Moreover, another bacterial preparation, Nocardia rubra cell wall skeleton and another low-dose chemotherapy, Mitomycin C could be used successfully instead of OK-432 or CPA. Transfer test of intraperitoneal cells (tumor cells plus host cells) of mice on day 11 post inoculation (on the day of AIT) revealed that OK-432 augmented the susceptibility of peritoneal cells to cultured lymphocytes in inhibition of transplantability, and that CPA after OK-432 augmented the anti-tumor effect of tumor-bearer-spleen cells which act synergistically with cultured lymphocytes. This therapy schedule seems to be the best model to augment the effect of AIT with minimal side effect.