MOPC-104E Cells Research Articles

Inoculation of Human Interleukin-17 Gene-Transfected Meth-A Fibrosarcoma Cells Induces T Cell-Dependent Tumor-Specific Immunity in Mice

Objective: The biological activities of interleukin-17 (IL-17), a newly cloned cytokine, have not been fully elucidated. The present study was designed to assess the in vitro and in vivo effect of transfecting the IL-17 gene into tumor cells. Methods: A complementary DNA (cDNA) encoding human IL-17 (hIL-17) was obtained by polymerase chain reaction amplification from the human CD4+ T cell cDNA library and inserted into the plasmid pRc/cytomegalovirus to construct an expression vector for the hIL-17 gene. Murine Meth-A fibrosarcoma cells were transfected with the hIL-17 gene using the lipofectin method. The hIL-17 gene-expressing clone (Meth-A/IL-17) was selected and analyzed for cytokine expression by Northern blot. Results: There was no significant difference in the in vitro proliferation rate among parent Meth-A, cells transfected with vector alone and Meth-A/IL-17 cells. When the tumor cells were transplanted subcutaneously into BALB/c nude (nu+/nu+) mice, there was no difference in in vivo growth rates among the three cell lines. Challenge with tumor cells in conventional BALB/c mice, however, resulted in the rejection of Meth-A/IL-17 cells, but the other two lines did grow. After immunization with Meth-A/IL-17 cells, the mice were rechallenged by parent Meth-A or syngeneic MOPC-104E plasmacytoma cells; the immunized mice rejected the Meth-A cells, but not the MOPC-104E cells. Injecting the anti-thy 1,2 (CD90), anti-CD4 or anti-CD8 monoclonal antibody into conventional BALB/c mice resulted in the resumption of in vivo growth of Meth-A/IL-17 cells, but injecting the anti-asialo GM1 antibody did not. Furthermore, flow cytometric analysis demonstrated a significant increase in the expression of major histocompatibility complex (MHC) class I and class II antigens and lymphocyte function-associated antigen-1 on Meth-A/IL-17 cells. Conclusion: Meth-A cells transfected with the hIL-17 gene can induce tumor-specific antitumor immunity by augmenting the expression of MHC class I and II antigens, and both CD4+ and CD8+ T cells may play important roles in inducing antitumor immunity, suggesting the possibility of developing a tumor vaccine incorporating IL-17-transfected tumor cells.

The effectiveness of active specific immunotherapy using interferon-gamma-gene-transduced tumor cells in a murine tumor model.

Active specific immunotherapy was examined in BALB/c mice using sonicated tumor extract(SE) from plasmacytoma MOPC104E or interferon-gamma-(IFN-gamma)-gene-transduced MOPC104E (Mu gamma), employing interleukin-1 (IL-1) as an adjuvant. Subcutaneous (s.c.) MOPC104E tumor growth was significantly suppressed in mice given a single preimmunization of IL-1 plus Mu gamma-SE, 9 days prior to inoculation, whereas the tumor growth in mice similarly pretreated with IL-1 alone or IL-1 plus MOPC104E-SE(MOPC-SE) was not affected; the mean tumor diameters on day 21 being 6.8 mm, 15.3 mm, and 13.2 mm, respectively. Two-dose preimmunization with Mu gamma-SE alone or IL-1 alone given 10 and 7 days prior to s.c. inoculation also resulted in profound suppression of tumor growth compared to the control. As postsurgical immunization, MOPC104E cells were injected into the foot pads of mice, followed by amputation of the tumor-bearing foot 20 days later, then treatment with IL-1 plus MOPC-SE or IL-1 plus Mu gamma-SE on days 4, 7, and 10 after the amputation. The mean survival of the mice treated with IL-1 plus Mu gamma-SE was significantly prolonged compared to that of the mice treated with IL-1 plus MOPC-SE, at 90.3 days vs 40.9 days, respectively (P < 0.05 by the Cox-Mantel test). These results suggest that SE prepared from IFN-gamma-gene-transduced MOPC104E is more effective for active specific immunotherapy than SE prepared from MOPC104E.

A new model of active specific immunotherapy using interleukin-1 and sonicated tumor supernatant in murine tumor system.

The possibility of active specific immunotherapy using interleukin-1 (IL-1) plus sonicated tumor supernatant (SS) was examined in a murine tumor model. The growth of intraperitoneally or subcutaneously inoculated plasmacytoma MOPC104E, which is syngeneic to BALB/c mice, was significantly suppressed by intraperitoneal pretreatment with IL-1 and SS from MOPC104E cells (MOPC-SS), on days 10, 7, and 4 before tumor inoculation. Pretreatment with IL-1 plus MOPC-SS or MethA-SS (SS from MethA cells) suppressed the growth of subcutaneous tumor of only the corresponding tumor cells, indicating the development of tumor-specific immunity in vivo. The splenic cells of immunized mice with IL-1 and MOPC-SS showed tumor neutralizing activity. However, their tumor neutralizing activity was abrogated when they were treated in vitro with anti-Thy1.2 or anti-L3T4 plus complement. Moreover, when combined with indomethacin per oral, IL-1 plus MOPC-SS significantly suppressed the growth of established subcutaneous tumor and prolonged survival of post-operative mice. These results suggest that this new type of active specific immunotherapy could be a useful method for cancer immunotherapy, especially when combined with oral indomethacin.

The induction of specific antitumor immunity by in vivo treatment with interleukin-1 and sonicated tumor extract in a murine model.

BALB/c mice were pretreated intraperitoneally with interleukin-1 (IL-1) and sonicated tumor extract (SE) from plasmacytoma MOPC104E, 10, 7, and 4 days prior to the intraperitoneal or subcutaneous inoculation of MOPC104E cells, following which significant suppression was observed. The mean survival time and tumor diameter on day 21 were 46.7 days and 0 mm, respectively, in contrast to the 20.9 days and 20.4 mm of control mice. Mice pretreated with IL-1 and SE from MOPC104E (MOPC-SE) were not suppressed following fibrosarcoma MethA inoculation, which indicates the tumor specificity of immunity in this model. This systemically operating antitumor immunity was also achieved by the intramuscular administration of IL-1, or when tumor challenge was performed on day 7 or 14. Moreover, MOPC104E-specific delayed-type hypersensitivity was detected in these mice. The results of this study suggest the possibilities of a new type of active specific immunotherapy, which could prove useful as postsurgical adjuvant therapy for cancer patients.

Host Response to Myeloma: Effect of Syngeneic Spleen Cells on the Growth and Functionof MOPC 104E Myeloma in Vitro

The effect of coculturing nonadherent and plastic adherent cells from the spleen with MOPC 104E KI81 for short (24 h) and long term (7 days) was investigated. In both culture systems, the effect of the spleen cells on the secretion of IgM by plaque-forming cells assay and growth of the plasmacytoma by cell counts and flow cytometry was measured. In these studies, a low effector:target (E:T) ratio which did not produce cytotoxicity to MOPC 104E cells was used. We observed that while nonadherent spleen cells from normal or MOPC 104E-primed mice inhibited secretion of IgM by the MOPC 104E cells, they stimulated the proliferation of MOPC 104E cells two times faster than MOPC 104E cells cultured alone. Plastic adherent cells from the spleens of normal mice or MOPC 104E-primed mice also inhibited secretion of IgM by the tumor cells as measured by plaque formation, and stimulated proliferation of MOPC 104E in 24 h coculture. Plastic adherent cells from normal nonprimed mice initially stimulated the myeloma to grow, but by 24 h, a large fraction of the population was in the G1 or possibly resting state. The effect of nonadherent and plastic adherent cells on the stem cell activity of MOPC 104E was also tested in 7-day colony-forming assays. Nonadherent cells had no effect on colony-forming units or plaque formation. Plastic adherent cells from normal spleen cells inhibited plaque formation by 68% but had no effect on colony formation. However, plastic adherent cells from spleens of mice primed in vivo with MOPC 104E tumor cells suppressed plaque formation by 98% and also reduced colony formation. The results showed that inhibition by macrophages of IgM production by MOPC 104E cells is independent of cell proliferation. The adherent macrophages from both normal and in vivo-primed spleen cells were Mac.1 positive after 7 days of coculture with MOPC 104E cells. However, the density of Mac.1 was greater on primed macrophages.

Host Response to Myeloma: I. Induction of Cytotoxic and Suppressor T Cells by In Vivo Immunization with MOPC 104E Plasmacytoma

Spleen cells from BALB/c mice immunized with mitomycin C-treated MOPC 104E plasmacytoma cells demonstrated negligible cytotoxic activity (less than 10% specific cytotoxic activity) in the 51Cr release assay. These cells exhibited increased cytotoxic activity when they were secondarily sensitized in vitro with mitomycin C-treated MOPC 104E cells. Spleen cells from normal mice showed tumor-specific cytotoxic activity when cocultured with mitomycin C-treated tumor cells at the optimal responder to stimulator ratio of 25:1. The level of cytotoxic activity obtained by in vivo primed and secondarily in vitro sensitized spleen cells did not exceed the level of activity obtained by in vitro primary sensitized cells. Significant suppression of the cytotoxic activity of in vitro primary sensitized cells was observed when cocultured with in vivo primed spleen cells during primary sensitization in vitro at a responder to suppressor cell ratio of 1:1. Suppressor cells of in vivo primed mice were removed by treatment with anti-Thy 1.2 and complement. These results suggest that spleen cells from in vivo primed mice consisted of at least two subpopulations of cells, a cytotoxic (prekiller) and suppressor T cells. Attempts to induce cytotoxic cells in vivo might have failed because of the appearance of suppressor T cells.

Some characteristics of the in vivo antitumor immunity exhibited by mice cured of a large MOPC-315 tumor by a low dose of melphalan.

BALB/c mice cured of a large MOPC-315 or MOPC-104E plasmacytoma following treatment with a low dose (2.5 mg/kg) of melphalan (L-PAM) were resistant to challenge with the other plasmacytoma but to a much lesser extent than to challenge with the autochthonous plasmacytoma. The resistance of the L-PAM-cured MOPC-315-tumor bearers to challenge with MOPC-104E tumor cells was increased when the MOPC-104E tumor cells were admixed with MOPC-315 tumor cells prior to their inoculation. This enhanced resistance to MOPC-104E cells was due to elimination of the MOPC-104E tumor cells through an innocent bystander killing effect since it did not render the mice more resistant to a subsequent challenge with MOPC-104E tumor cells alone. Administration of carrageenan to L-PAM-cured MOPC-315-tumor bearers 1 day after the challenge with the mixture of MOPC-104E and MOPC-315 tumor cells drastically reduced the ability of the mice to resist the tumor challenge. All of the tumors that developed in such mice were of MOPC-104E origin only (as judged by the binding specificity of the myeloma proteins secreted by the tumor cells as well as that present on their surface) even though (a) the tumor inoculum used consisted of up to 10-fold more MOPC-315 than MOPC-104E tumor cells and (b) the MOPC-315 tumor cells divide more rapidly. The same protocol of carrageenan treatment did not reduce the ability of normal BALB/c mice to develop in vivo a primary cell-mediated cytotoxic response nor a primary antibody response indicating that it has no effect on the initiation of an immune response. Therefore, it is conceivable that carrageenan treatment reduced the ability of L-PAM-cured MOPC-315-tumor bearers to reject a challenge with MOPC-315 and MOPC-104E tumor cells by interfering at the effector stage. The ability of the L-PAM-cured MOPC-315-tumor bearers to reject the MOPC-315 cells present in the challenge mixture was reduced when the mice were treated with anti-Thy 1.2 antibody but not with carrageenan, indicating that T-cells independent from carrageenan-sensitive effector cells are required for the rejection of the MOPC-315 tumor cells. Thus, at least two different effector mechanisms participate in the rejection of a challenge composed of MOPC-315 and MOPC-104E tumor cells by L-PAM-cured MOPC-315-tumor bearers.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytotoxic T lymphocyte clone recognizing self idiotype: its regulatory role in antibody production

MOPC 104E idiotype-specific cytotoxic T lymphocyte (CTL) clones were established. The clone KA1.02 specifically killed MOPC 104E cells and inhibited anti-dextran antibody production in an idiotype-specific manner. Both cytotoxicity and antibody inhibition were restricted to the cross-reactive idiotype and class I histocompatibility antigen. The data presented here provide definitive evidence that the self idiotype is the immunogenic determinant recognized by T lymphocytes in association with the major histocompatibility complex product. A regulatory role of CTL in antibody response is proposed.

Immunoglobulin gene expression in a coupled transcription/translation system from mouse plasmacytoma cell-free extracts

Mouse plasmacytoma cytoplasmic extracts and sonicated nuclei have been incubated under conditions which resulted in translation and transcription, respectively. When the cell-free systems were combined, incorporation of radioactive precursors into RNA and protein was enhanced and extended. Coupling of transcription and translation was indicted by the inhibition of protein synthesis, and specifically immunoglobulin synthesis, by actinomycin D and alpha-amanitin. When immunoglobulin synthesis was investigated in MOPC-104E cells Which contain both lambda and K mRNAs but secrete only lambda light chains, the extracts synthesized both K and Lambda light chains. These results indicated that the unexpressed MOPC-104E K mRNA could be translated on homologous ribosomes under the appropriate conditions and suggested that postranscriptional controls may play a role in k-chain gene expression in MOPC-104E cells.

Steady-State Kinetics of Mouse DNA Polymerase α&lt;xref ref-type="fn" rid="fn1"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;

Steady-state kinetic studies of DNA polymerase alpha purified from mouse myeloma MOPC104E cells have been carried out. The results of initial velocity analysis with or without sodium pyrophosphate, a product inhibitor, indicated that the reaction mechanism of this enzyme can be categorized as an ordered Bi Bi type where the concentration of the ternary complex is very low.

T Lymphocyte-Mediated Suppression of Myeloma Function in Vitro

Abstract Alloreactive cells generated by in vitro stimulation of C57BL/6 (H-2b) spleen lymphocytes with irradiated MOPC 315 or MOPC 104E (H-2d) cells were shown to lyse 51Cr-labeled myeloma targets at high effector:target ratios under conditions of maximal cell contact. After coculture at low effector:target ratios under conditions of inefficient cell contact, the alloreactive cells cause variable and frequently minimal lysis of myeloma targets but markedly suppress antibody secretion even by viable myeloma cells. The suppressor cells are radioresistant T cells lacking I-J subregion-encoded surface determinants; their precursors are insensitive to cyclophosphamide; suppression is H-2 specific and not mediated by secreted factors; and the suppression is blocked by Cytochalasin B, a known inhibitor of T cell-mediated cytolysis. These properties are typical of cytolytic T lymphocytes (CTL) and not of defined suppressor T cells, suggesting that inhibition of myeloma function probably represents a pre-lytic effect of the alloreactive CTL, although a CTL-like suppressor cell effect cannot be definitively excluded. These results are discussed with reference to the possible relationships between suppressor and cytolytic T lymphocytes.

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

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.

Suppression of myeloma establishment in adult mice with anti-μ antiserum

Uniform suppression of MOPC-104E tumor development was observed in adult BALB/c mice to which 0.4 ml of high titered anti-μ antiserum had been administered intraperitoneally or intravenously one day before subcutaneous tumor implantation. In contrast, when MOPC-104E cells were exposed to anti-μ antiserum in vitro for 10 min and subsequently injected into adult BALB/c mice, inhibition of tumor development was observed in only about half of the subject animals. Nude mice treated intraperitoneally with anti-μ antiserum were also uniformly refractory to MOPC-104E challenge. Anti-μ antiserum exhibited virtually no cytotoxicity against MOPC-104E cells in vitro. These observations suggest that neither complement-mediated cytotoxicity nor T cell-mediated immunity is likely to be the primary mechanism for anti-μ-mediated suppression of myeloma development in adult BALB/c mice. More plausible explanations for this type of suppression include macrophage arming, some type of antibody-dependent cell-mediated cytotoxicity, and/or opsonization.

Biosynthesis and assembly of IgM. Addition of J chain to intracellular pools of 8S and 19S IgM

The addition of J chain to intracellular and secreted IgM has been studied in mitogen-stimulated mouse spleen cells and two mouse myelomas (MOPC 104E and Y5781). The cells of Y5781 contain a large pool of intracellular 19S IgM and it is shown that this IgM is assembled by two pathways, one via the addition of J chain to the 8S IgM subunit and the second by direct addition of J chain to 19S IgM. The absence of J chain on the intracellular IgMs of MOPC 104E and mitogen-stimulated spleen cells suggests that IgM may be assembled in these cells predominantly by the second pathway. The detection in Y5781 cells of a population of 19S IgM molecules without J chain, to which J chain is added shortly before or during secretion, demonstrates that J chain is not necessary for the polymerisation of IgM in the intact cell.