An essential role for macrophage migration inhibitory factor (MIF) in angiogenesis and the growth of a murine lymphoma.

BACKGROUND: Macrophage migration inhibitory factor (MIF) has been shown to counterregulate glucocorticoid action and to play an essential role in the activation of macrophages and T cells in vivo. MIF also may function as an autocrine growth factor in certain cell systems. We have explored the role of MIF in the growth of the 38C13 B cell lymphoma in C3H/HeN mice, a well-characterized syngeneic model for the study of solid tumor biology. MATERIALS AND METHODS: Tumor-bearing mice were treated with a neutralizing anti-MIF monoclonal antibody and the tumor response assessed grossly and histologically. Tumor capillaries were enumerated by immunohistochemistry and analyzed for MIF expression. The effect of MIF on endothelial cell proliferation was studied in vitro, utilizing both specific antibody and antisense oligonucleotide constructs. The role of MIF in angiogenesis also was examined in a standard Matrigel model of new blood vessel formation in vivo. RESULTS: The administration of anti-MIF monoclonal antibodies to mice was found to reduce significantly the growth and the vascularization of the 38C13 B cell lymphoma. By immunohistochemistry, MIF was expressed predominantly within the tumor-associated neovasculature. Cultured microvascular endothelial cells, but not 38C13 B cells, produced MIF protein and required its activity for proliferation in vitro. Anti-MIF monoclonal antibody also was found to markedly inhibit the neovascularization response elicited by Matrigel implantation. CONCLUSION: These data significantly expand the role of MIF in host responses, and suggest a new target for the development of anti-neoplastic agents that inhibit tumor neovascularization.     

Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma

Follicular lymphoma (FL) is a disease that responds to current treatment regimens; however, patients in general relapse with increasingly refractory disease. Idiotype-based vaccines are currently under trial for the treatment of FL. These vaccines comprise the patient’s BCR idiotype (Id) as the tumor antigen conjugated to the protein carrier Keyhole Limpet Hemocyanin (KLH); however, other protein carriers may enhance the immune response to the lymphoma Id. In this study we investigated whether an alternate carrier, Listeriolysin O (LLO), would amplify the immune response to Id protein and provide better protection against challenge by 38C13 murine lymphoma. The Id-LLO vaccine compared favorably against Id-KLH in tumor-protection studies and both vaccines provided systemic immunity against 38C13 lymphoma. However, the immune response to the two conjugates was different in that Id-LLO induced a more powerful Th1 response characterized by high titer IgG2a anti-Id antibodies after one immunization and the presence of CD4 cells secreting IFN-γ. In vivo studies demonstrated that immune serum contributed to the anti-lymphoma efficacy seen following Id-LLO immunization. Interestingly, Id-LLO immunized mice, when challenged twice with 38C13 lymphoma provided better protection against challenge by the BCR loss variant 38C13-V2, suggesting that Id-LLO immunized mice have more potential to develop epitope spreading than Id-KLH. In conclusion, Id-LLO compared favorably against Id-KLH in its anti-lymphoma efficacy. Furthermore, Id-LLO induced a more potent humoral and cell-mediated immune response and promoted epitope spreading after lymphoma challenge. Thus, anti-Id vaccines incorporating LLO may be a better therapeutic option for treatment of B-cell lymphoma. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synergistic antitumor effect of interferon and anti-idiotype monoclonal antibody in murine lymphoma

Both IFN-alpha and anti-idiotype monoclonal antibody therapy have significant antitumor activity in vivo in a murine B cell lymphoma model. Combination therapy with syngeneic anti-idiotype antibody of the IgG2a or IgG2b isotype (a single i.p. injection of 100 micrograms) and recombinant human hybrid interferon-alpha A/D (10(4) to 10(6) U three times weekly for 3 wk) synergistically increased median survival time in mice challenged with a lethal dose of tumor cells compared with the sum of the median survival times of the two individual treatments. IFN-alpha has direct antiproliferative activity against 38C13 in vitro and enhances in vitro macrophage anti-idiotype antibody-specific cytolysis for IgG2a, IgG2b, and IgG1 isotypes.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular responses elicited by tumor-derived immunoglobulin M and its molecular subunits

C3H/HeN mice were immunized with idiotypic immunoglobulin M (IgM) and its molecular subunits from the syngeneic 38C13 lymphoma. Immunization with idiotypic IgM (38C-Id) resulted in idiotype-specific humoral and cellular immunity and protection against a lethal tumor cell challenge. Heavy (H38C) and light (L38C) chains were isolated by electroelution from preparative polyacrylamide gels. Both of these immunogens induced significant resistance to a subsequent tumor challenge. Variable region immunogens, in the form of trpE-fusion proteins, were obtained by cloning heavy and light chain variable region genes into the expression plasmid pATH-11. Of these, only the trpE-VH38C immunogen yielded immune resistance to tumor challenge. Finally, the nucleic acid sequence of 38C-Id light chain was determined and, based on the corresponding amino acid sequence and an analysis of predicted secondary structure, a region of potential antigenicity in complementarity-determining region 3 was chosen for the production of a synthetic peptide. Vaccination with this synthetic peptide resulted in significant suppression of tumor growth. Analysis of the humoral and cellular immunity generated by these vaccines revealed the presence of antibodies reactive with native idiotypic IgM only in 38C-Id, H38C, and trpE-VH38C immune sera, although the latter two were not idiotype-specific. Idiotype-specific lymphocytes, which proliferated in response to native 38C-Id, were observed in all immune animals. With the exception of the fusion protein immunogens, conjugation to an immunogenic carrier protein (keyhole limpet hemocyanin or thyroglobulin) was required for optimal humoral and cellular responses.     

Anti-idiotypic antibodies recognizing stable epitopes limit the emergence of idiotype variants in a murine B cell lymphoma.

The emergence of Id variants is a major escape mechanism from anti-Id therapy of human B cell malignancies and of the murine B cell lymphoma 38C13. To determine what impact the epitope specificity of anti-Id antibodies has on the prevention of emergence of such Id variants in the 38C13 lymphoma, anti-Id mAb of varying epitope specificity for the Id of 38C13 tumor cells were produced and studied. Some antibodies, produced by immunizing mice with both the wild-type 38C13 IgM and variant IgM, cross-reacted with wild-type 38C13 IgM and with all four members of a panel of variant IgM. These anti-Id did not react with separated 38C13 IgM H or L chains by Western blot, but did react with the cytoplasmic H chain of the surface Ig- variant cell line T2D that expresses the same H chain as wild-type 38C13 in its cytoplasm but does not express any associated L chain. In contrast, anti-Id of narrower specificity did not react with this H chain. This indicated that the broadly cross-reactive antibodies recognized a stable epitope on 38C13 H chain. When a broadly cross-reactive antibody MS11G6 was compared to S1C5, an antibody of narrower specificity, MS11G6, was superior at preventing tumor growth in mice inoculated with 38C13 cells. Moreover, no surface Ig+ variants emerged in escaping tumors in the MS11G6-treated group, whereas such variants were common in the S1C5 treated group. Both anti-Id were of equal efficacy in eliminating wild-type 38C13 cells by using 38C13 cells in tumor inoculums that had just been cloned in vitro, but MS11G6 was also capable of preventing the growth of several surface Ig+ variant cell lines in vivo. We conclude that anti-Id recognizing more stable Id determinants can limit the emergence of Id variants and therefore be more effective therapeutic agents. This finding is of additional importance as additional in vivo and immunophenotypic studies demonstrated that the generation of Id variants was an ongoing process both in cloned parental 38C13 cells and its variants.     

Defective helper T-cell activity in LPS-unresponsive C3H/HeJ mice

C3H/HeJ mice, unresponsive to LPS, exhibit a defective ability to mount antibody responses to T-dependent immunogens. The anti-TNP antibody response to TNP-HRBC, a T-dependent immunogen, was found to be lower in these mice as compared to LPS-responsive C3H/HeN mice, whereas the anti-TNP antibody response to TNP-Ficoll, a T-independent immunogen, was of the same magnitude in C3H/HeJ and C3H/HeN mice. An impaired helper activity of C3H/HeJ HRBC-primed spleen cells was demonstrated in a titration assay in which graded numbers of C3H/HeJ or C3H/HeN HRBC-primed spleen cells were added to cultures containing a constant number of unprimed spleen cells from either C3H/HeJ or C3H/HeN mice and the immunogen TNP-HRBC. The reduced helper T-cell activity of C3H/HeJ HRBC-primed spleen cells appears to be independent of macrophage defects, since C3H/HeJ and C3H/HeN macrophages were found equally effective in antigen presentation as evaluated by an in vitro antigen-specific T-cell proliferation assay. The difference in helper T-cell activity between these two substrains probably reflects a lower number and/or proliferation rate of antigen-responsive T cells in C3H/HeJ mice.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular requirements for the full expression of antitumor immunity

The roles of humoral and cellular antitumor immune responses induced by immunization with tumor-derived idiotypic IgM were studied in a syngeneic, transplantable B cell lymphoma (38C13) of C3H mice. Id vaccination with keyhole limpet hemocyanin-conjugated Id induced protection against a subsequent lethal tumor challenge. Such immunizations elicited anti-idiotypic antibodies that were cytotoxic in in vitro antibody-dependent cellular cytotoxicity assays as well as in vivo passive transfer experiments. L3T4+ T cells, which proliferated in vitro in response to the specific Id protein, were also induced. However, cells mediating direct cytotoxicity, either in vitro or in vivo, were not observed in the lymph nodes, spleens, or peritoneal cavity of immune mice or at the site of tumor regression as demonstrated by using a tumor sponge implantation model. In addition, in vitro sensitization of immune lymphocytes against 38C13 tumor cells failed to induce cytotoxicity. Immunization with lipid conjugated Id also elicited a T cell proliferative response but failed to induce anti-idiotypic antibodies and did not confer resistance to tumor growth. These results suggest that anti-idiotypic antibodies play the major role in the destruction of 38C13 tumor cells. However, in vivo depletion of L3T4+ or Lyt-2+ cells from 38C-Id-keyhole limpet hemocyanin-immunized mice resulted in diminished protection against a tumor challenge. Thus, although humoral responses appear to play the predominant part in tumor destruction, cellular responses are also required for the full expression of antitumor immunity in this system.     

Photodynamic therapy-generated vaccine for cancer therapy

A target tumor-derived whole cancer cell therapeutic vaccine was developed based on an in vitro pre-treatment by photodynamic therapy (PDT) and was investigated using a poorly immunogenic tumor model. The vaccine was produced by incubating in vitro expanded mouse squamous cell carcinoma SCCVII cells for 1 h with photosensitizer benzoporphyrin derivative (BPD), then exposing to light (690 nm, 1 J/cm²) and finally to a lethal X-ray dose. Treatment of established subcutaneous SCCVII tumors growing in syngeneic C3H/HeN mice with 2x10⁷ PDT-vaccine cells per mouse by a peritumoral injection produced a significant therapeutic effect, including growth retardation, regression and cures. Tumor specificity of this PDT-generated vaccine was demonstrated by its ineffectiveness when prepared from a mismatched tumor cell line. Vaccine cells retrieved from the treatment site at 1 h postinjection were intermixed with dendritic cells (DC), exhibited heat shock protein 70 on their surface, and were opsonized by complement C3. Tumor-draining lymph nodes treated by the PDT-vaccine contained dramatically increased numbers of DC as well as B and T lymphocytes (with enlarged memory phenotype fraction in the latter), while high levels of surface-bound C3 were detectable on DC and to a lesser extent on B cells. The PDT-vaccine produced no therapeutic benefit against tumors growing in C3-deficient hosts. It is suggested that surface expression of heat shock proteins and complement opsonization are the two unique features of PDT-treated cells securing avid immune recognition of vaccinated tumor and the development of a strong and effective antitumor adaptive immune response.     

Targeting IFN-alpha to B cell lymphoma by a tumor-specific antibody elicits potent antitumor activities

IFN-alpha, a cytokine crucial for the innate immune response, also demonstrates antitumor activity. However, use of IFN-alpha as an anticancer drug is hampered by its short half-life and toxicity. One approach to improving IFN-alpha's therapeutic index is to increase its half-life and tumor localization by fusing it to a tumor-specific Ab. In the present study, we constructed a fusion protein consisting of anti-HER2/neu-IgG3 and IFN-alpha (anti-HER2/neu-IgG3-IFN-alpha) and investigated its effect on a murine B cell lymphoma, 38C13, expressing human HER2/neu. Anti-HER2/neu-IgG3-IFN-alpha exhibited potent inhibition of 38C13/HER2 tumor growth in vivo. Administration of three daily 1-microg doses of anti-HER2/neu-IgG3-IFN-alpha beginning 1 day after tumor challenge resulted in 88% of the mice remaining tumor free. Remarkably, anti-HER2/neu-IgG3-IFN-alpha demonstrated potent activity against established 38C13/HER2 tumors, with complete tumor remission observed in 38% of the mice treated with three daily doses of 5 microg of the fusion protein (p = 0.0001). Ab-mediated targeting of IFN-alpha induced growth arrest and apoptosis of lymphoma cells contributing to the antitumor effect. The fusion protein also had a longer in vivo half-life than rIFN-alpha. These results suggest that IFN-alpha Ab fusion proteins may be effective in the treatment of B cell lymphoma.     

Differences in androgen-dependent induction of mk1, true tissue kallikrein in C3H/HeN and ICR mouse submandibular gland.

Androgen-dependent induction of mk1, true tissue kallikrein, in submandibular gland was studied in C3H/HeN and ICR mice and their F1 progeny. By injection of 5alpha-dihydrotestosterone (DHT), total esteroproteinase activities of female mice were increased to the level of male mice in both C3H/HeN and ICR strains. The mk1 content measured by the radioimmunoassay with anti-mk1 antiserum was decreased in ICR mice, but markedly increased in C3H/HeN mice after DHT injection. We examined the kallikrein isozyme pattern in SMG of two strains using isoelectric focusing. Female ICR mice expressed mainly mk1, mk13 and mk22, and slight mk9. Female C3H/HeN mice expressed mk1, mk9 and pI 6.6-kallikrein. Injection of DHT did not induce any additional kallikrein isozyme in C3H/HeN mice. Furthermore, we made an F1(C3H/HeN) mouse expressing mk13 and mk22 by mating (female C3H/HeN x male ICR). F1(C3H/HeN); these mice showed an androgen response similar to that observed in the ICR mice: mk1 induction in F1(C3H/HeN) mice was decreased by injection of DHT. We suggest the possibility that androgen-dependent mk1 biosynthesis might interact with the expression of other kallikrein isozymes.     

Clustering of extensive somatic mutations in the variable region of an immunoglobulin heavy chain gene from a human B cell lymphoma

Following treatment of a human B cell lymphoma with an anti-idiotype antibody, a subpopulation of tumor cells remained that had lost the tumor-specific heavy chain idiotypic determinant. Nucleotide sequence analyses of eight independent heavy chain variable region isolates showed extensive point mutations, so that no two sequences were identical. Comparison of pretreatment and posttreatment sequences implicated an amino acid in CDR2 as being involved in the idiotypic determinant. Apparently the malignant B cells escaped the therapeutic effects of the anti-idiotype antibody through an ongoing process of somatic mutation in their immunoglobulin genes. Non-random clustering of amino acid replacements in CDR2 suggested that growth of the tumor may have been influenced by endogenous selective forces interacting with the tumor cell-surface immunoglobulin.     

A murine B cell lymphoma expressing human HER2/neu undergoes spontaneous tumor regression and elicits antitumor immunity

 In the present study we describe a novel murine tumor model in which the highly malignant murine B cell lymphoma 38C13 has been transduced with the cDNA encoding human tumor-associated antigen HER2/neu. This new cell line (38C13-HER2/neu) showed stable surface expression but not secretion of human HER2/neu. It also maintained expression of the idiotype (Id) of the surface immunoglobulin of 38C13, which serves as another tumor-associated antigen. Surprisingly, spontaneous tumor regression was observed following s.c. but not i.v. injection of 38C13-HER2/neu cells in immunocompetent syngeneic mice. Regression was more frequently observed with larger tumor cell challenges and was mediated through immunological mechanisms because it was not observed in syngeneic immunodeficient mice. Mice that showed complete tumor regression were immune to challenge with the parental cell line 38C13 and V1, a variant of 38C13 that does not express the Id. Immunity could be transferred with sera, suggesting that an antibody response mediated rejection and immunity. Continuously growing s.c. tumors as well as metastatic tumors obtained after the i.v. injection of 38C13-HER2/neu maintained expression of human HER2/neu, which can serve as a target for active immunotherapy. As spontaneous tumor regression has not been observed in other human murine models expressing human HER2/neu, our results illustrate the enormous differences that can exist among different murine tumors expressing the same antigen. The present model provides a useful tool for the study of the mechanisms of protective immunity to B cell lymphoma and for the evaluation of different therapeutic approaches based on the stimulation or suppression of the immune response. Received: 2 August 2000 / Accepted: 20 September 2000     

CD38 expression enhances sensitivity of lymphoma T and B cell lines to biochemical and receptor-mediated apoptosis

CD38 has been widely characterised both as an ectoenzyme and as a receptor. In the present paper, we investigated the role of CD38 as possible modulator of apoptosis. CD38-positive (CD38(+)) and negative (CD38(-)) fractions, obtained by sorting CD38(+) cells from lymphoma T (Jurkat) and lymphoma B (Raji) and by transfecting lymphoma LG14 and myeloid leukemia K562 cell lines, were used. Cellular subpopulations were exposed to different triggers (H(2)O(2), UV-B, alpha-TOS and hrTRAIL) and the extent of apoptosis was determined by Annexin V-FITC/PI assay. Our data showed that, in lymphoma cells, propensity to apoptosis was significantly linked to CD38 expression and that, remarkably, such response was independent of the nature of the trigger used. Inhibition of CD38 expression by antisense oligonucleotides treatment resulted in CD38-silenced fractions which were as prone to apoptosis as CD38(-) ones. Notably, susceptibility of K562 to apoptosis-inducing challenges was not affected by CD38 expression.     

Treatment of murine B cell lymphoma with bispecific monoclonal antibodies (anti-idiotype x anti-CD3).

It has previously been reported that T lymphocytes can be targeted by using bispecific antibodies consisting of anti-target antibody and anti-CD3. In the present study, a bispecific mAb was developed by somatic hybridization of mouse hybridomas, one producing a mAb against the Id determinant of the mouse B cell lymphoma 38C13 and the other a mAb against a polymorphic determinant on murine CD3. The bispecific antibody, anti-38C13 x anti-CD3, is bi-isotypic (IgG1 x IgG2a) and was purified by ion exchange and affinity chromatography. The dual specificity of the hybrid hybridoma-produced mAb could be demonstrated by flow cytometry, the induction of T cell proliferation, the induction of IL-2 secretion by polyclonal T cells, and redirected lysis of the relevant target cells. The hybrid (bi-isotypic) Fc part of the bispecific antibodies was nonfunctional in FcR-dependent redirected lysis. In vivo studies demonstrate that this bispecific mAb could efficiently target T cells towards the tumor cells, resulting in long term survival and cure of the lymphoma.     

Bispecific anti-idiotype/anti-CD3 antibody therapy of murine B cell lymphoma.

Retargeting of T cells by bispecific IgG which binds to both CD3 and a tumor-associated Ag can induce T cell lysis of target cells irrespective of TCR specificity. The current studies were designed to further explore the efficacy and specificity of bispecific IgG-directed therapy in an immunocompetent animal model, and to evaluate the mechanisms responsible for bispecific IgG-directed inhibition of tumor cell growth by using the 38C13 murine lymphoma system. In vitro, proliferation of activated T cells in the presence of bispecific IgG was increased when the relevant, but not the irrelevant target cells were present. Bispecific IgG specifically induced activated T cell mediated lysis of cells expressing the target Ag, but not of cells expressing an irrelevant Ag, even when the irrelevant cells were in the same cell mixture, indicating contact between target cells and T cells plays a major role in bispecific IgG-mediated lysis. Bispecific IgG was less effective than anti-Id at inducing target cell lysis when peritoneal macrophages were used as effectors, suggesting bispecific IgG Fc is not responsible for cytotoxicity in this system. In vivo, bispecific IgG was significantly superior to anti-Id, anti-CD3, or a combination of anti-Id and anti-CD3 in preventing tumor growth in immunocompetent mice inoculated with syngeneic lymphoma. Phenotypic evaluation of tumors that emerged despite therapy indicated bispecific IgG selects for the emergence of Id variant lymphoma cells. In separate studies, 38C13 tumor inocula containing cells recognized by the therapeutic antibody were supplemented with a small number of 38C13 cells which expressed a distinct Id not recognized by the therapeutic antibody. Untreated mice inoculated with this mixture developed tumors containing cells of both phenotypes, whereas tumors emerging from mice treated with bispecific IgG contained only cells expressing the nonreactive Id. These studies demonstrate bispecific IgG-directed lysis is therapeutically superior to monospecific anti-Id therapy in the 38C13 tumor model, and that tumor lysis is mediated largely by cell-cell contact. As with other forms of anti-Id based therapy, Id variants can emerge as resistant cell populations after bispecific IgG therapy.     

Heterogeneity of a murine B cell lymphoma. Isolation and characterization of idiotypic variants

mAb directed toward the idiotype of the 38C13 murine B cell lymphoma can be used to treat and cure a high percentage of mice challenged previously with an otherwise lethal dose of tumor cells. Tumors developing in animals despite antibody therapy were examined by immunofluorescence and found to demonstrate either loss of surface Ig, or expression of an altered idiotype that no longer bound the antibody used for treatment. Further immunofluorescence analysis of the variant tumors revealed individual patterns of cross-reactivity with anti-38C13 idiotype mAb other than that used for therapy. The variant tumor cells were fused to myeloma cells and hybrids were isolated which secreted large quantities of the altered idiotype proteins. Polyclonal antibodies and mAb prepared against the mutant proteins demonstrated cross-reactivity with the original 38C13 protein and its other variants. But the variants and wild type cells could be distinguished from each other by their patterns of reactivity with the panels of anti-idiotype antibodies. Differences in apparent m.w. were demonstrated in the L chains of each of the mutant proteins. Southern blot analysis of the H chain locus of these mutants established that they were all clonally related; however, the L chain loci were grossly different. Thus, rare cells with alteration in their Ig L chain genes and expressed proteins can give rise to idiotype variants in this B cell tumor.     

In vivo anti-tumor immunity detected by leukocyte adherence inhibition

Summary The immune response of mice to a transplacentally induced alveolar cell tumor was studied with the leukocyte adherence inhibition (LAI) assay. The lung tumor, designated 85, was induced in a C3HfB/HeN (C3Hf) mouse by l-ethyl-l-nitrosourea (ENU). While a dose of 10⁵ cells of this tumor does not grow in syngeneic C3Hf mice, it does grow readily in (A×C3Hf)F₁ hybrid mice. The tumor possesses a tumor associated transplantation antigen (TATA) which cross-reacts with a normal tissue alloantigen in strain A/HeN (A) mice. Normal mice, tumor-immunized C3Hf mice, and tumor-bearing (A×C3Hf)F₁ mice possessed peritoneal cells, the majority of which adhered rapidly to glass and resisted gentle washing. When incubated with an extract of the 85 tumor, peritoneal cells from tumor-immunized mice demonstrated marked inhibition of adherence (62.4%) compared to similarly incubated peritoneal cells of either normal mice (30.3%) or tumor bearing mice (37.1%). Specificity of the reactivity in the LAI assay was demonstrated with a neuroblastoma extract and peritoneal cells from neuroblastoma-immunized C3Hf mice. Peritoneal cells from lung tumor-immunized mice, but not tumor-bearing mice, responded to a lung extract from strain A mice. In contrast to the microcytotoxicity assay, the LAI assay is capable of distinguishing the effective anti-tumor response of tumor-immunized C3Hf mice from the ineffective immune response of tumor-bearing (A×C3Hf)F₁ mice.     

The mechanism of tumor growth inhibition by tumor-specific Lyt-1+2-T cells. I. Antitumor effect of Lyt-1+2-T cells depends on the existence of adherent cells

In the present study we establish an assay system of tumor growth inhibition with the use of a diffusion chamber and investigate the mechanism by which tumor-specific Lyt-1+2-T cells exhibit their inhibiting effect on tumor cell growth. When a diffusion chamber containing X5563 plasmacytoma cells together with normal syngeneic C3H/HeN spleen cells was implanted in the peritoneal cavity of C3H/HeN mice, these tumor cells continued to proliferate at least 7 to 9 days. In contrast, spleen cells from C3H/HeN mice that had acquired X5563-specific immunity by intradermal (i.d.) inoculation of viable tumor cells, followed by surgical resection of the tumor, exhibited an appreciable inhibitory effect on the growth of X5563 tumor cells admixed in the chamber. This antitumor effect was mediated by Lyt-1+2-T cells and was tumor-specific, because the growth of X5563 or another syngeneic MH134 hepatoma cells was inhibited by spleen cells from C3H/HeN mice immunized to the respective tumor cell types. Most important, these tumor-specific Lyt-1+2-T cells lost their antitumor activity by depleting an adherent cell population contained in spleen cells, indicating that adherent cells are required for the Lyt-1+2-T cell-mediated antitumor effect. This was substantiated by the fact that immune spleen cells depleted of adherent cells could regain their tumor-inhibiting effect when normal spleen cells were added back as an adherent cell source, or more directly by adding back a splenic or peritoneal resident adherent cell population. These results indicate that tumor-specific Lyt-1+2-T cells mediate the tumor growth inhibition and that their antitumor effect depends on the coexistence of an adherent cell population.     

Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection.

Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.     

C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.