Immunization Of Syngeneic Mice Research Articles

Preventive cancer stem cell-based vaccination modulates tumor development in syngeneic colon adenocarcinoma murine model.

Cancer stem cells (CSCs), a rare sub-fraction of tumor cells, with the capability of self-renewal and strong oncogenicity are tightly responsible for chemo and radio resistance and tumor metastasis in colorectal cancer. Hence, CSCs targeting would improve the efficacy of therapeutic strategies and clinical outcomes. Here, using three-dimensional CSC spheroids and syngeneic mice model, we evaluated the cancer preventive impact of CSCs-based vaccination. CSCs enrichment was performed via colonosphere formation from CT-26 cell line and CT-26-derived tumor biopsy and characterized by confirming high expression of key stemness genes (OCT4, SOX2, and NANOG) and CSC-related surface biomarkers (CD166, DCLK1, and CD133) via real-time PCR and flow cytometry, respectively. Then, the stemness phenotype and self-renewal in CSC-enriched spheroids were further confirmed by showing serial sphere formation capacity, clonogenicity potential, and enhanced in vivo tumorigenic capacity compared to their parental counterparts. CSCs lysates were used as vaccines in prophylactic settings compared to the parental cell lysate and PBS groups. Immunization of syngeneic mice with CSCs lysates was effective in the prevention of tumor establishment and significantly decreased tumor growth rate accompanied by an improvement in survival rate in tumor-bearing mice compared to groups subjected to parental cells lysate and PBS. These results, for the first time, showed that mice immunized with cell lysate from tumor biopsy-derived spheroids are resistant to tumor induction. Immunofluorescence staining indicated that only the serum antibodies from CSC-vaccinated mice reacted with colonospheres. These findings represent CSCs lysate-based vaccination as a potential approach to hampering immunotherapy failure of colorectal cancer which along with other traditional therapies may effectively apply to prevent the establishment of aggressive tumors harboring stemness features.

Abstract 4580: Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model

Abstract Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. Using the syngeneic mouse models, we demonstrated that although both orthotopically-implanted murine 4T1 and EMT6 tumors are capable of disseminating into secondary organs, only 4T1 tumors develop overt metastasis. However, EMT6 tumors induce an anti-tumor immunity in syngeneic mice and that eradicates disseminated tumor cells (DTC) in distant organs. Following the complete removal of primary EMT6 tumors, mice do not develop detectable metastasis and generate an immunological memory that leads to complete elimination of repeatedly injected tumor cells via tail vein. Conversely, these cells readily grow and metastasize in immuno-deficient athymic or Rag2- mice, and when g-MDSCs from 4T1 tumor-bearing mice were co-injected into immunocompetent EMT6 primed mice. In contrast to complete resection, mice with residual tumors following surgery exhibited an enhanced growth of local and concomitant growth of DTCs at metastatic site with increased g-MDSCs accumulation in lung and spleen. Together, our results suggest that some tumors are capable of inducing anti-tumor immunity against the DTCs when complete resection of primary tumor cures animals. However, in the presence of residual tumors, inflammation induced by surgical procedure promote the growth of DTCs. Citation Format: Raziye Piranlioglu, Eunmi Lee, Maria Ouzuonova, Riley Rodier, Adam Greer, Feyzanur Bayraktar, Omer Can Durmus, Ali S. Arbab, Muthushamy Thangaraju, Max S. Wicha, Esteban Celis, Hasan Korkaya. Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4580.

Nano-enabled pancreas cancer immunotherapy using immunogenic cell death and reversing immunosuppression

While chemotherapy delivery by nanocarriers has modestly improved the survival prospects of pancreatic ductal adenocarcinoma (PDAC), additional engagement of the immune response could be game changing. We demonstrate a nano-enabled approach for accomplishing robust anti-PDAC immunity in syngeneic mice through the induction of immunogenic cell death (ICD) as well as interfering in the immunosuppressive indoleamine 2,3-dioxygenase (IDO) pathway. This is accomplished by conjugating the IDO inhibitor, indoximod (IND), to a phospholipid that allows prodrug self-assembly into nanovesicles or incorporation into a lipid bilayer that encapsulates mesoporous silica nanoparticles (MSNP). The porous MSNP interior allows contemporaneous delivery of the ICD-inducing chemotherapeutic agent, oxaliplatin (OX). The nanovesicles plus free OX or OX/IND-MSNP induce effective innate and adaptive anti-PDAC immunity when used in a vaccination approach, direct tumor injection or intravenous biodistribution to an orthotopic PDAC site. Significant tumor reduction or eradication is accomplishable by recruiting cytotoxic T lymphocytes, concomitant with downregulation of Foxp3+ T cells.

Abstract 5296: The role of SOCS-1 in stimulating melanoma development through growth factor receptors and MAPK pathways

Abstract Knowledge on the regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now recognized that multiple signaling pathways that affect motility, growth control, invasion, metabolism, cytokine release and the ability to escape the immune response, regulate melanoma development. Silencing of SOCS-1 protein, a negative regulator of Jak/Stat pathway, leads to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS-1 silencing with shRNAi inhibits in vitro growth by cell cycle regulation with S phase arrest and increased size of cells and nuclei. It also inhibited tumor cell migration and invasion. Down-regulation of SOCS-1 decreased the expression of epidermal growth factor receptor (mainly the phosphorylated-R), Ins-Rα, and fibroblast growth factor receptors. Based on the results of SOCS-1 silencing, the present work aims at analyzing the expression of proteins relevant to tumor development, searching for signaling pathways related with SOCS-1. A protein microarray analysis of murine melanoma cells silenced for SOCS-1 by shRNAi SOCS-1 lentivirus transduction was undertaken using as controls, cells transduced with the empty vector. Among the 614 differentially expressed genes some cytokine/growth factor receptors were down regulated, e.g. c-kit, met and EphA3. Western blotting of B16 shR-SOCS-1 cell extracts showed a significant decrease in the expression of these receptors, indicating that SOCS-1 positively regulates the MAPK pathways in these cells. We analyzed the main MAPK pathways that were up regulated in melanoma and observed a decrease in the phosphorylation of mediators of ERK1/2 and p38 pathways and of STAT3 (S727). These results demonstrate that SOCS-1 can play an important role in tumor development and progression up regulating a number of growth factor receptors in murine melanoma cells. Interestingly, p-CREB (S133) was up regulated in the SOCS-1-deficient B16F10-Nex2 cells. In vivo experiments showed that using a prophylactic protocol, the subcutaneous immunization of syngeneic mice with B16 shR-SOCS-1 viable cells, rendered a protective effect against subcutaneously grafted and metastatic murine melanoma, with significant reduction in tumor growth and metastates in the lungs. A flow cytometry assay showed decreased expression of PD-L1 at the surface of B16 shR-SOCS-1 cells. This might explain the protective effect of subcutaneous immunization with these cells, rather than with wild-type B16F10-Nex2 cells that overexpress PD-L1, which negatively regulates T-cell immune response. Down regulation of PD-L1 is compatible with inactive ERK. The present results show the important role of SOCS-1 in murine melanoma stimulating cell growth and migration, allowing for the invasive phenotype. Citation Format: Rodrigo Berzaghi, Felipe V. Pereira, Vera S.C. Maia, Jorge B. Scutti, Luiz R. Travassos. The role of SOCS-1 in stimulating melanoma development through growth factor receptors and MAPK pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5296. doi:10.1158/1538-7445.AM2014-5296

Genetically Targeted T Cells Eradicate Established Breast Cancer in Syngeneic Mice

The purpose of the present study was to evaluate the capacity and mechanisms of genetically modified erbB2-specific T cells to eradicate erbB2+ tumors in syngeneic mice. Primary mouse T cells were modified to target the breast tumor-associated antigen erbB2 through retroviral-mediated transfer of a chimeric antigen receptor, termed single-chain antibody (scFv)-CD28-zeta. Antitumor efficacy of scFv-CD28-zeta-modified T cells was analyzed in mice bearing D2F2/E2 breast tumors. The scFv-CD28-zeta-modified T cells were shown to specifically secrete T cytotoxic-1 cytokines and lyse erbB2+ breast tumor cells following receptor stimulation in vitro. Treatment with scFv-CD28-zeta-modified T cells was able to lead to long-term, tumor-free survival in mice bearing erbB2+ D2F2/E2 breast tumors. Importantly, the surviving mice developed a host memory response to D2F2/E2 tumor cells, and this host response was able to protect against a rechallenge with erbB2+ D2F2/E2 tumor cells and parental erbB2(-) D2F2 tumor cells. In addition, scFv-CD28-zeta T-cell expression of perforin and interferon-gamma were essential for complete antitumor efficacy. Treatment with scFv-CD28-zeta-modified T cells was able to induce a host antitumor immunity in syngeneic mice. Complete tumor elimination by scFv-CD28-zeta-modified T cells required T cell-derived interferon-gamma and perforin, indicating that cytotoxicity and cytokine secretion play a role in the in vivo response.

Engrafting costimulator molecules onto tumor cell surfaces with chelator lipids: a potentially convenient approach in cancer vaccine development.

The genetic modification of cells to develop cell-based vaccines and to modulate immune responses in vivo can be risky and inconvenient to perform in clinical situations. A novel chelator lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA) that, via the NTA group has high affinity for 6His peptide, was used to directly anchor recombinant forms of T cell costimulatory molecules containing a C-terminal 6-His sequence onto tumor cell surfaces. Initial experiments using murine P815 tumor cells established the optimum conditions for incorporating NTA-DTDA onto the membranes of cells. P815 cells with incorporated NTA-DTDAbound hexahistidine-(6His)-tagged forms of the extracellular domains of murine B7.1 and CD40 (B7.1-6H and CD40-6H) at very high levels (fluorescence 200-300-fold above background), and both proteins could be anchored onto the cells simultaneously. Significant loss of the anchored or "engrafted" protein occurred through membrane internalization following culture of the cells under physiological conditions, but P815 cells with engrafted B7.1-6H and/or CD40-6H stimulated the proliferation of allogenic and syngeneic splenic T cells in vitro, and generated cytotoxic T cells when used as vaccines in syngeneic animals. Furthermore, the immunization of syngeneic mice with P815 cells engrafted with B7.1-6H or with B7. 1-6H and CD40-6H induced protection against challenge with the native P815 tumor. The results indicate that the use of chelator lipids like NTD-DTDA to engraft costimulatory and/or other molecules onto cell membranes could provide a convenient alternative to transfection in the development of cell-based vaccines and for modulation of immune function.

Novel cross-reactive anti-idiotype antibodies with properties close to the human intravenous immunoglobulin (IVIg).

The most important link between the immune network theory and clinically useful therapies so far is the use of human intravenous immunoglobulin (IVIg) in the treatment of autoimmune diseases. Although still controversial, one of the main mechanisms that has been postulated for the in vivo effects of IVIg, is the selection of immune repertoires through idiotypic interactions. We describe here anti-idiotype IgG monoclonal antibodies (MAbs), which were obtained by immunization of syngeneic mice (Balb/c) with an anti-ganglioside antibody. These anti-idiotype MAbs show multiple idiotypic connections and share some of the properties of the IVIg pool. The antiidiotype (Ab2) MAbs B7 and 34B7 showed heterogeneous binding with the idiotypes of several anti-ganglioside antibodies, MAbs obtained from splenocytes of nonimmunized newborn mice, F(ab')2 fragments of IgG human myeloma proteins, and nonimmunoglobulin antigens. The recognition pattern of the B7 MAb to the idiotypes of human immunoglobulins was also studied using a phage display library obtained from the variable region genes of an asymptomatic AIDS patient and also F(ab')2 fragments obtained from an IVIg pool of healthy human donors. We also demonstrated that these MAbs produced some of the in vitro effects reported for the human IVIg pool, such as the inhibition of cell proliferation of human B and T cell lines and of normal human lymphocytes activated with different mitogens. Another striking property of the MAb B7 was its ability to induce a dose-dependent specific antibody T-cell response in vivo in syngeneic mice. Both anti-idiotype MAbs showed anti-metastatic effect in vivo when injected intravenously to mice inoculated with MB16-F10 melanoma cells. The antimetastatic effect of the antiidiotype MAbs was not observed in athymic mice inoculated with the same tumor. This kind of antibody can become an interesting tool for further exploration of the role of idiotypic network connections in the regulation of the immune system and to study the effects of interventions on network connectivity in experimental autoimmune disease, using a reagent better chemically defined than the IVIg pool.

Cutting Edge: Cross-Priming of CTL Responses In Vivo Does Not Require Antigenic Peptides in the Endoplasmic Reticulum of Immunizing Cells

Abstract It has been proposed that the cross-priming of CTL responses in vivo involves the transfer to host APCs of heat shock protein glycoprotein 96-chaperoned antigenic peptides released from the endoplasmic reticulum (ER) of dying or infected cells. We have tested this possibility directly using TAP-deficient cell lines lacking antigenic ER peptides derived from two model Ags, the human adenovirus type 5 early regions E1A and E1B. Although both proteins were well expressed, the cells were not recognized by E1A- or E1B-specific CTLs unless the relevant epitope was either provided exogenously as a synthetic peptide or targeted to the ER in a TAP-independent fashion. Despite the absence of these ER peptides, the TAP1−/− cells were able to efficiently cross-prime E1A- and E1B-specific CTLs following immunization of syngeneic mice. These results indicate that, although purified peptide/glycoprotein 96 complexes are potent immunogens, the mechanism of CTL cross-priming in vivo does not depend upon antigenic peptides in the ER of immunizing cells.

GM-CSF and B7-1 (CD80) co-stimulatory signals co-operate in the induction of effective anti-tumor immunity in syngeneic mice.

B7-1 (CD80) co-stimulatory molecule gene-transduced Lewis lung carcinoma (LLC) cells (LLC/B7 cells) resulted in remarkable loss of tumorigenicity in syngeneic C57BL/6 mice (87.5% rejection) compared to B7-negative, wild-type LLC (LLC/wt) cells (0% rejection). However, mice that had rejected LLC/B7 cells developed almost no systemic immunity protective against challenge with wild-type tumor cells after 4 weeks (11.8% rejection). Enhancement of MHC class I (H-2Kb) expression of LLC/B7 cells with in vitro interferon-gamma treatment did not result in enhancement of protective immunity. In vivo depletion assay revealed that abrogation of tumorigenicity in LLC/B7 depended on CD8+ T cells but not on CD4+ T cells. However, vaccination of C57BL/6 mice with irradiated LLC cells transduced with GM-CSF (LLC/GM) led to the induction of potent, specific immunity against challenge with the LLC/wt cells after 2 weeks (80.8% rejection). Next, we established a double transfectant of LLC cells expressing both B7-1 and GM-CSF (LLC/GM + B7). The tumorigenicity of these clonal cells was also remarkably suppressed (90% rejection) to the same degree as LLC/B7, whereas that of LLC/GM was not suppressed (0% rejection). Interestingly, mice that had rejected LLC/GM+B7 cells developed enhanced protective immunity against challenge with LLC/wt cells after 4 weeks (55.6% rejection) compared to the results of LLC/B7 cells (11.8%). To evaluate whether co-expression of GM-CSF and B7-1 enabled the tumor cells to activate cytotoxic T cells more efficiently than B7-1 alone, we performed an in vitro killing assay. We found that immunization with LLC/GM+B7 cells resulted in a 3-fold stronger cytotoxic response than that with LLC/B7. Our data indicate that co-transfection of the B7-1 co-stimulatory molecule and GM-CSF genes may be more effective for the induction of stronger protective immunity in this experimental system.

GM‐CSF and B7‐1 (CD80) co‐stimulatory signals co‐operate in the induction of effective anti‐tumor immunity in syngeneic mice

B7-1 (CD80) co-stimulatory molecule gene–transduced Lewis lung carcinoma (LLC) cells (LLC/B7 cells) resulted in remarkable loss of tumorigenicity in syngeneic C57BL/6 mice (87.5% rejection) compared to B7-negative, wild-type LLC (LLC/wt) cells (0% rejection). However, mice that had rejected LLC/B7 cells developed almost no systemic immunity protective against challenge with wild-type tumor cells after 4 weeks (11.8% rejection). Enhancement of MHC class I (H-2Kb) expression of LLC/B7 cells with in vitro interferon-γ treatment did not result in enhancement of protective immunity. In vivo depletion assay revealed that abrogation of tumorigenicity in LLC/B7 depended on CD8+ T cells but not on CD4+ T cells. However, vaccination of C57BL/6 mice with irradiated LLC cells transduced with GM-CSF (LLC/GM) led to the induction of potent, specific immunity against challenge with the LLC/wt cells after 2 weeks (80.8% rejection). Next, we established a double transfectant of LLC cells expressing both B7-1 and GM-CSF (LLC/GM + B7). The tumorigenicity of these clonal cells was also remarkably suppressed (90% rejection) to the same degree as LLC/B7, whereas that of LLC/GM was not suppressed (0% rejection). Interestingly, mice that had rejected LLC/GM+B7 cells developed enhanced protective immunity against challenge with LLC/wt cells after 4 weeks (55.6% rejection) compared to the results of LLC/B7 cells (11.8%). To evaluate whether co-expression of GM-CSF and B7-1 enabled the tumor cells to activate cytotoxic T cells more efficiently than B7-1 alone, we performed an in vitro killing assay. We found that immunization with LLC/GM+B7 cells resulted in a 3-fold stronger cytotoxic response than that with LLC/B7. Our data indicate that co-transfection of the B7-1 co-stimulatory molecule and GM-CSF genes may be more effective for the induction of stronger protective immunity in this experimental system. Int. J. Cancer 73:556–561, 1997. © 1997 Wiley-Liss, Inc.

Use of combinatorial peptide libraries to construct functional mimics of tumor epitopes recognized by MHC class I-restricted cytolytic T lymphocytes.

Identification of cytolytic T lymphocyte (CTL) epitopes presented by major histocompatibility complex (MHC) class I molecules on tumor cells is critical for the design of active immunotherapy. We describe the use of combinatorial peptide libraries with defined amino acids in two MHC anchor positions to search for epitopes that are recognized by H-2Db- and Kb-restricted CTL specific for the mouse lymphoma EL4. An iterative strategy was used for screening libraries in which 16 amino acids were divided into 3 groups and 3 subgroups: alpha (AL, VT, FY); beta (GS, P, DE); gamma (KR, H, NQ). The proportions of each group and subgroup at individual peptide positions were changed in the library synthesis, and the effect of these changes on CTL activity was measured in a sensitive RMA-S cell assay. A single H-2Db epitope mimic was deduced from the original library that contained > 2 x 10(8) potential peptides and was at least 9 logs more potent than the original library. Immunization of syngeneic mice with this peptide elicited CTL that lysed EL4 cells as well as RMA-S cells pulsed with peptides isolated from Db molecules of EL4 cells, indicating functional similarity between the mimicking peptide and the naturally processed CTL epitope. Furthermore, adoptive transfer of such a CTL line had a therapeutic effect in mice with EL4 established as an ascites tumor. Two H-2Kb-restricted epitope mimics of the same tumor were also identified. Our method represents a novel approach for the construction of MHC class I-restricted targets that can serve as immunogens for active immunotherapy of cancer.

Induction of anti-tumour immunity in syngeneic mice by a leukaemic cell line.

Induction of anti-tumour immunity in syngeneic mice by LBC cell line derived from a non-immunogenic T cell leukaemia was studied. The immunization of BALB/c mice with LBC irradiated cells induced in them anti-tumour spleen cells, cytotoxic T lymphocytes and anti-LBC antibodies. The anti-LBC antibodies reacted with components of 14, 16 and 27 kDa present on LB tumour cells, LBC cell line and normal thymocytes, but not with normal lymph node cells. Furthermore, immunization of the autologous hosts with LBC cells partially protected them against subsequent challenge with the original LB leukaemic cells. These findings demonstrate that culture conditions induced modifications in the antigenic properties of the leukaemic cells, allowing LBC cells to stimulate an immune response directed against components expressed at early stages during T cell maturation. These results also suggest that the immune response is responsible for the prolongation of the survival time of the mice inoculated with the parental leukaemic cells.

Membranes Activate Tumor- and Virus-Specific Precursor Cytotoxic T Lymphocytes In Vivo and Stimulate Tumor-Specific T Lymphocytes In Vitro

Plasma membranes contain the entire antigenic repertoire of a cell in the form of processed antigens presented as peptides by major histocompatibility complex (MHC) class I molecules. We report here that plasma membranes but not internal membranes of cognate tumors stimulate murine fibrosarcoma and human melanoma-specific cytotoxic T lymphocyte (CTL) clones in vitro in an antigen-specific. MHC class I-restricted manner. Although stimulation of CTLs by class I-peptide complexes on reconstituted membranes has been documented before, this is the first demonstration of stimulation of cloned CTLs by natural, endogenously processed MHC class I-peptide complexes on plasma membranes. In addition to their ability to stimulate CTLs in vitro, immunization of syngeneic mice with membranes derived from ultraviolet-induced tumor cells, SV40 transformed fibroblasts, or influenza-infected fibroblasts elicits an antigen-specific, MHC class I restricted primary CTL response. To the best of our knowledge, this is also the first demonstration of the ability of cellular membranes to prime an MHC class I-restricted CTL responses in vivo. The ability of membranes to prime a CTL response in vivo suggests that they may be used as T-cell vaccines against tumors or infectious viruses. This approach circumvents the difficulties in generation of human tumor cell lines and identification of CTL-recognized determinants for vaccination and avoids some of the risks associated with whole-cell vaccination such as inoculation of patients with immunosuppressive factors, transforming DNA, or infectious viruses.

Distinct immunologic specificity of tumor regression mediated by effector cells isolated from immunized and tumor-bearing mice

Sensitized T lymphocytes can mediate potent antitumor effects when transferred to tumorbearing animals. Employing the MCA 105 and MCA 106 sarcomas, we were able to generate antitumor effector cells by immunization of syngeneic mice with tumor cells admixed with Corynebacterium parvum. These immune splenocytes could be further sensitized and expanded in culture by the in vitro sensitization (IVS) method utilizing tumor stimulator cells and IL-2. Adoptive immunotherapy of pulmonary metastases mediated by noncultured splenocytes from immunized mice or immune IVS cells showed exquisite specificity between the two sarcomas. These results demonstrate the presence of tumor-specific antigens on MCA 105 and MCA 106 tumor cells which can serve as target molecules for immunotherapy. Recently, we have generated therapeutic T lymphocytes from mice bearing progressively growing tumors by the IVS method. However, IVS cells from tumor-bearing mice showed cross-reactivity between the MCA 105 and 106 sarcomas in adoptive immunotherapy experiments. Since these IVS cells did not affect other control tumors, the limited cross-reactivity suggests the presence of common tumor-associated antigens on MCA 105 and MCA 106 tumor cells which can also serve as the target for tumor rejection. Therefore, immune responses to progressive tumor growth and to immunization are distinct with respect to antigen recognition by T lymphocytes.

Monoclonal antibodies against murine IFN-gamma abrogate in vivo tumor immunity against RSV-induced murine sarcomas.

Previous work has shown that immunization of syngeneic mice with v-src-induced sarcomas gives rise to specific protection against a lethal dose of v-src-transformed fibroblasts. This immune response is mediated by Lyt-1+, Lyt-2,3+ T lymphocytes, with no apparent involvement of cytotoxic T cells, as shown in Winn-type assays. Immune cells mediating tumor rejection in this system have now been further characterized, and it was found that L3T4+ T lymphocytes alone provided full protection against v-src-induced sarcomas. Moreover, the role of interferon-gamma (IFN-gamma) in the tumor rejection was analyzed. A monoclonal antibody directed against this lymphokine was able to reverse the protective effect displayed by immune T lymphocytes, by eliciting highly effective T suppressor cells. It was thus concluded that T cells with L3T4 surface marker are the main thing responsible for the adoptive immunity in this tumor system, and the activity of these cells is positively modulated by lymphokines such as IFN-gamma.

Idiotype vaccination against murine B cell lymphoma. Inhibition of tumor immunity by free idiotype protein.

A murine B cell lymphoma (38C13) was used as a model to study the induction of idiotype (Id)-specific tumor immunity. Immunization of syngeneic mice with Id protein derived from the tumor resulted in the production of anti-Id antibodies by the host and in the induction of a state of resistance to tumor growth. Tumor immunity could be established only if the Id protein was conjugated to a strongly immunogenic carrier protein such as keyhole limpet hemocyanin or thyroglobulin, and if the conjugate was administered at least 1 week prior to tumor challenge. Free Id protein, such as that present in tumor bearing animals, was found to inhibit tumor immunity in a dose-dependent manner. Although tumor immunity could be induced in animals with pre-existent serum Id protein, the expression of the immune state was inhibited by the presence of the soluble protein.

Monoclonal antibodies to endogenous galactose-specific tumor cell lectins.

A monoclonal antibody, 5D7, was obtained after immunization of syngeneic mice with B16 melanoma cell extracts enriched for endogenous lectin activity and screening for inhibition of lectin-mediated hemagglutination. Binding of this antibody to affinity-purified B16 melanoma galactoside-specific lectin was revealed by solid-phase radioimmunoassay and binding to the surface of viable B16 cells was demonstrated by indirect immunofluorescence. Inhibition of lectin activity and cell surface labeling by 5D7 antibody were also found with several types of cultured human and murine cells including melanoma, sarcoma and carcinoma. This monoclonal antibody should be useful for evaluating the role of tumor cell surface lectins in intercellular interactions and metastasis.

Immunization of syngeneic mice against malignant melanoma with subcellular membrane fractions of a bromodeoxyuridine-grown variant

Syngeneic C57BL/6 mice immunized with non-tumorigenic B16 melanoma cells and crude membrane fractions are able to reject challenge with the tumorigenic B559 parent clone. The immunogenic C3471 variant was derived from the malignant B559 clone by continuous growth in the presence of 1 μg 5-bromodeoxyuridine (BrdUrd)/ml. Fifty-one percent (35 of 69) of mice immunized by three inoculations of 106 cell equivalents of C3471 crude membranes (CM) isolated by nitrogen cavitation remained tumor-free for at least 50 days after challenge with a tumorigenic dose of B559 cells. This compares favorably with the virtually 100% protection evoked by 106 viable C3471 cells. For those CM-immunized mice failing to reject B559 challenge, the mean latent period for tumor formation was significantly increased (P≥0.001) over controls. In addition, mice immunized with cultured C3471 cells were able to reject, with equal efficiency, challenge with either cultured or tumor-derived B559 cells, indicating that the immunogen(s) present on C3471 cells and CMs was (were) not a tissue culture artifact. Freshly prepared syngeneic fascia cells and membranes as well as CM prepared from cultured malignant B559 cells had no tumor rejection activity. In vivo tumor rejection activity in plasma membrane vesicles prepared from C3471 cells by formaldehyde treatment also demonstrated tumor rejection activity. The host response to CMs, as to C3471 cells, could be transferred by lymphoid cells from mice immunized with C3471 CMs or cells. Co-injection of leucocytes from CM-immunized mice together with a tumorigenic dose of B559 cells into immunocompetent syngeneic mice resulted in abrogation of B559 tumorigenicity. The tumor rejection antigen(s) induced or increased by growth in BrdUrd has not yet been characterized biochemically, but is likely to involve a cell surface component common to both cell types and is retained by crude membrane fractions. The use of subcellular fractions from a spontaneous melanoma grown with BrdUrd, which elicits immunity against the malignant tumor, represents a model with immunoprophylactic potential for human neoplasia.

Cytolytic antibodies to methylcholanthrene-induced sarcomas elicited by immunization of syngeneic mice

Cytolytic antibodies were tested in sera of C 3H/HeHa mice grafted twice with solid syngeneic sarcoma or immunized with an MC-sarcoma converted into an ascitic form. These sera reacted with syngeneic tumor as well as with normal and malignant allogeneic cells. Absorption experiments suggested that these antisera contained two populations of antibodies. One population combined with antigen(s) shared by the MC-sarcomas of C 3H/HeHa mice, by normal and malignant cells of C 3H/St mice, and by normal and malignant cells of C 57BL/ 6 mice. The other antibody population failed to react with cells of C 57BL/ 6 mice but it reacted with C 3H/HeHa MC-sarcoma and with normal and malignant cells of C 3/St origin.

Studies on the antigenicity of radiation-induced murine osteosarcomata.

The immunogenicities of 15 murine osteosarcomata induced with a bone seeking radioisotope ((90)Sr) in normal and chimaeric CBA mice were studied. Attempts were made to induce tumour-specific immunity in syngeneic mice by treatment with x-irradiated (15,000 rad) tumour or surgical excision of developing subcutaneous tumour grafts. Resistance was evoked against 6 tumours and this was relatively weak. With the remaining tumours, no resistance against the immunizing tumour could be demonstrated, even though the transplantation tests were made highly sensitive by the use of inocula of as few as 2 × 10(3) cells in pre-irradiated (400 rad) hosts. Sera from mice immunized against each of the tumours were tested against viable cells of the immunizing tumour by indirect immunofluorescence. In no instance did tumour antisera give a convincing reaction with tumour cells although alloantisera raised by hyperimmunization of H-2 identical and H-2 different donors with osteosarcomata consistently gave strongly positive reactions. The results are interpreted as illustrating the weak tumour specific antigenicity of radiation-induced murine osteosarcomata. The possibility that antigenic deficiency is a consequence of immunosurveillance in this tumour system is discussed.