Reduced efficacy of allogeneic versus syngeneic fibroblasts modified to secrete cytokines as a tumor vaccine adjuvant

We examined the relative efficacy of allogeneic versus syngeneic fibroblasts admixed with tumor cells as a vaccine to induce antitumor T-cell reactivity. Allogeneic (3T3) or syngeneic (BLK) fibroblasts transfected to secrete equivalent amounts of GM-CSF were admixed with either D5 melanoma or MCA 207 sarcoma and inoculated s.c. into the flanks of C57BL/6 mice. Vaccine-primed lymph node (LN) cells were examined for in vivo antitumor reactivity in an adoptive transfer model. At fibroblast: tumor cell ratios of < or=1, allogeneic and syngeneic granulocyte macrophage colony-stimulating factor-secreting fibroblasts enhanced T-cell reactivity to tumor cells. However, at ratios of 2.4, the adjuvant effect induced by granulocyte macrophage colony-stimulating factor was not evident. Instead, we observed increased alloreactivity of primed LN cells against 3T3 targets as assessed by cytotoxicity and cytokine release assays, which was not observed with syngeneic fibroblasts. Moreover, with increasing numbers of allogeneic fibroblasts, there was a skewing of the T-cell Vbeta repertoire. These latter cells responded to tumor stimulation with the release of greater amounts of interleukin 10, which may account for the diminished antitumor reactivity observed in vivo. Allogeneic fibroblasts transduced to secrete interleukin 2 or IFN-gamma also induced diminished tumor reactivity of primed LN cells. Syngeneic fibroblasts are superior to allogeneic fibroblasts as vehicles to deliver cytokines in tumor vaccines.     

Does recurrent acute pancreatitis lead to chronic pancreatitis? Sequential morphological and biochemical studies

BACKGROUND: For the relatively nonimmunogenic B16-F10 murine melanoma, it has been found that genetically engineered expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin (IL)-2, IL-4, or interferon-gamma (IFN-gamma) resulted in a vaccine that could induce resistance to rechallenge. Because T cells from lymph nodes draining the sites of some progressive tumors can mediate tumor regression after in vitro activation, it seemed possible that even apparently nonimmunogenic melanoma cells might induce similar preeffector cells in the vaccine-draining lymph nodes (DLNs). METHODS: C57BL/6 mice were vaccinated with B16-F10 cells that were either unmodified or genetically modified to produce IL-2, IL-4, GM-CSF, or IFN-gamma. DLNs were harvested 10 days after vaccination for adoptive immunotherapy (AIT). The DLN cells were activated with bryostatin 1 and ionomycin (B/I), expanded for 10 days in culture, and transferred to mice with 3-day pulmonary metastases. Pulmonary nodules were counted 14 days after AIT. RESULTS: Adoptive transfer of expanded DLN lymphocytes sensitized by inoculation of WT B16-F10, or IL-4, GM-CSF, or IFN-gamma expressing cells significantly reduced pulmonary metastases. Despite the spontaneous regression of IL-2-transduced B16-F10 tumors, DLN from mice inoculated with IL-2 producing B16 cells had little or no antitumor activity. CONCLUSIONS: B16-F10 vaccination strategies that apparently do not induce systemic immunity can effectively sensitize DLN preeffector cells.     

Eliciting T cell immunity against poorly immunogenic tumors by immunization with dendritic cell-tumor fusion vaccines

Dendritic cells (DCs) are the most effective APCs and are being studied as natural adjuvants or Ag delivery vehicles to elicit T cell-mediated antitumor immunity. This study examined whether inoculation of DCs fused with poorly immunogenic tumor cells elicited tumor-reactive T cells for adoptive immunotherapy. DCs derived from bone marrow of C57BL/6 (B6) mice were fused with syngeneic B16 melanoma or RMA-S lymphoma cells by polyethylene glycol. The B16/DC and RMA-S/DC fusion hybrids expressed MHC class I, class II Ags, costimulatory molecules, as well as DC-specific and tumor-derived surface markers. The tumor/DC hybrids were capable of processing and presenting tumor-derived Ags, and immunization of B6 mice with irradiated B16/DC or RMA-S/DC vaccine elicited tumor-specific CTL activities. Vaccination of B6 mice with irradiated B16/DC fusion preparations induced partial host protective immunity against B16 tumor challenge. Reduced tumor incidence and prolonged survival time were observed. Adoptive transfer of T cells derived from B16/DC vaccine-primed lymph nodes into B16 tumor-bearing mice greatly reduced the number of established pulmonary metastases with or without in vivo administration of IL-2. Moreover, adoptive transfer of RMA-S/DC vaccine-primed, cultured lymph node T cells eradicated disseminated FBL-3 tumor. The results demonstrate that tumor/DC fusion products are effective cellular vaccines for eliciting T cell-mediated antitumor immunity.     

Divergent effects of TNF alpha in the adoptive immunotherapy of a murine sarcoma

We have previously described an in vitro sensitization (IVS) procedure which enabled the generation of therapeutic T cells from tumor-bearing mice for adoptive immunotherapy. The procedure involved culture of tumor-draining lymph node (TDLN) cells with irradiated tumor in the presence of interleukin-2 (IL-2). The availability of many recombinant cytokines affords an opportunity to examine their effects on the immune response to tumor. In this study, we investigated the effect of tumor necrosis factor-alpha (TNF alpha) on the generation and function of IVS cells utilized in adoptive immunotherapy of the murine MCA 106 sarcoma. TNF alpha administered iv at nontherapeutic doses was found to enhance the antitumor efficacy mediated by IVS cells plus IL-2 in the treatment of pulmonary metastases. In contrast, TNF alpha administration to mice bearing progressive footpad tumors had inhibitory effects on the sensitization of tumor-reactive cells in TDLN since IVS cells generated from these animals displayed a diminished antitumor effect. This effect appeared to be due to a reduced number of tumor-reactive lymphoid cells in the TDLN since TNF alpha added to IVS cultures did not alter the antitumor efficacy of the resultant IVS effector cells. These findings indicate the divergent effects of TNF alpha on the immune response to tumor and adoptive immunotherapy with IVS cells.     

Tumor growth alters T cell and macrophage production of and responsiveness to granulocyte-macrophage colony-stimulating factor: partial dysregulation through interleukin-10

Tumor growth induces phenotypic and functional changes among splenic T cells and macrophages (M phi) that contribute to the immunosuppression observed in tumor-bearing hosts (TBH). These changes partly arise through alterations in immune cell production of and responsiveness to cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important T cell- and M phi-derived cytokine that is produced during normal host immunogenic challenge, but it's involvement during cancer is poorly defined. In contrast, interleukin-10 (IL-10) is an inhibitory cytokine that is produced by immune cells as a deactivation factor. IL-10 can disrupt GM-CSF synthesis and may be associated with tumor-induced changes in cytokine synthesis. We determined if tumor growth alters T-cell and M phi synthesis of and responsiveness to GM-CSF, and if these alterations occur because tumor growth heightens immune cell sensitivity to IL-10. Tumor growth significantly decreased T-cell synthesis of GM-CSF during activation by concanavalin A, and TBH T cells were more susceptible to GM-CSF synthesis inhibition by IL-10 than their normal host (NH) counterparts. This suppression was observed using both unseparated splenic lymphocyte preparations and purified CD4+ and CD8+ T cells. Similarly, TBH M phi (both splenic and peritoneal) produced less GM-CSF than NH M phi during activation by lipopolysaccharide. Tumor growth also altered major histocompatibility complex (MHC) class II- M phi GM-CSF synthesis. TBH M phi were more susceptible to GM-CSF synthesis inhibition by IL-10 than their NH counterparts. Although TBH T cells demonstrate less proliferation than NH T cells during activation, tumor growth did not compromise T-cell responsiveness to GM-CSF. However, tumor growth did increase TBH T-cell susceptibility to inhibition of proliferation by IL-10. Tumor growth suppressed M phi responsiveness to GM-CSF, and IL-10 further decreased M phi responsiveness to GM-CSF. Collectively, these results suggest that T cell and M phi production of and responsiveness to GM-CSF is disrupted during tumor growth, and that TBH T cells and M phi are more susceptible to the suppressor activity of IL-10 than their NH counterparts.     

Allogeneic cell therapy for a murine mammary carcinoma

The effect of allogeneic cell therapy on tumor growth was studied in a murine model of mammary carcinoma (4T1) as an experimental model of solid tumors in humans. i.v. inoculation of 4T1 (H-2d) cells into syngeneic mice [BALB/c or (BALB/cXC57BL/6)F1] (F1) carrying the H-2d histocompatible antigens results in tumor colonies in the lungs that finally cause the death of all of the mice. Sublethally irradiated F1 mice were inoculated with 4T1 cells to simulate minimal residual disease and with immunocompetent splenocytes derived from naive donors of F1 (syngeneic), BALB/c (syngeneic to the tumor but semiallogeneic to the host), or C57BL/6 (allogeneic to the tumor and semiallogeneic to the host) mice. The survival of F1 tumor-bearing mice that were treated with allogeneic C57BL/6 splenocytes was significantly prolonged (P < 0.02) compared with hosts given F1 or BALB/c-derived splenocytes that are syngeneic to 4T1 tumor cells. Adoptive transfer of lung cells that were isolated from F1 primary mice inoculated with 4T1 cells and syngeneic BALB/c or F1 splenocytes led to local tumor growth and death in secondary recipients. In contrast, only 1 of 22 secondary recipients developed tumors when inoculated with lung cells derived from F1 mice given allogeneic C57BL/6 splenocytes. All of the 21 secondary hosts survived disease-free for a follow-up time of >200 days. These results indicate that immunocompetent cells allogeneic to the mammary carcinoma cells were able to inhibit tumor development in the primary hosts and to prevent tumor growth in the adoptive recipients, which suggests that allogeneic cell therapy may be an efficient antitumor tool to eradicate minimal residual disease in human solid tumors.     

Genetic modification of a carcinoma with the IL-4 gene increases the influx of dendritic cells relative to other cytokines

Tumor cells genetically modified with certain cytokine genes gain immunogenic properties that allow the development of systemic anti-tumor immunity. Whether different cytokines may influence infiltration of transduced tumors by dendritic cells (DC) has not been investigated. Therefore, we analyzed the C26 murine colon carcinoma genetically modified to release interleukin (IL)-2, IL-4, IL-12, granulocyte colony-stimulating-factor (CSF) or granulocyte-macrophage (GM)-CSF for immunostaining with the monoclonal antibody NDLC145 recognizing the DEC205 determinant which, on tumor sections, is virtually restricted to DC. Infiltrating leukocytes were also characterized for expression of co-stimulatory molecules like CD54, CD86 and major histocompatibility complex class II. The intratumoral DC content was dependent on the type of transduced cytokines with C26/IL-4 being the most abundant in DEC205+ cells. The effect of IL-4 in recruiting DC did not depend on the type of tumor since it was confirmed in the TSA mammary carcinoma. In comparison with C26/GM-CSF, C26/IL-4 had more B7.2+ cells but less Ia+ cells. Furthermore, the hypertrophic skin overlaying tumors producing GM-CSF showed numerous Langerhans cells stained by NDLC145 and the draining lymph nodes showed abundance and paucity of DC in C26/GM-CSF and C26/IL-4, respectively. When injected into the ear pinna, C26/GM-CSF stimulated, whereas C26/IL-4 inhibited DC-mediated priming of delayed-type hypersensitivity reaction by 2,4-dinitro-1-fluorobenzene. These findings prove that transduced cytokines differently influence DC recruitment at the tumor site and DC function in nearby tissues. Along with the other leukocytes and their secondary produced cytokines, DC create an environment in which T cells can be differently modulated. Such a phenomenon may have implications on genetic modification of tumor cells to be used as cancer vaccine.     

Clinical observations on adoptive immunotherapy with vaccine-primed T-lymphocytes secondarily sensitized to tumor in vitro

The adoptive immunotherapy of human malignancy requires reliable methods to sensitize and expand patients' T-cells reactive to autologous tumors. In animal studies, we have generated therapeutic effector cells against a poorly immunogenic tumor by a two-step procedure: vaccination of the host followed by the secondary stimulation of vaccine-primed lymph node (LN) cells by in vitro sensitization (IVS) with tumor in the presence of interleukin 2 (IL-2). Based on these observations, we performed a clinical trial in patients with advanced cancer to evaluate the antitumor efficacy of vaccine-primed LN cells which were similarly activated in vitro. Patients were vaccinated with irradiated autologous tumor admixed with Bacillus Calmette-Guérin and had draining LN excised 10 days later for IVS culture. During IVS culture, LN cells expanded up to 14-fold (average of 8.4-fold). A mean of 6.7 x 10(9) cells was infused in ten patients (seven melanoma, three renal cell cancer) along with the concomitant i.v. administration of IL-2 (180,000 IU/kg every 8 h for 5 days). Phenotype analysis of IVS-LN cells revealed 78 +/- 4% CD3+ T-cells which were predominantly CD4+ (67 +/- 5%) with expression of HLA-DR and IL-2 receptor. IVS-LN cells displayed relative specificity of autologous tumor lysis in four of ten cases compared to zero of seven IVS-peripheral blood lymphocytes derived from the same patients as measured by the 51Cr release assay. One mo after therapy, seven of nine patients treated with IVS-LN cells and IL-2 developed delayed-type hypersensitivity reactivity to autologous tumor compared to zero of nine patients treated with tumor vaccination and IL-2 only (P < 0.002). These observations suggest that antitumor reactivity was passively transferred with the IVS-LN cells. Major toxic side effects including fever, hepatic dysfunction, and weight gain associated with the capillary leak syndrome were associated with exogenous IL-2 administration. Tumor vaccination and cell transfer were well tolerated without significant complications. Of the ten patients treated with IVS-LN cells and IL-2, there were one partial and one minor response, and one patient has had stable disease for 27+ mo. There was no evidence of tumor response in ten patients treated with tumor vaccination and IL-2 only. Further clinical studies evaluating the antitumor reactivity of vaccine-primed LN cells are warranted.     

TCR vaccines against T cell lymphoma: QS-21 and IL-12 adjuvants induce a protective CD8+ T cell response

Tumor-specific TCR can serve as an effective target for active immunotherapy of T cell malignancies. Using the murine T cell tumor model C6VL, vaccination with C6VL TCR protected mice from a subsequent lethal dose of tumor cells. This study characterizes the immune mechanisms involved in the tumor protection, and the influence of immunologic adjuvants in inducing a protective immune response. Immune responses induced by TCR vaccines formulated with various adjuvants: QS-21, IL-12, SAF-1, CD40L, and GM-CSF were compared. QS-21, IL-12, and SAF-1 biased the humoral immune response toward Th1-type, reflected by the induction of IgG2a and IgG2b anti-C6VL TCR Abs. CD40L and GM-CSF exclusively produced IgG1 Abs, reflecting a Th2-type immune response. In our tumor model system, only vaccines containing adjuvants that induced a Th1-type immune response favored tumor protection. Furthermore, we demonstrated that CD8+ T cells were necessary and sufficient for tumor protection using anti-CD8 mAb depletion and adoptive cell transfer experiments. Transfer of hyperimmune serum containing anti-C6VL TCR Abs into na ive mice had modest anti-tumor effects and was not sufficient to prevent tumor growth. TCR-vaccinated B cell-deficient mice were not protected against C6VL tumor, and tumor protection was not completely restored after hyperimmune serum transfer. Thus, B cells may serve as important APCs in inducing a protective immune response. Based on these results future TCR vaccines should be designed to maintain native TCR conformation, as well as induce a strong Th1-type immune response.     

Antigen-specific regression of established tumors induced by active immunization with irradiated IL-12- but not B7-1-transfected tumor cells

Transfection of modestly immunogenic tumors to express B7 family co-stimulator molecules results in their rejection by syngeneic mice, suggesting a possible clinical application in cancer patients. Immunization of naive mice with irradiated B7-1-transfected P1.HTR cells is sufficient to induce specific cytolytic T lymphocytes (CTL) and to protect against tumor challenge. However, patients to be treated will have an existing tumor burden; thus, preclinical models should examine therapeutic efficacy in an established tumor setting. Contrary to expectations, immunization of mice with irradiated B7-1-transfected P1.HTR cells had no impact on the growth of pre-established control-transfected tumors. Mice bearing control-transfected P1.HTR tumors successfully rejected living B7-1 transfectants on the contralateral flank, demonstrating the ability of tumor-bearing mice to respond to B7 co-stimulation. Inasmuch as IL-12 is another important factor for CTL maturation, P1.HTR transfectants expressing B7-1 and/or IL-12 were then constructed. Remarkably, regression of pre-established tumors was achieved following immunization with irradiated IL-12 transfectants, even without co-expression of B7-1. Rejection required a shared antigen with the tumor used for immunization, could not be reproduced with rIL-12 alone, depended on host T lymphocytes and correlated with a high IFN-gamma-producing T cell phenotype. In addition, IL-12-facilitated tumor rejection required co-operation with a CTLA-4 ligand provided by the host, and correlated with up-regulation of B7-1 and B7-2 on host antigen-presenting cells. Thus, active immunization in the established tumor setting is benefitted greatly by the provision of IL-12, which may recruit participation of sufficient B7 co-stimulation from the host that it need not be provided exogenously.     

Cytokine and hepatitis B virus DNA co-immunizations enhance cellular and humoral immune responses to the middle but not to the large hepatitis B virus surface antigen in mice

Genetic immunization is a potentially useful strategy to prevent or treat hepatitis B virus (HBV) infection. We have previously shown that HBV envelope proteins are highly immunogenic using this technique. The large envelope protein (LHBs), however, induced significantly weaker humoral and cellular immune responses when compared with the middle envelope protein (MHBs). We studied the effect of co-immunizations with cytokine DNA expression constructs encoding for interleukin (IL)-2 and (GM-CSF) on the immunogenicity of LHBs at the B-and T-cell level. Co-immunizations of mice with plasmids encoding for MHBs and IL-2 or GM-CSF increased anti-HBs responses, helper T-cell proliferative activity, and cytotoxic T lymphocyte (CTL) killing. In contrast, co-immunizations of plasmids encoding for LHBs and IL-2 or GM-CSF had no effect on humoral and cellular immune responses. LHBs did not inhibit the production or secretion of IL-2 and GM-CSF. In addition, IL-2, tumor necrosis factor alfa (TNF-alpha), and interferon gamma (IFN-gamma) had no suppressive effect on HBV envelope protein expression in vitro. Based on these data, MHBs, but not LHBs, genetic immunization can be augmented by IL-2 or GM-CSF cytokines.     

Multicomponent gene therapy vaccines for lung cancer: effective eradication of established murine tumors in vivo with interleukin-7/herpes simplex thymidine kinase-transduced autologous tumor and ex vivo activated dendritic cells

Multiple antitumor modalities may be necessary to overcome lung tumor-mediated immunosuppression and effectively treat non-small cell lung cancer (NSCLC). To evaluate a multimodality gene therapy approach for control of local tumor growth, a weakly immunogenic murine alveolar cell carcinoma, L1C2, was transduced with either the interleukin-7/hygromycin-herpes simplex thymidine kinase (IL-7/HyHSVtk) internal ribosome entry site (IRES) retroviral vector or a vector containing the HyHSVtk, but not the IL-7 gene. Of the many cytokines available for gene transfer, IL-7 was chosen for these studies because it both stimulates CTL responses and down-regulates tumor production of the immunosuppressive peptide TGF-beta. Following selection in hygromycin, IL-7 transduction was confirmed by ELISA. Clones produced 1.25 to 10 ng of IL-7/ml/10(6) cells per 24 h. In vitro, genetically modified tumor cells were significantly more sensitive to ganciclovir (GCV) than unmodified parental tumor cells. The in vivo growth of ex vivo modified L1C2 cells was evaluated. There was a dose-response relationship between the amount of IL-7 secreted in vitro and the growth of genetically modified murine tumor in vivo. Transduced tumor cells regressed in mice following GCV therapy. Although ex vivo gene modification of tumor cells led to complete resolution of the tumor following implantation in vivo, IL-7 and HSVtk gene modified tumor cells were not effective in treating established parental tumors. However when 5 x 10(5) bone marrow-derived, in vitro activated dendritic cells (DC) were administered in combination with transduced tumor and GCV, 5 day old established tumors were eradicated in 80% of mice. These studies suggest that multicomponent vaccines may facilitate improved host responses by replacing host immune deficits and thus could have a role in adjuvant therapy and local control of NSCLC.     

Adoptive immunotherapy with vaccine-primed lymph node cells secondarily activated with anti-CD3 and interleukin-2

PURPOSE: In preclinical studies, we have reported the ability to induce immune T cells in lymph nodes (LN) primed by in vivo vaccination with tumor cells admixed with a bacterial adjuvant. These LN cells can be activated and expanded ex vivo for the successful immunotherapy of established tumors. We have applied these methods to generate vaccine-primed LN in patients with advanced melanoma and renal cell cancer (RCC) for therapy. MATERIALS AND METHODS: Irradiated autologous tumor cells admixed with bacille Calmette-Guérin (BCG) were used to vaccinate patients. Seven days later, draining LN were removed for activation with anti-CD3 monoclonal antibody (mAb) followed by expansion in interleukin-2 (IL-2). Activated LN cells were administered intravenously (IV) with the concomitant administration of IL-2. RESULTS: A total of 23 patients were evaluated (11 melanoma and 12 RCC). Vaccine-primed LN were expanded ex vivo with a mean of 8.4 x 10(10) cells administered per patient. Among 20 patients assessed, 15 demonstrated minimal cytotoxicity of autologous tumor cells by the activated LN cells, with the remaining mediating nonspecific cytotoxicity. By contrast, a majority of the activated LN cells showed highly specific release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFN-gamma) to autologous but not allogeneic tumor stimulation. This tumor-specific cytokine release was found to be major histocompatibility complex (MHC) class I-restricted, which indicates the involvement of CD8+ cells. Among 11 melanoma patients, one had a partial tumor response. Among 12 RCC patients, two had complete and two partial responses. A trend (P = .066) between the enhancement of delayed-type hypersensitivity (DTH) reactivity to autologous tumor after therapy and tumor regression was observed. CONCLUSION: Tumor vaccines can be used to induce immunologically specific T-cell responses against melanoma and RCC in draining LN. Anti-CD3/IL-2 activation of primed LN cells can be reliably performed for clinical therapy and appears to have activity in patients with metastatic RCC.     

Interleukin 6 and its relationship to clinical parameters in patients with malignant pleural mesothelioma

We explored the potential therapeutic benefit of introducing GM-CSF, IFN-gamma or a combination of both factors into CT26 tumor cells. CT26 cells secreting either GM-CSF or IFN-gamma exhibited delayed tumorigenicity; however, cells expressing both GM-CSF and IFN-gamma did not form tumors. Even when wild type CT26 cells were introduced into a distant site of mice that had been inoculated with CT26/GM-CSF/IFN-gamma cells, no tumors were generated. Furthermore, when we injected GM-CSF + IFN-gamma cells into animals bearing established tumors, the tumors were either rejected or their development was delayed, suggesting that synergistic effects were induced against these tumors via a systemic immune response. Histopathological examination of the tumors injected with cells expressing GM-CSF and IFN-gamma combined showed necrosis and few signs of malignancy. The growth of tumors from mice treated with CT26/GM-CSF/IFN-gamma cells exhibited a delay in tumor formation and no effects were seen in athymic nude mice, which are deficient in T lymphocytes, or in splenectomized nude mice, which are deficient in natural killer (NK) cells, respectively. Our data indicate a dual role for T and NK cells in mediating the anti-tumor activity of this therapy. Our results suggest that transduction of tumor cells with both GM-CSF + IFN-gamma results in a powerful synergistic effect of the 2 cytokines that is of greater therapeutic benefit than transduction with either cytokine alone.     

A new mouse model of experimental melanoma for vaccine and lymphokine therapy

The annual incidence of malignant melanoma is estimated at 10-12 per 100,000 inhabitants in countries of central Europe and the United States, and alarmingly there has been a dramatic upward trend in that estimate. The B16 murine melanoma is a rapidly growing metastatic tumor of spontaneous origin, as are human malignant melanomas. Melanoma cells produce specific antigens which are uniquely different from normal cellular antigens, and the expression of such antigens is the cornerstone for preparation of anti-melanoma vaccines. One major problem in evaluating the effectiveness of vaccination and other biologic therapies is the variability of experimental tumor models. A new metastatic model of experimental melanoma which was developed in our laboratory imitates the major clinical stages of malignant metastatic melanoma: stage I, primary (local) tumor growth and bone marrow invasion; stage II, regional lymph node involvement; and stage III, metastasis to distant organs, such as the lungs. This model has been used successfully for screening vaccines constructed in our laboratory. Immunization with formalinized vaccines (of extracellular antigens, intact melanoma cells, or B700 antigen) or irradiated vaccines (of intact melanoma cells) partially inhibit primary melanoma tumor growth, reduce metastasis to regional lymph nodes and lungs, and significantly increase mean survival time. These anti-tumor effects were improved when polyvalent and monovalent vaccines were combined with IL-2 therapy. We also compared the immunogenic activity of vaccines made from B16 melanoma cells transfected with genes encoding murine IL-2 or GM-CSF, and effects on tumor bearing mice were compared with or without therapy using the corresponding lymphokines. In sum, comparison of antibody production, growth of primary melanoma tumors, number of surviving mice, mean survival time, and percent of mice with lung metastases, showed that the best course of immunotherapy involves vaccination of mice with irradiated B16 melanoma cells transfected to secrete GM-CSF, coupled with GM-CSF therapy.     

Canarypox virus-mediated interleukin 12 gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity

The antitumoral activity of recombinant canarypox virus vectors (ALVAC) expressing murine interleukin 12 (IL-12) was evaluated in the syngeneic, nonimmunogenic murine mammary adenocarcinoma model (TS/A). Seven-day preestablished subcutaneous tumors (5- to 6-mm mean diameters) were injected on days 7, 10, 14, 17, 21, and 24 with the vector ALVAC-IL12 at 2.5 x 10(5) TCID50 (50% tissue culture infective dose). Total tumor regression occurred in 40 to 50% of the treated mice. Furthermore, 100% of the cured mice were protected against a contralateral subsequent challenge with the TS/A parental cells on day 28. The ALVAC-IL12 treatment is not effective in nude mice, suggesting the critical role of T cells. CD4 and CD8 T cells infiltrated the tumors treated with ALVAC-IL12 in the BALB/c model. Furthermore, in vivo depletion of CD4+ T cells totally abrogated the induction of the long-term antitumoral immune response by ALVAC-IL12. Interestingly, some tumor growth inhibition was also observed with ALVAC-betaGal treatment and a vaccinal effect was found in 33% of the treated animals, suggesting an adjuvant effect of the vector itself. Other ALVAC vectors expressing murine cytokines (IL-2, GM-CSF, IFN-gamma) were evaluated in the same model. Major antitumoral activity was observed with ALVAC-GM-CSF. However, a combination of ALVAC-GM-CSF and ALVAC-IL12 had no synergistic effect. These results suggest that in vivo gene transfer with canarypox virus expressing IL-12 may provide an effective and safe strategy for the treatment of human cancers.     

Synergistic regulation of inducible nitric oxide synthase gene by CCAAT/enhancer-binding protein beta and nuclear factor-kappaB in hepatocytes

Intratumoral (i.t.) injection of a plasmid DNA vector encoding the murine interleukin-2 (IL-2) gene was used to treat established renal cell carcinoma (Renca) tumors in BALB/c mice. Tumor regression was observed in 60-90% of mice that were injected i.t. for 4 days with IL-2 plasmid DNA complexed with the cationic lipid DMRIE/DOPE ((+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propa naminium bromide/dioleoylphosphatidylethanolamine). The mice remained tumor-free until the conclusion of the study, which was 4 months after tumor challenge. In a rechallenge experiment, mice that were rendered tumor-free for 6 months by IL-2 plasmid DNA treatment rejected a subsequent challenge of Renca cells but could not reject a challenge with the unrelated, syngeneic CT-26 tumor. Spleen cells from cured mice contained Renca-specific cytotoxic T lymphocytes, and adoptive transfer of mixed lymphocyte cultures into naive mice at 2 days after challenge with Renca cells prevented tumor growth. In vivo depletion of T-cell subsets at the time of i.t. injection with IL-2 plasmid DNA demonstrated that CD8+ T cells, but not CD4+ T cells, were the primary effectors of the antitumor response.     

Induction of antitumor immunity by interleukin-2 gene-transduced mouse mammary tumor cells versus transduced mammary stromal fibroblasts

BACKGROUND: Tumor cell-targeted cytokine gene transfer has been used to generate tumor cell vaccines, but this approach is limited by the need to establish and implant live tumor cells. PURPOSE: The purpose of this study was to determine if stromal fibroblasts could be used as an alternative vehicle for delivery of the cytokine interleukin-2 (IL-2) into the tumor microenvironment. We attempted to establish the feasibility of (a) genetic immunotherapy in a mammary tumor system and (b) engineering stromal fibroblasts as well as tumor cells. We compared the effects of tumor cell-mediated and stromal fibroblast-mediated local IL-2 expression on the generation of antitumor immune responses. METHODS: Retroviral vectors containing a human IL-2 gene were used to transduce a mouse mammary tumor line, 4TO7, and an immortalized but nontumorigenic fibroblast line established from syngeneic mammary fatpads. Expression of the IL-2 gene in transduced cells was determined by measuring IL-2 secretion, by RNA-polymerase chain reaction, and by immunochemistry. Groups of 5-12 BALB/c mice were injected with either 4TO7 cells or various doses of IL-2-secreting 4TO7 cells (4TO7-IL-2); tumor growth was monitored. To test whether local IL-2 expression by transduced cells could influence the growth of unmodified tumor cells, we determined tumor development in groups of mice treated with 4TO7 cells co-injected with either 4TO7-IL-2 cells or IL-2-secreting fibroblasts. RESULTS: 4TO7-IL-2 cells induced active immunity able to reject the immunizing tumor and to resist challenge with parental 4TO7 cells on the contralateral side. Mice pretreated with 4TO7-IL-2 were significantly protected compared with untreated control animals or mice pretreated with irradiated 4TO7 cells. The immunity induced by 4TO7-IL-2 cells did not protect against challenge with another subline, 4T1, which was derived from the same spontaneously arising mammary tumor as 4TO7. Co-injection of 4TO7 cells with 4TO7-IL-2 cells reduced tumorigenicity, whereas co-injection of 4TO7 cells with IL-2 secreting fibroblasts did not. CONCLUSION: Our results suggest that induction of anti-tumor immune response by local IL-2 production is most effective when the helper cytokine is secreted by the tumor cell. IMPLICATION: Our studies caution against the use of IL-2 gene-transduced syngeneic stromal cells as an alternative strategy of gene therapy for cancer. However, they may allow study of the mechanisms of tumor antigen recognition and the possible involvement of co-stimulatory signals for effective tumor vaccination by gene-modified cells.     

Demonstration of mRNA editing and localization of guide RNA genes in kinetoplast-mitochondria of the plant trypanosomatid Phytomonas serpens

The adoptive transfer of naive CD4(+) T cell receptor (TCR) transgenic T cells was used to investigate the mechanisms by which the adjuvant lipopolysaccharide (LPS) enhance T cell clonal expansion in vivo. Subcutaneous administration of soluble antigen (Ag) resulted in rapid and transient accumulation of the Ag-specific T cells in the draining lymph nodes (LNs), which was preceded by the production of interleukin (IL)-2. CD28-deficient, Ag-specific T cells produced only small amounts of IL-2 in response to soluble Ag and did not accumulate in the LN to the same extent as wild-type T cells. Injection of Ag and LPS, a natural immunological adjuvant, enhanced IL-2 production and LN accumulation of wild-type, Ag-specific T cells but had no significant effect on CD28-deficient, Ag-specific T cells. Therefore, CD28 is critical for Ag-driven IL-2 production and T cell proliferation in vivo, and is essential for the LPS-mediated enhancement of these events. However, enhancement of IL-2 production could not explain the LPS-dependent increase of T cell accumulation because IL-2-deficient, Ag-specific T cells accumulated to a greater extent in the LN than wild-type T cells in response to Ag plus LPS. These results indicate that adjuvants improve T cell proliferation in vivo via a CD28-dependent signal that can operate in the absence of IL-2.     

Social mobility and 21 year mortality in a cohort of Scottish men

Effective immunization against the murine B16 melanoma by a nonviral approach in which a gene gun is used to transfer GM-CSF cDNA into tumor cells has been described. We have extended this nonviral approach by using the poorly immunogenic murine myeloma MPC11 model. Vaccination with the transfected, GM-CSF-expressing MPC11 cells induced a potent antitumor cytotoxic T lymphocyte response associated with tumor rejection in the majority of the test mice. Furthermore, nearly 100% (27 of 28) of the tumor-free mice were able to reject a tumor rechallenge. While this approach is clinically attractive because of minimal tissue manipulation/culturing and the absence of infectious agents, a number of tested human primary tumors, including myeloma cells, have failed to produce high levels of GM-CSF after gene gun transfection. To circumvent the low transfection efficiency in certain human tumor cells, we showed that combining irradiated tumor cells to provide tumor antigens together with gene gun-transfected fibroblasts to provide GM-CSF induced effective tumor rejection. We also report that normal human skin fibroblasts transfected by the gene gun produce high levels of human GM-CSF (250 ng/10(6) cells/24 hr). These results suggest that combining irradiated tumor cells with gene gun-transfected fibroblasts results in antitumor immune responses and may allow for a wider application of this approach to cancer immunotherapy.