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.     

Generation of cytotoxic immune responses during the progression of a rat glioma

Cytotoxic T lymphocytes specific for tumor-associated antigens are produced by exposing animals to tumor cells and stimulating lymphocytes from animals immunized in vitro with tumor cells and small amounts of interleukin-2 (IL-2). This study was designed to determine whether a fast-growing immunogenic avian sarcoma virus-induced glioma produces primed cytotoxic T lymphocyte precursors during its progression. Lymphocytes from intracerebral glioma-bearing rats generally failed to proliferate in vitro in response to immunization with tumor cells and IL-2 and, when proliferative responses were observed, the lymphocytes were not cytotoxic for glioma cells. However, when the same tumor was growing subcutaneously, lymphocytes proliferated and exhibited glioma-specific cytotoxicity when stimulated in vitro with autologous tumor cells and IL-2. Subcutaneous immunization of intracerebral glioma-bearing rats with tumor cells and adjuvant induced strong cytotoxic T lymphocyte responses. The results demonstrated that, while intracerebral tumor progression itself does not induce an anti-glioma immune response, immune responses to tumor-associated antigens may be induced by systemic immunization of tumor-bearing animals. The results suggest that the immunogenicity of brain tumors is masked by the immunologically privileged status of the brain, not by the induction of generalized immune suppression during tumor progression.     

Tumor microenvironment can restrict the effectiveness of activated antitumor lymphocytes

Transgenic mice expressing the oncogene SV40 T antigen (Tag) in the insulin-producing beta cells of the pancreas develop islet cell carcinomas. Expression of the oncogene beginning in adult life leads to autoimmunity and lymphocytic infiltration of premalignant lesions. Nevertheless, Tag-expressing solid tumors escape the immune surveillance and are devoid of infiltrating lymphocytes. Attempting to elicit a tumor inflammatory response, we have both expressed a potent costimulator in oncogene-expressing beta cells and increased the abundance of reactive T cells. Coexpression of the costimulator B7.1 and the Tag oncoprotein leads to destruction of normal and premalignant islets and severe diabetes. Nevertheless, Tag+ tumors eventually develop, evidencing significantly reduced B7.1 expression and no infiltration. Another approach, whereby the abundance of reactive T cells was increased in double transgenic mice expressing Tag and a Tag-specific, CD4+-restricted T-cell receptor, was similarly unable to elicit tumor infiltration and destruction. Thus, neither costimulatory tumor cells nor hyperactivated antitumor lymphocytes were sufficient to produce an effective tumor immune response. In contrast, adoptive transfer of lymphocytes activated ex vivo did result in modest tumor infiltration with a limited induction of high endothelial venules on tumor vasculature, provided that T cells were transferred into irradiated recipients. However, adoptive transfer of ex vivo activated lymphocytes did not produce the dramatic inflammation seen in premalignant lesions. Thus, in addition to the parameters of activation and abundance of antitumor lymphocytes, the tumor microenvironment is evidently a critical parameter that can suppress lymphocyte extravasation and/or function inside tumors, likely in part via distinctive properties of the tumor vasculature.     

Eradication of rat malignant gliomas by retroviral-mediated, in vivo delivery of the interleukin 4 gene

Overexpression of interleukin 4 (IL-4) can impair the tumorigenicity of glioma cells, but direct evidence of its antitumor efficacy after in vivo gene transfer into malignant gliomas has not been provided. To test this, we first injected into the brain of Sprague Dawley rats a 1:1 mixture of C6 rat glioblastoma cells and psi2.L4SN20 or E86.L4SN50 retroviral producer cells (RPCs), secreting 20 and 50 ng of IL-4/5 x 10(5) cells/48 h, respectively. Twenty-seven and 56% of rats receiving injections with these low- or medium-level IL-4 RPCs, respectively, survived tumor injection, whereas control rats died in about 1 month. E86.L4SN50 RPCs coinjected with 9L gliosarcoma cells into syngeneic Fischer 344 rats yielded similar results. A novel IL-4 RPC clone expressing higher levels of IL-4, E86.L4SN200, coinjected with 9L cells increased to 75% the fraction of long-term survivors and induced tumor regression in 50% of rats when injected into established 9L gliosarcomas. Cured rats developed an immunological memory because they rejected a challenge of wild-type 9L cells into the contralateral hemisphere. Magnetic resonance imaging was used to monitor 9L and C6 gliomas and gave direct evidence for tumor rejection in treated rats. Immunohistology showed inflammatory infiltrates in IL-4-treated tumors in which CD8+ T lymphocytes were more abundant, although CD4+ T lymphocytes, B lymphocytes, and macrophages were also present. Overall, these findings suggest that IL-4 gene transfer is a new, promising approach for treating malignant gliomas.     

Effect of desialylation of highly purified isoforms of human luteinizing hormone on their bioactivity in vitro, radioreceptor activity and immunoactivity

The effect of intracarotid infusion of the bradykinin analog, RMP-7, on blood-to-tumor and blood-to-brain transport of three cytokines were investigated. Wistar rats with RG2 gliomas were utilized and a unidirectional transfer constant, Ki, was determined using quantitative autoradiography. Interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-2 (IL-2) were labeled with 125Iodine for quantitative transport studies using autoradiography. Radiolabeled cytokines were injected as an intravenous bolus. Intracarotid infusion of RMP-7 (0.1 microgram kg-1 min-1) increased the selective transport to tumors of IFN-gamma by 3.97-fold (p < 0.005), of TNF-alpha by 5.30-fold (p < 0.005), and of IL-2 by 4.34-fold (p < 0.005), compared to intracarotid saline infusion. To determine whether the increased IFN-gamma or TNF-alpha transport to tumors with RMP-7 could enhance expression of intercellular adhesion molecule 1 (ICAM-1) in tumors, ICAM-1 expression in RG2 glioma was evaluated by immunohistochemistry. Both IFN-gamma and TNF-alpha increased ICAM-1 expression of RG2 cells in vitro. In vivo, intracarotid infusion of IFN-gamma combined with RMP-7 significantly enhanced ICAM-1 expression in intracerebral RG2 gliomas compared to infusion of IFN-gamma without RMP-7. Expression of ICAM-1 was not enhanced by TNF-alpha combined with RMP-7. Intracarotid infusion of RMP-7 is a novel method of cytokines delivery to brain tumors.     

Importance of cyclophosphamide-induced bystander effect on T cells for a successful tumor eradication in response to adoptive immunotherapy in mice

Cyclophosphamide (CTX) increases the antitumor effectiveness of adoptive immunotherapy in mice, and combined immunotherapy regimens are now used in some clinical trials. However, the mechanisms underlying the synergistic antitumor responses are still unclear. The purpose of this study was (a) to evaluate the antitumor response to CTX and adoptive immunotherapy in mice bearing four different syngeneic tumors (two responsive in vivo to CTX and two resistant); and (b) to define the mechanism(s) of the CTX-immunotherapy synergism. Tumor-bearing DBA/2 mice were treated with a single injection of CTX followed by an intravenous infusion of tumor-immune spleen cells. In all the four tumor models, a single CTX injection resulted in an impressive antitumor response to the subsequent injection of spleen cells from mice immunized with homologous tumor cells independently of the in vivo response to CTX alone. Detailed analysis of the antitumor mechanisms in mice transplanted with metastatic Friend leukemia cells revealed that (a) the effectiveness of this combined therapy was dependent neither on the CTX-induced reduction of tumor burden nor on CTX-induced inhibition of some putative tumor-induced suppressor cells; (b) the CTX/immune cells' regimen strongly protected the mice from subsequent injection of FLC, provided the animals were also preinoculated with inactivated homologous tumor together with the immune spleen cells; (c) CD4(+) T immune lymphocytes were the major cell type responsible for the antitumor activity; (d) the combined therapy was ineffective in mice treated with antiasialo-GM1 or anti-IFN-alpha/beta antibodies; (e) spleen and/ or bone marrow cells from CTX-treated mice produced soluble factors that assisted in proliferation of the spleen cells. Altogether, these results indicate that CTX acts via bystander effects, possibly through production of T cell growth factors occurring during the rebound events after drug administration, which may sustain the proliferation, survival, and activity of the transferred immune T lymphocytes. Thus, our findings indicate the need for reappraisal of the mechanisms underlying the synergistic effects of CTX and adoptive immunotherapy, and may provide new insights into the definition of new and more effective strategies with chemotherapy and adoptive immunotherapy for cancer patients.     

The Friedreich's ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis

Interleukin 12 (IL-12) exhibits anti-tumor activity in a variety of laboratory models. Although IL-12 itself activates strong anti-tumor activity, the combination of vaccine therapy with IL-2-transduced tumor cells and systemic rIL-12 has been shown to cure tumor-bearing mice more effectively than either rIL-12 or IL-2-transduced tumor vaccines alone. In the present study, regression of brain tumors established in naive mice was obtained by combined administration of an intratumoral injection of a single dose of IL-2-producing glioma cells (SR/IL-2 cells) and recombinant IL-12. Intraperitoneal rIL-12 administration substantially delayed the growth of s.c. inoculated gliomas, but not of gliomas located in the brain. Although vaccination with SR/IL-2 cells alone was not effective against s.c. inoculated gliomas, the combination therapy of vaccination with irradiated SR/IL-2 cells and systemic rIL-12 was more effective than rIL-12 alone. In our brain-tumor model, intratumoral administration of irradiated SR/IL-2 cells and of rIL-12 remarkably prolonged survival as compared with untreated mice. Efficacy was reduced when studies were performed in mice depleted of CD8+ cells or NK cells. Mice cured of their intracerebral tumors by combined administration of SR/IL-2 cells and rIL-12 demonstrated protective immunity upon rechallenge. In summary, the therapeutic potential for control of tumor growth by intratumoral administration of IL-2-producing glioma cells and rIL-12 may be useful in the development of treatment for patients with glioma.     

Enhancement of interleukin-4-mediated tumor regression in athymic mice by in situ retroviral gene transfer

Intratumoral grafting of genetically engineered cells that produce interleukin-4 (IL-4) has been shown to produce tumor regression as well as prolong survival of mice harboring intracerebral gliomas. We sought to determine whether retroviral-mediated gene delivery into tumor cells in situ resulted in enhanced tumor regression by IL-4. Two mouse fibroblast lines were obtained: they both secreted similar levels of IL-4 but one produced a retrovirus vector bearing the IL-4 gene (CRE-MFG-IL-4 cells), whereas the other did not (NIH3T3-IL-4 cells). In mixed transplantation assays in the subcutaneous flanks of athymic mice, CRE-MFG, IL-4 cells were more effective than NIH3T3-IL-4 cells in inhibiting the growth of rat C6 glioma cells (p < 0.005, ANOVA). Subcutaneous tumors injected with fibroblasts that produced a control retrovirus vector without producing IL-4 (CRE-MFG-LacZ cells) did not inhibit subcutaneous tumor growth. An intracranial assay was used to evaluate survival of athymic mice harboring intracranial gliomas. Three days after implanting rat C6 glioma cells into the right frontal lobes of athymic mice, NIH3T3-IL-4 cells (n = 10) or CRE-MFG-IL-4 cells (n = 10) were stereotactically inoculated into the tumor bed. The average survival of mice treated with CRE-MFG-IL-4 cells was 38 days (+/- 2.4, SE), whereas that of mice treated with NIH3T3-IL-4 cells was 31 days (+/- 0.8, SE) (p < 0.005, ANOVA; p < 0.001, log-rank analysis).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene therapy for malignant gliomas using replication incompetent retroviral and adenoviral vectors encoding the cytochrome P450 2B1 gene together with cyclophosphamide

Cyclophosphamide is an inactive prodrug which is converted by hepatic cytochrome P450 2B1 to cytotoxic metabolites which produce interstrand DNA cross-linking in a cell cycle-independent fashion. The limited ability of these metabolites to cross the blood-brain barrier contributes to the poor activity of cyclophosphamide against brain tumors. In this study we demonstrate that replication deficient retroviral and adenoviral vector-mediated gene transfer of cytochrome P450 2B1 into 9L glioma cells significantly increases the sensitivity of these tumor cells to cyclophosphamide in vitro, and prolongs the survival of animals bearing intracerebral 9L tumors treated with cyclophosphamide in vivo. Attempts to improve the effectiveness of retrovirally mediated transduction of the P450 2B1 gene by increasing the concentration of cyclophosphamide delivered to the tumors using intracarotid and intratumoral injections did not prolong animal survival, although survival was increased when a second treatment with P450-expressing retroviral vectors and cyclophosphamide was administered. These results suggest that in situ transduction of tumor cells with the P450 2B1 gene using retroviral and adenoviral vectors increases their sensitivity to cyclophosphamide and may have a potential role in the therapy of malignant gliomas.     

RG-2 glioma growth attenuation and severe brain edema caused by local production of interleukin-2 and interferon-gamma

Two aspects of cytokine therapy of intracerebral tumors are considered in this study: modulation of tumor growth in vivo and central nervous system toxicity. Coimplantation of RG-2 glioma cells and retroviral vector producer cell lines was performed to provide a local source of interleukin-2 (IL-2) or IFN-gamma within the tumor and coinitiate an antitumor immune response. We demonstrated that local intratumoral production of IL-2 and IFN-gamma generates a cell-mediated antitumor response in vivo. This response was manifest as a diffuse infiltration of monocytes/macrophages, CD4+ and CD8+ T cells, and activation of microglial OX42+ cells in intracerebral RG2 tumors. The cell-mediated antitumor immune response resulted in the early suppression of intracranial and subcutaneous tumor growth, but the effect was not sustained and there were no tumor regressions. The absence of increased survival of animals with intracranial tumors is explained in part by the severe central nervous system toxicity caused by local production of IL-2 and IFN-gamma. Central nervous system toxicity induced blood-brain barrier disruption, vasogenic brain edema, and dislocation of the brain midline structures, as observed by dynamic magnetic resonance imaging and direct measurements of tissue water content. The clinical application of IL-2 and IFN-gamma gene transfer therapy for intracerebral tumors must consider the potential for severe vasogenic brain edema associated with intracerebral production of these cytokines.     

CD4+ lymphocytes provide MUC1-specific tumor immunity in vivo that is undetectable in vitro and is absent in MUC1 transgenic mice

A C57BL/6 mouse transgenic for human MUC1 (MUC1.Tg) was developed to evaluate MUC1-specific tumor immunity in an animal that expresses MUC1 as a normal self protein. Previous studies showed that MUC1.Tg mice, challenged with syngeneic tumors expressing MUC1 (B16.MUC1), developed progressively growing MUC1-positive tumors, whereas wild-type C57BL/6 (wt) mice developed MUC1-negative tumors at a significantly slower rate. The results of a limiting dilution CTL frequency assay were not informative, in that similar numbers of MUC1-specific CTL precursors (CTL) were detected in MUC1.Tg and wt mice. Tumor immunity in vivo was characterized by an adoptive transfer method to evaluate the degree of MUC1 or non-MUC1 tumor immunity in wt or MUC1.Tg mice. The results revealed that wt mice developed protective tumor immunity mediated by MUC1-specific CD4+ lymphocytes, while MUC1.Tg mice were functionally tolerant to MUC1 in vivo. The potential of adoptive immunotherapy to provide immunity to tumors expressing MUC1 and to produce undesirable autoimmunity in recipient MUC1.Tg mice expressing MUC1 as a self Ag was evaluated. Adoptive transfer of immune cells from wt mice primed in vivo with B16.MUC1 tumor cells into MUC1.Tg recipients resulted in significant increases in the survival of MUC1.Tg recipients compared with unmanipulated control MUC .Tg mice challenged with B16.MUC1 tumor cells. This response was specific for MUC1 since control tumors developed at equivalent rates in recipient or control MUC1.Tg mice. No gross or histologic evidence of autoimmunity was observed in recipient MUC1.Tg mice, indicating that tumor immune responses mediated by MUC1-specific CD4+ lymphocytes spare nontransformed epithelia-expressing MUC1.     

Eradication of pre-established lymphoma using herpes simplex virus amplicon vectors

Herpes simplex virus amplicon vectors expressing RANTES (HSVrantes) and the T-cell costimulatory ligand B7.1 (HSVB7.1) were studied for their ability to elicit a tumor-specific T-cell response in a murine lymphoma model. HSVB7.1- and HSVrantes-transduced EL4 cells expressed high levels of B7.1 and RANTES as analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. Inoculation of ex vivo HSVB7.1 transduced cells in syngeneic mice resulted in regression of both transduced cells and nontransduced cells inoculated contralaterally. Direct intratumoral injection of HSVB7.1 and/or HSVrantes alone or in combination into established EL4 tumors led to complete tumor regression in injected tumors as well as in nontransduced contralaterally implanted tumor, whereas control tumors or tumors injected with HSVlac expressing beta-galactosidase did not regress. Maximal protection was achieved with combined injection of HSVB7.1 and HSVrantes; mice showing tumor regression were resistant to rechallenge with parental EL4 cells, and tumor cell-specific cytolytic T-cell activity was observed in mice demonstrating regression. HSV amplicon-mediated delivery of immune effector molecules may represent a useful strategy for immunotherapy in the setting of pre-existing tumor.     

Localization of tumor-reactive lymph node lymphocytes in vivo using radiolabeled monoclonal antibody

BACKGROUND: Lymph node lymphocytes vary in their responsiveness to tumor. A technique has been developed that uses radiolabeled monoclonal antibody (MoAb) against the tumor-associated mucin, TAG-72, and a gamma-detecting probe by which lymph nodes containing microscopic tumor and/or shed TAG-72 can be identified in vivo. The immunologic characteristics of these lymph nodes were examined. METHODS: Patients with colon cancer received 125I-labeled MoAb CC49 by intravenous injection preoperatively. During laparotomy lymph nodes that appeared normal on inspection and palpation but which contained radiolabeled MoAb were identified using a hand-held gamma-detecting probe. These lymph nodes and other lymph node and tumor specimens were resected for analysis. RESULTS: Lymph nodes identified by the probe were found by immunohistochemical studies to contain microscopic tumor and/or shed antigen associated with germinal centers. They were characterized by greater CD4+:CD8+ ratios, rates of expansion, and cytolytic activity compared with lymphocytes from lymph nodes with macroscopic tumor, noninvolved lymph nodes, and tumors. All lymph node lymphocytes identified by the probe demonstrated significant proliferative responses to autologous tumor and, in contrast to lymphocytes from noninvolved lymph nodes, significant proliferative responses to allogeneic TAG-72+ tumor cells and to soluble TAG-72+ mucin. CONCLUSIONS: By locating lymph nodes with microscopic tumor and/or shed antigen, the use of radiolabeled MoAb in vivo can be used to reproducibly identify tumor-reactive lymph node lymphocytes. This technique may be useful in identifying cells for use in adoptive immunotherapy programs and in studying the regulation of immune responses in vivo.     

Human tumor antigens recognized by T cells

The ability of tumor-reactive T cells to mediate in vivo tumor regression has been demonstrated in murine tumor models and by the clinical responses to adoptive immunotherapy with tumor-infiltrating lymphocytes isolated from human melanomas. Investigations carried out in the past several years have resulted in the isolation of a number of the genes encoding antigens recognized by melanoma-reactive T cells. The ability of these products to serve as tumor regression antigens has now begun to be evaluated in clinical vaccine trials.     

Intravenous RMP-7 increases delivery of ganciclovir into rat brain tumors and enhances the effects of herpes simplex virus thymidine kinase gene therapy

Herpes simplex virus thymidine kinase (HSV-tk) gene therapy for brain tumors depends on ganciclovir (GCV) and its transport across the blood-brain tumor barrier (BBTB). We examined whether RMP-7, the bradykinin analog and potent BBTB permeabilizer, could enhance the efficacy of GCV treatment of brain tumors by increasing the BBTB delivery of GCV. In vitro, a significant bystander cytocidal effect of GCV was shown in mixed HSV-tk-transduced (HSV-tk+) and control vector-transduced (HSV-tk-) C6 glioma cultures. A dose-dependent cytotoxic effect of GCV on untransformed C6 cells was also shown. In vivo, rats with 100% HSV-tk+ or 100% HSV-tk- intracerebral C6 gliomas were treated for 7 days with intravenous infusions of GCV alone or with GCV and RMP-7 (2.5 microg/kg/day). The growth of HSV-tk+ and HSV-tk- gliomas decreased with increasing doses of GCV. A high dosage (100 mg of GCV/kg/day) eradicated all HSV-tk- and HSV-tk+ tumors. An intermediate dosage (5 mg of GCV/kg/day) reduced the growth of HSV-tk- gliomas by 42% if given alone, and by 88% in combination with RMP-7. A low dosage (0.5 mg of GCV/kg/day) in combination with RMP-7 enhanced the regression of HSV-tk+ gliomas by 87% compared with GCV alone. Low-dose GCV was ineffective in HSV-tk- tumors. RMP-7 increased [3H] GCV tumoral uptake by 2.6- and 1.7-fold in the tumor center and periphery, respectively. We conclude that RMP-7 could be an important adjunctive treatment for suicide gene therapy of brain tumors, while an RMP-7/GCV combination may also have a significant antitumor effect in untransfected gliomas.     

Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein

We have previously reported that bryostation 1 (Bryo 1) induces differentiation of chronic lymphocytic leukemia (CLL) in vitro to a hairy cell (HC) stage. This study tests the hypothesis that Bryo 1-differentiated CLL cells are more susceptible to 2-chlorodeoxyadenosine (2-CdA) than parent CLL cells. A recently established EBV-negative CLL line (WSU-CLL) from a patient resistant to chemotherapy including fludarabine was used to test this hypothesis. Both Bryo 1 (10-1000 nM) and 2-CdA (5.6-22.4 microM) exhibited a dose-dependent growth inhibitory effect on the WSU-CLL cell line. In vitro, the sequential exposure to Bryo 1 (100 nM for 72 h) followed by 2-CdA (11.2 microM) resulted in significantly higher rates of growth inhibition than either agent alone. Changes in immunophenotype, enzymes, lipids, proteins, and the DNA of WSU-CLL cells were studied before and after Bryo 1 treatment. Bryo 1 induced a positive tartrate-resistant acid phosphatase reaction and two important markers, CD11c and CD25, after 72 h of culture, confirming the differentiation of CLL to HC. The Fourier transformation infrared spectroscopic analysis showed that the amount of membrane lipids significantly increased in Bryo 1-treated cells compared to controls after 24 h, whereas the protein content, as well as the DNA content, decreased. This finding supports the change of CLL to HC. To evaluate the in vivo efficacy of Bryo 1 and 2-CdA, we used a xenograft model of CLL in WSU-CLL-bearing mice with severe combined immune deficiency. s.c. tumors were developed by injection of 10(7) WSU-CLL cells, and fragments were then transplanted into a new batch of severe combined immunodeficient mice. Bryo 1 and 2-CdA at the maximum tolerated doses (75 micrograms/kg i.p. and 30 mg/kg s.c., respectively) were administered to the mice at different combinations and schedules. The survival in days, the tumor growth inhibition ratio, the tumor growth delay, and the log10 kill of the mice treated with Bryo 1 followed by 2-CdA were significantly better than the control and other groups. We conclude that the sequential treatment with Bryo 1 followed by 2-CdA resulted in higher antitumor activity and improved animal survival.     

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.     

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.     

Differences in the recognition of tumor-specific CD8+ T cells derived from solid tumor, metastatic lymph nodes and ascites in patients with gastric cancer

We established gastric cancer-specific CD8+ T-cell (T(CD8+)) lines derived from different lymphocyte sources in the same patients by repeated stimulation with mitomycin-C-treated autologous tumor cells with low-dose interleukin-2, and we compared recognition patterns among the T(CD8+) derived from solid tumor, lymph node metastasis and ascites in the same patient (n = 3) to determine their similarities and differences for therapeutic purposes. We confirmed that gastric cancer-specific T(CD8+) lines can be isolated, in a MHC class I-restricted manner, from solid tumors, metastatic lymph nodes and malignant ascites. T(CD8+) lines derived from tumor-infiltrating lymphocytes (TIL) in solid tumor recognized autologous tumor cells derived from solid tumor, but not autologous tumor cells derived from ascites or metastatic lymph node, while T(CD8+) lines derived from tumor-associated lymphocytes (TAL) in malignant ascites recognized autologous tumor cells derived from ascites, but not tumor cells from solid tumor or metastatic lymph node. Furthermore, T(CD8+) lines derived from regional lymph node lymphocytes (RLNL) recognized autologous tumor cells derived from metastatic lymph nodes, but not tumor cells derived from ascites. No significant differences were seen in MHC class I expression among the tumors derived from solid tumor, lymph node metastasis or ascites in the same patient. This suggests that there are differences of recognition patterns among the TILs, TALs and RLNLs in the same patient and that it is important to consider the source of lymphocytes, e.g., a combination of TILs, TALs and RLNLs, for adoptive immunotherapy in gastric cancer patients.