Phase II evaluation of gefitinib in patients with newly diagnosed Grade 4 astrocytoma: Mayo/North Central Cancer Treatment Group Study N0074.

PURPOSE: Amplification of the epidermal growth factor receptor (EGFR) gene represents one of the most frequent gene alterations in glioblastoma (GBM). In the current study, we evaluated gefitinib, a potent EGFR inhibitor, in the treatment of adults with newly diagnosed GBM. METHODS AND MATERIALS: Ninety-eight patients (96 evaluable) were accrued between May 18, 2001, and August 2, 2002. All were newly diagnosed GBM patients who were clinically and radiographically stable/improved after radiation treatment (enrollment within 5 weeks of radiation completion). No prior chemotherapy was permitted. EGFR amplification/mutation, as assessed by fluorescence in situ hybridization and immunohistochemistry, was not required for treatment with gefitinib but was studied when tissues were available. Gefitinib was administered at 500 mg each day; for patients receiving dexamethasone or enzyme-inducing (CYP3A4) agents, dose was escalated to a maximum of 1,000 mg QD. Treatment cycles were repeated at 4-week intervals with brain magnetic resonance imaging at 8-week intervals. RESULTS: Overall survival (OS; calculated from time of initial surgery) at 1 year (primary end point) with gefitinib was 54.2%, which was not statistically different compared with that of historical control population (48.9%, data from three previous Phase III North Central Cancer Treatment Group studies of newly diagnosed GBM patients). Progression-free survival (PFS) at 1 year post-RT (16.7%) was also not significantly different to that of historical controls (30.3%). Clinical outcome was not affected by EGFR status (amplification or vIII mutation). Fatigue (41%), rash (62%), and loose stools (58%) constituted the most frequent adverse events, the majority of these being limited to Grade 1/2. Of note, the occurrence of drug-related adverse effects, such as loose stools was associated with improved OS. CONCLUSIONS: In our evaluation of nearly 100 patients with newly diagnosed GBM, treatment with adjuvant gefitinib post-radiation was not associated with significant improvement in OS or PFS. However, patients who experienced gefitinib-associated adverse effects (rash/diarrhea) did demonstrate improved OS.

T cell-mediated tumor rejection displays diverse dependence upon perforin and IFN-gamma mechanisms that cannot be predicted from in vitro T cell characteristics.

Experimental pulmonary metastases have been successfully treated by adoptive transfer of tumor-sensitized T cells from perforin knockout (KO) or Fas/APO-1 ligand(KO) mice, suggesting a prominent role for secretion of cytokines such as IFN-gamma. In the present study we confirmed that rejection of established methylcholanthrene-205 (MCA-205) pulmonary metastases displayed a requirement for T cell IFN-gamma expression. However, this requirement could be obviated by transferring larger numbers of tumor-sensitized IFN-gamma (KO) T cells or by immunosensitizing sublethal irradiation (500 rad) of the host before adoptive therapy. Extrapulmonary tumors (MCA-205 s.c. and intracranial) that required adjunct sublethal irradiation for treatment efficacy also displayed no requirement for host or T cell expression of IFN-gamma. Nonetheless, rejection of MCA-205 s.c. tumors and i.p. EL-4 tumors, but not MCA-205 pulmonary or intracranial tumors, displayed a significant requirement for T cell perforin expression (i.e., CTL participation). The capacity of T cells to lyse tumor targets and secrete IFN-gamma in vitro before adoptive transfer was nonpredictive of the roles of these activities in subsequent tumor rejection. Adoptive therapy studies employing KO mice are therefore indispensable for revealing a diversity of tumor rejection mechanisms that may lack in vitro correlation due to delays in their induction. Seemingly contradictory KO data from different studies are reconciled by the capacity of anti-tumor T cells to rely on alternative mechanisms when treated in larger numbers, the variable participation of CTL at different anatomic locations of tumor, and the apparent capacity of sublethal irradiation to provide a therapeutic alternative to host or T cell IFN-gamma production.

Systemic adoptive T-cell immunotherapy in recurrent and metastatic carcinoma of the head and neck: a phase 1 study.

OBJECTIVE: To evaluate the feasibility and toxic effects of systemic adoptive T-cell immunotherapy in patients with unresectable squamous cell carcinoma of the head and neck (SCCHN). DESIGN: Nonrandomized phase 1 clinical trial. SETTING: Academic tertiary care hospital. PATIENTS: Between April 1, 1996, and September 30, 1998, 17 patients with confirmed recurrent and metastatic SCC of the upper aerodigestive tract were enrolled. Two patients did not receive T cells because of poor vaccine response. Fifteen patients were successfully treated with T-cell immunotherapy. INTERVENTION: Patients were vaccinated on the thigh with irradiated autologous tumor cells admixed with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by 3 additional daily injections of GM-CSF at the vaccination site. Eight to 10 days later, tumor cell vaccine-draining inguinal lymph nodes were resected, and lymph node lymphocytes were activated with staphylococcal enterotoxin A and expanded in interleukin 2 in vitro. Resulting cultured cells were infused into patients peripherally on an outpatient basis. RESULTS: Toxic effects of infusion were limited to grade 2 reactions in 3 of 16 treatments. One patient required overnight hospitalization for fever and emesis. Median cell expansion was 37 times (range, 4-416 times), and median cell dose was 7.5 x 10(9) (range, 1.3 x 10(8) to 4.2 x 10(10)). Infused cells were predominantly CD3+ (>97%), being a mixture of CD4+ and CD8+ cells. Three patients demonstrated stabilization of previously progressive disease. Two patients experienced favorable clinical courses after adoptive T-cell transfer, including 1 patient with no evidence of disease 4 years after surgical resection of a vertebral body metastasis. CONCLUSIONS: Adoptive immunotherapy is a technically feasible and safe treatment with low toxicity and may demonstrate therapeutic activity in patients with unresectable SCCHN.

Expression of major histocompatibility complex antigens in squamous cell carcinomas of the head and neck: effects of interferon gene transfer.

The effect of retroviral-mediated interferon-gamma (IFN-gamma) gene transfer on major histocompatibility complex (MHC) class I and II antigen expression was investigated in 13 head and neck squamous carcinoma cell lines. Six cell lines exhibited increased MHC class I expression, and 10 exhibited increased MHC class II expression after IFN-gamma gene transfer. Differences in MHC antigen expression between parental and transduced cell lines were significant (P = 0. 002) only for cell lines that upregulated MHC class II expression. After incubation in medium containing 100 U/mL recombinant IFN-gamma, or in medium from IFN-gamma retrovirus-transduced NIH 3T3 cells, 12 cell lines significantly upregulated MHC class I expression, and 9 significantly upregulated MHC class II expression. Only cell lines that exhibited increased MHC class II expression after retroviral transduction also upregulated class II expression with exogenous IFN-gamma treatment. Thus some head and neck squamous carcinoma cell lines can upregulate MHC class I and II expression after exogenous application of either IFN-gamma or IFN-gamma retroviral transduction. These are promising findings for head and neck cancer immunotherapy and gene therapy.

Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules.

The adoptive transfer of tumor-draining lymph node (LN) T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56lck and p59fyn and proteins of the NF-kappaB family were analyzed in tumor-draining LN T cells. The levels of p56lck and p59fyn were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56lck and p59fyn and protein tyrosine kinase activity increased. Interestingly, the levels of p56lck, p59fyn, and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear kappaB DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-kappaB. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function.

Systemic T cell adoptive immunotherapy of malignant gliomas.

OBJECT: To determine the feasibility, toxicity, and potential therapeutic benefits of systemic adoptive immunotherapy, 10 patients with progressive primary or recurrent malignant glioma received this treatment. Adoptive immunotherapy, the transfer of immune T lymphocytes, is capable of mediating the regression of experimental brain tumors in animal models. In animal models, lymph nodes (LNs) that drain the tumor vaccine site are a rich source of tumor-immune T cells. METHODS: In this clinical study, patients were inoculated intradermally with irradiated autologous tumor cells and granulocyte macrophage-colony stimulating factor as an adjuvant. Cells from draining inguinal LNs, surgically resected 7 days after vaccination, were stimulated sequentially with staphylococcal enterotoxin A and anti-CD3, and a low dose of interleukin-2 (60 IU/ml) was used to expand the stimulated cells. The maximum cell proliferation was 350-fold over 10 days of culture. The activated cells were virtually all T cells consisting of various proportions of CD4 and CD8 cells. These cells were given to patients by intravenous infusion at doses ranging from 9 x 10(8) to 1.5 x 10(11). There were no Grade 3 or 4 toxicities associated with the treatment. Following T-cell transfer therapy, radiographic regression that lasted at least 6 months was demonstrated in two patients with recurrent tumors. One patient demonstrated stable disease that has lasted for more than 17 months. The remaining patients had progressive disease; however, four of the eight patients with recurrent tumor remain alive more than 1 year after surgery for recurrence. Three patients required intervention with corticosteroid agents or additional surgery approximately 1 month following cell transfer. CONCLUSIONS: These intriguing clinical observations warrant further trials to determine whether this approach can provide therapeutic benefits and improve survival.

Chronic lymphocytic leukemia: a brief review.

Chronic lymphocytic leukemia (CLL), the most common type of leukemia, is often discovered incidentally when a complete blood count is performed during a routine examination. This disease varies in its course, eventually requiring treatment in most patients, but remaining indolent without therapy in a lucky minority. This paper reviews the pathology, diagnosis, and treatment of CLL.

Targeted gene transfer for adenocarcinoma using a combination of tumor-specific antibody and tissue-specific promoter.

We have developed a highly specific gene transfer method for adenocarcinoma using a monoclonal antibody against tumor-specific antigen coupled with a plasmid containing the carcinoembryonic antigen (CEA)-specific promoter. The chimeric CEA promoter (CC promoter), which contained an enhancer from the immediate early gene of cytomegalovirus and the CEA promoter, achieved 4- to 5-fold higher transgene expression in CEA-producing cells than the original CEA promoter while maintaining CEA specificity. Furthermore, a complex of a monoclonal antibody against Lewis Y antigen (LYA), the CC promoter-containing plasmid and cationic liposomes (DOTAP) achieved specific gene expression in CEA-producing and LYA-positive adenocarcinoma cell lines that was 200-fold more efficient than in CEA-non-producing and LYA-negative cell lines during a short in vitro incubation. This strategy may be applicable for clinical gene therapy.

Tumor immunology.

Malignant tumors express antigens that may stimulate and serve as targets for antitumor immunity. Virally induced tumors usually contain integrated proviral genomes in theircellulargenomes and often express viral genome-encoded proteins that may stimulate specific host immune responses. Antigens unique to individual tumors that stimulate specific rejection of transplanted tumors have been demonstrated only in experimental animals. Other tumor antigens that potentially can stimulate immune responses are shared by different tumors. These include products of mutated or rearranged oncogenes or tumor-suppressor genes. Tumors may also overexpress tissue differentiation antigens or embryonic antigens, which also have the potential to be recognized by the immune system. The recent identification of tumor antigens recognized by cytotoxic T cells opens up new possibilities for constructing chemically defined antigens for specific immunotherapy. Treatment of malignant tumors in humans by immunologic approaches, although theoretically attractive, has not yet succeeded on a large scale. Important progress in immunotherapy of cancer is emerging with several different treatment modalities.

Cytokines as an adjuvant to tumor vaccines: efficacy of local methods of delivery.

BACKGROUND: We examined alternative methods of delivering cytokines as an adjunct for priming lymph node (LN) cells draining sites of vaccine inoculation for the purpose of generating immune cells for adoptive immunotherapy. METHODS: Using syngeneic murine tumors we examined the ability of IL-2, IL-4, or GM-CSF delivered locally to a site of tumor inoculum to induce antitumor reactive draining LN cells. Mice were inoculated subcutaneously with tumor cells transduced to secrete cytokine; tumor cells admixed with fibroblasts transduced to secrete cytokine; or intralesional inoculation of cytokine in established tumor to induce sensitized LN cells capable of mediating tumor regression in adoptive transfer. RESULTS: Both IL-4 and GM-CSF cytokines were effective in enhancing the antitumor reactivity of vaccine-primed LN cells compared to IL-2, which was ineffective. The local delivery of GM-CSF by autocrine or paracrine secretion of genetically engineered cells, as well as direct intratumoral delivery was capable of upregulating LN sensitization compared to systemic administration, which did not. CONCLUSIONS: The local delivery of GM-CSF as an adjuvant for tumor vaccination can be accomplished by various methods, including direct injection, which avoids the need for gene transfer.

Monocyte chemoattractant protein inhibits the generation of tumor-reactive T cells.

The adoptive transfer of tumor-sensitized T cells can eradicate disseminated malignancy in murine animal models. T cells must be sensitized to tumor antigens in vivo to acquire antitumor reactivity. T-cell sensitization has been demonstrated to be dependent on host antigen-presenting cells. Tumor-associated macrophages are a heterogeneous population of cells that may have both inhibitory and stimulatory influences on the sensitization of naive T cells. Here we demonstrate that a weakly immunogenic tumor, the MCA 205 sarcoma, produces substantial amounts of murine monocyte chemoattractant protein 1 (MCP-1). Neutralization of MCP-1 during in vivo T-cell sensitization resulted in T cells that possessed enhanced therapeutic activity against established pulmonary metastases. These T cells sensitized during MCP-1 depletion also exhibited enhanced production of IFN-gamma upon recognition of tumor targets. These results demonstrate that MCP-1 can have a potent inhibitory influence on the development of tumor-reactive T cells.

Treatment of subcutaneous tumor with adoptively transferred T cells.

Adoptive immunotherapy with T cells directed at tumor antigens has been demonstrated to result in the regression of malignant tumors in humans. These encouraging results have prompted the further exploration of parameters necessary to treat tumor in various locations in animal models. We have demonstrated that T cells that are sensitized to tumor antigens and then ex vivo cultured are capable of eradicating pulmonary metastases. In this report, we demonstrate that these T cells are capable of eliminating subcutaneous tumor deposits. Critical to the successful treatment of subcutaneous tumor was treatment with a large number of adoptively transferred T cells and pretreatment of the mice with irradiation. The transfer of T cells from tumor-bearing mice into irradiated mice failed to inhibit the therapeutic effect of ex vivo cultured T cells, suggesting that irradiation was not acting only as an immunosuppressant. Irradiation resulted in increased expression of the F4/80 and 33D1 epitopes on antigen-presenting cells within the tumor. The therapeutic effect of the adoptively transferred T cells was eliminated if either CD4 cells or CD8 cells were depleted. Naive T cells subjected to the same culture conditions were completely ineffective at eliminating tumor. These results demonstrate that adoptively transferred T cells derived from tumor-bearing hosts can treat subcutaneous tumor deposits, and they define the conditions necessary for the elimination of tumor in this location.

Concurrent induction of CD4+ and CD8+ T cells during tumor growth with antitumor reactivity in adoptive immunotherapy.

We have previously reported that the poorly immunogenic D5 melanoma transduced to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) will elicit immunity in tumor-draining lymph node (TDLN) cells after subcutaneous inoculation. After in vitro activation with anti-CD3 and interleukin-2 (IL-2), these cells acquire in vivo antitumor reactivity to wild-type tumor in the adoptive immunotherapy of pulmonary metastases. Using monoclonal antibodies, depletion of CD4+ or CD8+ T cells immediately after the adoptive transfer of activated TDLN cells revealed that both subsets could mediate the regression of tumor in the absence of exogenous IL-2 administration. CD8+ cells were more potent than CD4+ cells in mediating tumor regression on a per cell basis. We found that the exogenous administration of IL-2 enhanced the antitumor efficacy of CD4+ T cells. Purified CD4+ and CD8+ TDLN cells that were activated separately in culture released GM-CSF and interferon-gamma in response to wild-type tumor in vitro and mediated tumor regression in vivo. Last, the induction of either immune CD4+ or CD8+ T-cell subset during growth of the GM-CSF-secreting melanoma was found to be unaffected by the depletion of the alternate T-cell subset before tumor inoculation. These findings demonstrate that both CD4+ and CD8+ T cells can independently acquire therapeutic reactivity and presumably recognize two separate epitopes involved in tumor rejection.

Secretion of biologically active superantigens by mammalian cells.

The genetic modification of tumor cells to secrete immune regulatory molecules can elicit a potent antitumor immune response. Bacterial superantigens are among the most potent T cell mitogens. Activation of tumor-sensitized T cells by bacterial superantigens can lead to immune effector cells with potent and specific in vivo antitumor activity. Retrovirus vectors encoding for the bacterial superantigens SEA and SEC2 were constructed, and recombinant retrovirus stocks were generated. SEA and SEC2 could be detected in the culture supernatant of tumor cells after a single exposure to retrovirus. Molecular analysis of the genetically modified cells revealed intact proviral DNA and abundant vector-derived superantigen RNA. Biologic activity was apparent for both superantigens. Secretion of biologically active superantigen by mammalian cells has not been reported previously, and this will enable investigating the potential for superantigen gene therapy.

Tumor reactivity of immune T cells in short-term culture.

The adoptive transfer of immune T cells is capable of mediating the regression of established neoplasms in a variety of animal tumor models. The antitumor activity is invariably proportional to the number of cells transferred, thus methods to expand immune cell number while maintaining therapeutic efficacy have been extensively investigated. Here we demonstrate that a short-term culture of immune T cells can amplify the T cell number and enhance the therapeutic reactivity against established pulmonary tumor, while maintaining immunological specificity. In contrast, the therapeutic reactivity of immune T cells against established subcutaneous tumor is diminished by short-term culture. While cultured immune T cells are not cytotoxic in a 4-h Cr-release assay, they do specifically secrete interferon gamma upon stimulation with tumor cells. T cells cultured after a single exposure to tumor are even more active against pulmonary tumor than T cells cultured from mice immunized repeatedly. This culture system can rapidly induce T cell proliferation and differentiation into mature effector cells, and the resulting cells demonstrate an enhanced ability to treat visceral metastases, but a decreased ability to treat subcutaneous tumor. Thus T cells cultured after a single exposure to tumor represent an ideal population of cells for use in human adoptive immunotherapy trials.

Isolation based on L-selectin expression of immune effector T cells derived from tumor-draining lymph nodes.

The ability to generate a large number of tumor-reactive T lymphocytes is the most critical requirement for adoptive immunotherapy. Our laboratory has previously demonstrated that cells from tumor-draining lymph nodes (LNs) are an excellent source of tumor-reactive T lymphocytes. After activation with anti-CD3, these cells readily proliferate in low concentrations of interleukin 2 and acquire effector functions. The adoptive transfer of these cells is capable of mediating the regression of tumors established in the lung as well as in the brain. Here, we analyzed several adhesion molecules on the tumor-draining LN T cells and separated them based on L-selectin expression. The homing receptor L-selectin mediates adhesion to the luminal surface of specialized high endothelial venules, thus regulating lymphocyte recirculation through peripheral LNs. In response to progressive tumor growth, a small population of draining LN T cells down-regulated L-selectin and increased the expression of CD44 and lymphocyte function-associated antigen 1. In adoptive immunotherapy, purified T cells with low L-selectin (L-selectin-) expression constituted all the in vivo antitumor reactivity, whereas isolated high L-selectin (L-selectin+) cells were ineffective. Furthermore, reverse transcription-PCR analysis revealed that L-selectin- cells expressed interleukin 2, IFN-gamma and tumor necrosis factor alpha mRNA upon in vitro stimulation with specific tumor cells. These results suggest that highly potent immune T cells can be isolated based on their pattern of adhesion molecule expression. The ability of the immune effector cells to transcribe cytokine genes when stimulated with tumor cells provides a basis for identifying similar cells for adoptive immunotherapy of cancer in humans.

Tubulogenesis from isolated single cells of adult mammalian kidney: clonal analysis with a recombinant retrovirus.

The adult mammalian kidney tubule epithelium exists in a relatively dormant, slowly replicative state but has a large potential for regenerative morphogenesis following severe ischemic or toxic injury. Under selective serum-free growth conditions, which included epidermal growth factor and retinoic acid, a subpopulation of renal proximal tubule cells isolated from adult rabbit kidney were grown in cell culture. These cells possessed two important characteristics: 1) an ability to differentiate morphogenically into tubule structures when grown in three-dimensional collagen gels and 2) a high capacity for self-renewal, since cell lineage analysis with a recombinant retrovirus demonstrated that in vitro tubulogenesis arose from clonal expansion of a single cell. Thus individual cells in the adult kidney have retained the ability for kidney tubulogenesis in vitro.

Lack of plasminogen activator inhibitor-1 effect in a transgenic mouse model of metastatic melanoma.

Tumor cell invasion and metastasis is a complex, multistep process that is postulated to require degradation of extracellular matrix at several steps. Urokinase-type plasminogen activator (uPA) is expressed on the cell surface of B16 murine melanoma cells and is thought to contribute to the pericellular proteolysis necessary for tumor cell migration. In vitro modification of B16 melanoma cell surface uPA activity has been shown to alter the invasive and metastatic potential of these murine melanoma cells in vivo. Plasminogen activator inhibitor-1 (PAI-1), a rapid inhibitor of both uPA and tissue-type plasminogen activator (tPA) is the major physiologic regulator of plasminogen activator activity. To test the role of host PAI-1 in the invasive and metastatic capacity of B16 melanoma cells we analyzed local tumor growth and pulmonary metastasis in transgenic mice engineered to overexpress murine PAI-1 in multiple tissues including lung, and in mice completely deficient in PAI-1. No significant difference in the number of pulmonary metastases was observed after intravenous inoculation of tumor cells into PAI-1-overexpressing and PAI-1-deficient mice when compared with wild-type controls. Similarly, in a spontaneous metastasis model, PAI-1-overexpressing and PAI-1-deficient mice demonstrated no difference in primary tumor size or overall survival. These data demonstrate that wide variations of host PAI-1 expression, from complete absence to marked overexpression, does not significantly influence the metastatic potential of B16 melanoma cells in a murine model.

Diverse manifestations of tumorigenicity and immunogenicity displayed by the poorly immunogenic B16-BL6 melanoma transduced with cytokine genes.

We evaluated the in vivo response to the poorly immunogenic B16-BL6 (BL6) murine melanoma genetically altered to secrete interleukin-2 (IL-2), IL-4, interferon gamma (IFN gamma) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). Three parameters were evaluated: (1) tumorigenicity, (2) vaccination of naive animals, and (3) assessment of antitumor reactivity of T cells derived from tumor-draining lymph nodes (TDLN). Secretion of IL-2 abrogated the tumorigenicity of BL6, while IFN gamma and IL-4 partially reduced tumorigenicity, and GM-CSF had no effect. Protective immunity to wild-type tumor challenge could not be achieved by vaccination with irradiated cytokine-secreting tumors, although IL-2 and IL-4 secretion appeared to retard the growth of the challenge inoculum significantly. An alternative method to evaluate the immunogenicity of the cytokine-secreting tumors was to measure the ability of T cells obtained from TDLN to mediate regression of wild-type tumor in adoptive immunotherapy. Neither IL-2 nor IFN gamma secretion resulted in the induction of immune T cells. By contrast, GM-CSF and IL-4 secretion were found to induce immune T cells in the TDLN with GM-CSF being superior to IL-4. The combined secretion of GM-CSF and IL-4 did not lead to enhanced induction of immune T cells. GM-CSF secretion was found to upregulate B7-1 expression in TDLN, consistent with an increase in the population of antigen-presenting cells. These studies demonstrated that reduced tumorigenicity by cytokine secretion did not correlate with increased immunogenicity. With the cytokines examined, there was limited capability of developing protective immunity against the BL6 tumor. Nevertheless, GM-CSF and IL-4 secretion significantly enhanced T cell immune reactivity to the poorly immunogenic BL6 tumor.