A specific vaccine effective against stage I and stage II malignant disease in guinea pigs effect of variations in preparations and storage

Summary Guinea pigs, each with an established, syngeneic dermal line 10 tumor and lymph node metastases, were immunized by intradermal injection of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG. Immunization eradicated 7- or 10-day-old dermal tumors (about 10 or 12 mm in diameter, respectively) and prevented growth of microscopic lymph node metastases. Fourteen-day-old dermal tumors (about 15 mm in diameter) were not rejected by immunization.Guinea pigs with stage II disease (21-day-old dermal tumors and palpable metastases in the first draining lymph node) were treated by excision of the dermal tumor and the first draining lymph node, and by specific immunization. This treatment eliminated tumor cells remaining in the second draining lymph nodes. The surgical treatment alone was not curative, palpable metastases in the second draining lymph nodes progressed and the animals died (some with visible lung metastases).Emulsions containing killed BCG were good adjuvants even after prolonged storage at 4° C, but lost most of their adjuvant activity after autoclaving or freezing.     

Eradication of microscopic hepatic metastases by active specific immunization

Strain-2 guinea pigs, each with microscopic deposits of line 10 hepatocarcinoma in the liver, were treated by ID immunization with a mixture of irradiated tumor cells and an oil-in-water emulsion containing cell walls of Mycobacterium bovis strain Bacillus Calmette-Guérin (BCG CWE). Injection of line 10 hepatoma cells into the hepatic portal vein led to the development of tumor foci in the liver, metastasis in the hepatic lymph node, malignant ascites, and death. Active immunization using irradiated line 10 cells and BCG CWE was effective therapy when administered 1, 7, and 14 days after intraportal injection of line 10 cells. Effective immunization required both irradiated line 10 tumor cells and the BCG cell wall emulsion. Immunization with BCG CWE admixed with irradiated line 1 tumor cells, a hepatoma antigenically distinct from line 10, did not prevent outgrowth of line 10 deposits in the liver. Animals rendered free of disease could reject a challenge of line 10 tumor cells but not of line 1 tumor cells.     

Monocyte chemoattractant protein-1 (MCP-1) gene transduction: an effective tumor vaccine strategy for non-intracranial tumors

Recently, there has been renewed interest in the concept of tumor vaccines using genetically engineered tumor cells expressing a variety of cytokines to increase their immunogenicity. Human MCP-1 (JE) is a potent chemoattractant and activator of monocytes and T lymphocytes and thus a good candidate gene for a tumor vaccine. We therefore evaluated the efficacy of vaccines consisting of irradiated tumor cells transduced with the murine MCP-1 gene in the syngeneic 9L gliosarcoma brain tumor model. 9L cell lines stably expressing murine MCP-1 (9L-JE) and control cell lines expressing neomycin 3 phosphotransferase (9L-Neo) were generated by infection with a Moloney murine leukemia retroviral vector. Fisher 344 rats were immunized with intradermal injections of 5×10⁵ or 2×10⁶ irradiated (5000 cGy) 9L-JE, 9L-Neo, and wild-type 9L (9L-WT) cells. Two weeks later immunized an non-immunized animals were challenged with varyious doses of intradermal (5×10⁶–5×10⁷) or intracerebral (2×10⁴–5×10⁵) 9L-WT cells. Intradermal tumors grew in all non-immunized animals. No tumors grew in animals immunized with irradiated 9L-JE or 9L-Neo cells and challenged with inocula of fewer than 5×10⁵ 9L-WT cells. With higher inocula up to 10⁷ cells, tumors appeared in all the animals. Tumors in animals immunized with 9L-JE were always smaller than tumors in the other groups. In addition, only the 9L-JE vaccine protected against tumor inocula of 5×10⁷ cells. Thus vaccination with MCP-1-expressing cells was able to protect animals against at least a 100-fold larger number of challenge tumor cells than vaccination with control cells. In contrast to studies with intradermal tumors, immunization with 9L-JE and 9L-Neo produced only minimal protection against intracerebral tumors. There was no significant difference between the 9L-JE and 9L-Neo vaccines in intracerebral challenge. This study suggests that tumor vaccines expressing cytokine genes such as MCP-1 can increase the antitumor response. However, the protective effect of these vaccines appears to be largely limited to intradermal tumors rather than intracerebral tumors.     

The effect of gamma-irradiation on the toxicity of malathion in V79 Chinese hamster cells and Molt-4 human lymphocytes.

There is growing interest in the irradiation of food and agricultural products for insect disinfestation, sprout inhibition, delayed ripening and the reduction of microbiological loads. Extensive research has been done on this process, and irradiation to a maximum dose of 10 kGy is recognized as safe by national and international regulatory agencies. The question has been raised, however, whether irradiation of pesticide residues might produce radiation products that were more toxic or less toxic than the original pesticide. To address this question, we observed the effects of 10 kGy of gamma-radiation on malathion as measured by sister-chromatid exchange (SCE), micronuclei formation, cell survival, growth rate and polyploid formation. We found no significant differences between the effects of irradiated and unirradiated malathion on any of these end-points. Polyploid formation was the most dramatic effect of both irradiated and control malathion on V79 Chinese hamster cells. Cell survival, polyploid formation and growth rate were slightly better in cells treated with irradiated malathion. In Molt-4 human lymphocyte cells, micronuclei formation was not affected by irradiated or unirradiated malathion. Compared to malathion alone, the lack of such biological effects indicates that none of the presumed radiation-induced breakdown products increased or decreased the endpoints studied. The number of SCE was consistently, but not significantly, higher in the cells treated with irradiated malathion. There were no significant differences in cell survival or micronucleus formation in the human lymphocyte cell line Molt-4 treated with irradiated or control malathion. Thus, the irradiation of the pesticide malathion to 10 kGy, a recommended upper dose for most food irradiations, does not significantly alter its toxicity in these in vitro systems.     

Inhibition of pulmonary metastases of B16 melanoma with irradiated tumor cells and BCG

Summary When the tumor-bearing leg of C57BL/6J mice was amputated 16 days after SC inoculation of 10⁶B16 melanoma cells, all the amputated mice died of pulmonary metastases. Transfer of lungs from the amputated to normal syngeneic mice revealed tumor cells in the lungs just after amputation. Repeated weekly injections of BCG and irradiated tumor cells, beginning 24 h after amputation of the tumor-bearing limb, prolonged the survival only of mice presensitized to BCG. Injections of BCG or irradiated melanoma cells alone, of neuraminidase- and mitomycin C-treated tumor cells or of Levamisole had no effect, but injections of ConA-coated tumor cells slightly prolonged the survival of the amputated mice. Both BCG and B16 cells induced humoral and cell-mediated immunity but there was no cross-reactivity between BCG and B16 cells. Abbreviations used: ConA, concanavalin A; SC, subcutaneous; IP, intraperitoneal; IV, intravenous; ID, intradermal; IT, intratumoral; PBS, phosphate-buffered saline (0.01 M sodium phosphate, pH 7.1); VCN, Vibrio cholerae neuraminidase; HBSS, Hank's balanced salt solution; RPMIM, Roswell Park Memorial Institute medium     

Increased efficiency of immunotherapy using irradiated tumor cells

Summary The efficacy of irradiated tumor cells combined with chemotherapy or non-specific immunostimulation with complete Freund's adjuvant was tested in a model of minimal residual tumor-bearing syngeneic mice. Male C57BL/6J mice were innoculated in the right rear leg with live tumor cells from a methylcholanthrene induced fibrosarcoma. The tumor was resected when it reached 0.7 cm in diameter and animals were treated with doses of irradiated tumor cells (XTC) from the primary tumor ranging in number from 1×10³ to 9×10³. Best survival was noted using 5×10³ XTC combined with irradiated tumor cells of liver or pulmonary metastases origin, complete Freund's adjuvant or cytoxan. The combination of irradiated tumor cells of metastatic origin did not enhance the therapeutic effect of XTC alone. Freund's adjuvant was not of benefit in enhancing the efficacy of XTC. However, improved survival was noted when chemotherapy in the form of cytoxan was used to supplement XTC. Our data suggests that XTC is more efficacious as a mode of immunotherapy than are live tumor cells. The dose of XTC used is critical in determining its effect. Chemotherapy appears to enhance the benefit of XTC.Research fellow from Zhejiang Medical University, Hongzhou, ChinaSupported by the Brigham Surgical Group, Inc.     

Treatment of visceral tumor with BCG-tumor cell vaccine

Summary A vaccine containing irradiated tumor cells and BCG, previously shown to be effective against a contralateral, intradermal (ID) tumor inoculum, was also effective in suppressing or curing tumor deposited in the subcutaneous (SC) space or injected intravenously (IV).     

Irradiated tumor cell vaccine for treatment of an established glioma. I. Successful treatment with combined radiotherapy and cellular vaccination

Rats bearing a 5-day intracranial (i.c.) syngeneic glioma were treated with a subcutaneous (s.c.) vaccination consisting of irradiated glioma cells or a multimodality approach composed of radiotherapy plus s.c. vaccination. Vaccination of rats harboring a T9 glioma with 5 x 10(6) irradiated T9.F glioma cells (a clone derived from the T9 glioblastoma cell line) resulted in a marked enhancement of i.c. glioma growth and a significant decrease in survival. Histopathology of the tumors from vaccinated rats revealed a massive glioma composed of healthy tumor tissue lacking any marked inflammation, edema or hemorrhage. Analysis of the tumor-infiltrating mononuclear cells indicated that gliomas from vaccinated rats contained a 10-fold greater lymphoid infiltrate per milligram of tumor as compared to tumors from non-vaccinated rats, suggesting that the vaccination had induced immune cells to localize to the i.c. glioma. Combined treatment consisting of 15 Gy of whole head irradiation of the 5-day glioma followed by vaccination with T9.F cells resulted in a significant increase in survival compared to that of non-treated rats, 45% of which remained tumor-free. Microscopic evaluation in survivors of the tumor implantation site revealed the presence of hemosiderin-laden macrophages and other mononuclear cells, with the absence of tumor cells within the residual lesion. When survivors were challenged s.c. with viable T9.F glioma cells, a delayed-type hypersensitivity (DTH) reaction appeared at the challenge site. T cells purified from these rats proliferated and secreted Th(1)-associated cytokines when stimulated with irradiated T9.F glioma cells, and lysed T9.F target cells. In contrast, when these rats were challenged s.c. with the unrelated MadB106 adenocarcinoma, tumor formation was observed. These findings indicate that the treatment of an established i.c. glioma with a cellular vaccination alone may induce enhanced tumor growth; however, when the vaccination is combined with radiation therapy, the results are beneficial in terms of increased survival time or complete remission that is accompanied by the development of tumor-specific cellular immunity.     

The antimetastatic function of concomitant antitumor immunity. I. Host Ly-1+2+ effector T cells prevent the enumeration of metastatic tumor cells in a biological assay

A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2b X H-2d) host bearing a primary intradermal P815 tumor. The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2d) injected i.p. into mice and their mean survival time. It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2b serum and complement, in order to selectively destroy H-2bd host cells. This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2d P815 tumor cells when admixed with the tumor cells and injected i.p. into 800-rad irradiated test recipients. The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant.     

The improved effects of specific active immunotherapy on a rat fibrosarcoma by antitumor drugs

Summary We have tried to find out if the combination of a xenogenized tumor cell vaccine and antitumor drugs is able to induce a synergistic increase in the antitumor therapeutic effect. The degree of increase in the LTD₅₀ (50% lethal tumor dose) is expressed numerically, as a quantitative index designed to compare degrees of transplantation resistance to tumor cell challenge. A LTD₅₀ was achieved by an intradermal (i. d.) immunization with xenogenized tumor cells when challenged with tumor cells implanted intraperitoneally 2 weeks after the immunization: this LTD₅₀ value was 527 000 times higher than that of the non-immunized group. When we combined this type of immunization with appropriate doses of bleomycin (BLM) or cyclophosphamide (CY), which are able to augment antitumor immunity, the LTD₅₀ was 723 000–1190 000 times higher than that of the non-immunized group. This increase in the LTD₅₀ is definitely higher than that achieved by a single immunization with irradiated tumor cells (× 33 000) and combined with either BLM (× 93 000) or CY (×140 000). We also studied the therapeutic effect of a tumor cell vaccine combined with antitumor drugs BLM or CY in tumor-bearing rats. We observed a synergistic effect caused by BLM or CY after i. d. immunization with xenogenized tumor cells: this showed a significant increase when compared with the therapeutic effects obtained by chemotherapy alone (P <0.01). Nevertheless, there was no evidence that the above antitumor effects is superior to the effect achieved by irradiated tumor cells.     

Active specific immunotherapy with an autologous tumor cell vaccine in patients with resected non-small cell lung cancer

Eighteen postoperative patients with non-small cell lung cancer were actively immunized with a vaccine that included autologous cryopreserved irradiated tumor cells admixed with bacillus Calmette-Guerin. Patients received three weekly intradermal immunizations beginning 1-3 months after surgery (15 patients) or after completion of postoperative radiotherapy (3 patients). There was marked heterogeneity in the relative proportion of tumor cells versus host infiltrating cells within individual vaccines (range of percent tumor cells 7-75%). Five patients exhibited positive delayed cutaneous skin test reactivity (DCR) to autologous irradiated tumor cells prior to immunization, whereas 8 of 13 converted from skin test negative to positive. There were no correlations between DCR reactivity and in vitro lymphoproliferative responses to autologous tumor cells or to clinical outcomes, i.e., freedom from relapse. Possible explanations for the heterogeneity of the lung cancer vaccine and approaches for improving its immunogenicity are discussed.     

GM-CSF-secreting cancer immunotherapies: preclinical analysis of the mechanism of action

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A phase III comparison of methyl-CCNU + vincristine with or without BCG + allogeneic tumor cells in metastatic melanoma

Summary Sixty-two patients with metastatic malignant melanoma were randomized to treatment with either (a) methyl-CCNU (200 mg/m², PO every 8 weeks) plus vincristine (2 mg IV every 4 weeks), or (b) the same chemotherapy plus intradermal (ID) injections of irradiated (15,000 rads) allogeneic (fresh-frozen) melanoma cells (1–2×10⁸) admixed with BCG (Glaxo, 2–4.5×10⁶ organisms) every 2 weeks. Treatment cycles were repeated every 8 weeks until tumor progression. Seven (2 CR, 5 PR) objective remissions were noted among 31 patients (22.5%) treated with chemotherapy alone, whereas six (3 CR, 3 PR) objective remissions were noted among 31 patients (19%) treated with chemoimmunotherapy (P>0.05). The medians for remission duration (6 months) and survival (6.5 months) in the chemotherapy group did not differ significantly from the medians for remission duration (8 months) and survival (8 months) in the chemoimmunotherapy group. The patients manifested no unexpected toxicity. Hematologic toxicity was experienced by patients on both regimens; however, those receiving chemoimmunotherapy rebounded more quickly.     

TARC and RANTES enhance antitumor immunity induced by the GM-CSF-transduced tumor vaccine in a mouse tumor model

INTRODUCTION: Transduction of the granulocyte-macrophage colony stimulating factor (GM-CSF) gene into mouse tumor cells abrogates their tumorigenicity in vivo. Our previous report demonstrated that gene transduction of GM-CSF with either TARC or RANTES chemokines suppressed in vivo tumor formation. In this paper, we examined whether the addition of either recombinant TARC or RANTES proteins to irradiated GM-CSF-transduced tumor vaccine cells enhanced antitumor immunity against established mouse tumor models to examine its future clinical application. MATERIALS AND METHODS: Three million irradiated WEHI3B cells retrovirally transduced with murine GM-CSF cDNA in combination with either recombinant TARC or RANTES were subcutaneously inoculated into syngeneic WEHI3B-preestablished BALB/c mice. RESULTS: Vaccinations were well tolerated. Mice treated with GM-CSF-transduced cells and the chemokines demonstrated significantly longer survival than mice treated with GM-CSF-transduced cells alone. Splenocytes harvested from mice treated with the former vaccines produced higher levels of IL-4, IL-6, IFN-gamma, and TNF-alpha, suggesting enhanced innate and adaptive immunity. Immunohistochemical analysis of tumor sections after vaccination revealed a more significant contribution of CD4+ and CD8+ T cells to tumor repression in the combined vaccine groups than controls. CONCLUSIONS: TARC and RANTES enhance the immunological antitumor effect induced by GM-CSF in mouse WEHI3B tumor models and may be clinically useful.     

Lymphokine-activated tumor inhibition in mice. Ability of a nonapeptide of the human IL-1 beta to recruit anti-tumor reactivity in recipient mice

The ability of the VQGEESNDK synthetic peptide corresponding to fragment 163-171 of human IL-1 beta to trigger lymphokine-activated tumor inhibition (LATI) of a poorly immunogenic fibrosarcoma (CE-2) of BALB/c mice was compared to that of the whole IL-1 beta. Neither molecule inhibits in vitro proliferation of CE-2 cells. Administration at the tumor challenge site for 10 days of daily injections of 50 micrograms of peptide 163-171 induce a consistent, although limited, inhibition of tumor growth, whereas similar injections of 1 pg of IL-1 beta induced a more marked LATI. However, strong LATI was elicited when these injections were performed in mice challenged with tumor cells admixed at 1/10 cell ratio with nonreactive lymphocytes from CE-2-bearing mice. The L3T4+ lymphocyte subset is mainly responsible for this enhancement. This reaction is abolished when recipient mice are sub-lethally irradiated, treated with cyclosporin A, or when the reactivity of L3T4+ and asialo GM1+ cells is suppressed. A similarly efficient LATI is found on combining the daily peptide injections with that of 10 U of IL-2. LATI stemming from this association, too, is abolished when mice are irradiated or treated with anti-L3T4 antibody, whereas it is not affected by cyclosporin A or anti-asialo GM1 antibody. Finally, a tumor-specific immune memory is acquired by about 50% of mice after LATI induced by IL-1 beta or 163-171 peptide alone and by about 80% of mice after LATI induced by peptide and lymphocytes from tumor-bearing mice or peptide and IL-2. These findings could lead to the building of a molecularly defined system to induce efficient immune recognition of tumor cells by using a peptide that does not cause any of the several inflammation-associated changes induced by the whole IL-1 beta.     

Development of an in vitro colony formation assay for the evaluation of in vivo chemotherapy of a rat brain tumor.

An in vitro colony formation assay for the evaluation of in vivo brain tumor therapy has been developed. When plated, disaggregated cells derived from solid tumors proliferated to form relatively homogeneous colonies after a latency period of 2 to 6 days. Increasing concentrations of fetal calf serum enhanced colony-forming efficiency (CFE) with a plateau between 7 and 16%. Supplementation with either irradiated feeder cells (10(3) to 10(5) cells per dish), or medium conditioned by 1 to 3 days of in vitro incubation with the same cell line, doubled the CFE. The density of tumor cells (untreated or previously treated with chemotherapeutic agents) did not affect the CFE when a minimum of 10(4) total cells (tumor plus feeder) were plated. Therefore, in this system the optimal experimental conditions for evaluating chemotherapy and radiotherapy require incubation of disaggregated tumor cells for 12 days in medium containing 10% of fetal calf serum and enough feeder cells to provide a minimum of 10(4) cells per dish. The CFE for untreated tumors was 18 +/- 10% (+/-S.D.), demonstrating that there is significant biological variation. The assay appeared sensitive, with reproducible results, when applied to individual chemically treated tumors. An estimate of the percentage of clonogenic cells affected by in vivo chemotherapy may be obtained by comparing the CFE of cells from treated and untreated tumors. This assay can measure up to a 5 log(10) cell kill, and it should prove to be valuable in developing more effective regimens for the treatment of solid tumors in animals and man.     

Mechanism of natural delayed-type hypersensitivity reactions to tumor cells in nonimmunized syngeneic guinea pigs

Summary Nonimmunized 2/N guinea pigs respond to the presence of chemical carcinogen-transformed syngeneic tumorigenic cells with a sustained (delayed-hypersensitivity-type) 4-day intradermal induration consisting of predominantly polymorphonuclear leukocytes on day 1 and mononuclear cells by day 4, which is independent of the presence of tumor-specific antigens on the tumorigenic cells. Chemical carcinogen-induced morphologically transformed but nontumorigenic cells also induce a polymorphonuclear response by day 1, but neither induration nor a mononuclear response is present on day 4, demonstrating the specificity of the 4-day sustained indurative response for tumorigenic cells. Induration and cellular infiltrates are unaltered if tumor cells are treated prior to injection with the cytostatic lymphokine lymphotoxin or with x-irradiation to inhibit cell proliferation. The intradermal polymorphonuclear leukocyte host response on day 1, but not the mononuclear response on day 4, is also induced by mitomycin C-treated cells or a cytokine culture medium from the cells. No response is present on day 1 or day 4 when cell membranes or lyophilized cells are injected. Thus natural delayed-hypersensitivity-type skin reactivity is a mononuclear leukocyte response specifically directed against intact and metabolically active but not necessarily proliferating tumor cells.     

Effect of in vivo administration of prostaglandins and interferon on natural killer activity and on B-16 melanoma growth in mice

We investigated the effect on in vivo administration of 16,16-dimethyl-prostaglandin E2 (di-M-PGE2) alone and in combined treatment with alpha-beta interferon (IFN) on NK activity in normal, cyclophosphamide (CY)-treated, tumor bearing or irradiated and bone marrow reconstituted mice and on B-16 melanoma growth. Normal mice inoculated with IFN (30,000 U/mouse, 24 hr before testing) showed a significant increase in NK activity, while those treated with di-M-PGE2 (10 micrograms/mouse daily X 4 days) showed a suppressed NK response. No difference was observed between mice treated with di-M-PGE2 alone and those treated with di-M-PGE2 associated with IFN. In immunosuppressed animals single treatments were slightly (di-M-PGE2) or not (IFN) effective, while the combined administration of di-M-PGE2 and IFN caused a marked increase in NK activity. Moreover, di-M-PGE2 was able to accelerate the recovery rate of NK activity in bone marrow-reconstituted murine chimeras, suggesting that the synergistic effect of prostaglandins and IFN could derive from the action of di-M-PGE2 on progenitor pre-NK cells and of IFN on effector cells. Finally, we observed a good correlation between the enhancement of the NK activity and the tumor growth inhibition.     

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNgamma release from an M1-specific T-cell line, expansion of M1 peptide-specific Vbeta17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.