Cells Gene-Modified Tumor-Primed Lymph Node Specific Effector ...

Successful Adoptive Immunotherapy of Murine Poorly Immunogenic Tumor with Specific Effector Cells Generated from Gene-Modified Tumor-Primed Lymph Node Cells1 Hiroshi Tanaka,* Hirohisa Yoshizawa,2* Yoshifumi Yamaguchi,* Kazuhisa Ito,* Hiroshi Kagamu,* Eiichi Suzuki,* Fumitake Gejyo,* Hirofumi Hamada,† and Masaaki Arakawa* We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-g, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-g and B7-1 (MCA102/B7-1/IFN-g) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-g and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-g tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-g tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers. The Journal of Immunology, 1999, 162: 3574 –3582.

T

he T cell plays a crucial role in the host immune response to cancer. The adoptive immunotherapy of cancers with tumor-sensitized T cells has been well documented in several animal models (1–5). We previously reported that cells from lymph nodes (LN)3 draining progressively growing tumors could develop into mature effector cells after in vitro activation with anti-CD3 mAb and IL-2 (6 –9). Transfer of these activated cells into mice bearing established tumors resulted in tumor eradication in a tumor-specific manner. Although numerous successful adoptive immunotherapy approaches have been reported in animal models, only a limited number of patients have responded to the therapy in clinical settings (10 –16). The reason for the failure of adoptive immunotherapy of human cancer may be due in part to the weak immunogenicity of tumor cells and tumor-induced immunosuppression of the generation of immune effector cells. To enhance the immune response elicited in the host, exogenous ex*Department of Medicine (II), Niigata University Medical School, Niigata, Japan; and † Department of Molecular Biotherapy Research, Cancer Chemotherapy Center, Cancer Institute, Tokyo, Japan Received for publication August 10, 1998. Accepted for publication December 14, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by a Niigata Prefecture Research Grant, a Niigata University Research Grant, and a Tsukada Memorial Research Grant. 2 Address correspondence and reprint requests to Dr. Hirohisa Yoshizawa, Department of Medicine (II), Niigata University Medical School, 1-Asahimachidori, Niigata City, Niigata 951-8122, Japan. E-mail address: [email protected] 3 Abbreviations used in this paper: LN, lymph nodes; mIL, murine IL; hIL, human IL; CM, complete medium; LLC, Lewis lung carcinoma; PE, phycoerythrin.

Copyright © 1999 by The American Association of Immunologists

pression of a variety of cytokines in tumor cells has been explored in animal models and has been shown to reduce or abrogate tumorigenicity (17–34). Although a vaccination approach is successful in the presence of a minimal tumor burden, combination therapies may widen the stage of tumors that can be effectively treated. For example, vaccination with cytokine gene-transduced tumor cells can be combined with adoptive immunotherapy. We reported that tumor cells genetically modified to secrete Th cytokines could enhance the in vivo priming of precursor cells of tumor-specific effector T cells and could subsequently augment the antitumor efficacy of anti-CD3/IL-2-activated cells (35). Another approach to enhance the host immune response is the transfection of tumor cells with cDNA-encoding costimulatory molecules. It is clear that Ag recognition alone is not sufficient for T cell activation to effector functions. Second signals such as coligation of auxiliary molecules are also critical for generating T cell-mediated immunity. Ag recognition in the absence of these second signals can lead to tolerance or anergy (36). One of a large number of costimulatory molecules, B7-1 (CD80) plays an important role in antitumor immunity (37, 38). The expression of B7-1 in some murine immunogenic tumor cells has been shown to induce tumor regression, whereas it has limited effects on poorly immunogenic tumors, suggesting that tumor immunogenicity is critical to the outcome (38 – 44). Looking toward the treatment of human cancer, the present study aimed to establish a potent immunotherapy for tumors that lack apparent immunogenicity. MCA102, a poorly immunogenic fibrosarcoma of B6 origin, was transfected with cDNA for IL-2, IL-4, IL-6, IFN-g, and/or B7-1. Tumor cells transfected with both B7-1 and 0022-1767/99/$02.00

The Journal of Immunology IFN-g induced tumor regression when injected s.c. Transfection with both B7-1 and IFN-g also resulted in tumor regression in other poorly immunogenic tumors, B16 and Lewis lung carcinoma (LLC). Cells from LN draining these gene-transduced tumors were activated by an anti-CD3/IL-2 method and were adoptively transferred to mice bearing established pulmonary metastases. The coexpression of B7-1 and IFN-g gave the most efficient enhancement of the antitumor efficacy of anti-CD3/IL-2-activated cells. The mechanism of this enhancement effect was further explored, and we found that the coexpression of B7-1 and IFN-g can enhance the generation of precursor lymphocytes of sensitized T cells and subsequently enhance the antitumor reactivity of adoptive immunotherapy mediated by anti-CD3/IL-2-activated cells.

3575 mAb and flow cytometry

Female C57/BL6J (B6) mice (Central Laboratory of Experimental Research, Tokyo, Japan) were used for experiments at the age of 10 wk or older. They were maintained in specific pathogen-free conditions.

Hybridomas producing mAb against the murine CD3 chain (145-2C11), CD4 (GK1.5, L3T4), CD8 (2.43, Lyt-2), and the murine L-selectin (MEL14) were obtained from the American Type Culture Collection (Manassas, VA). Anti-CD3 mAb was harvested as a supernatant of an in vitro culture with hybridoma cells and then partially purified by 50% ammonium sulfate precipitation, and the IgG content was determined by ELISA. AntiCD4 mAb, anti-CD8 mAb, and anti-L-selectin mAb were produced as ascites fluid from sublethally irradiated (500 rad) DBA/2 mice. For in vivo depletion of CD41/CD81 T cells, mice were given i.v. injections of 0.15– 0.2 ml of ascites fluid diluted to 1.0 ml with HBSS. This procedure has been previously shown to be effective in long-term T cell depletion (8). FITC-conjugated anti-B7-1 (16 –10A), FITC-conjugated anti-H-2Kb (AF688.5), FITC-conjugated anti-H-2Db (KH95), phycoerythrin (PE)-conjugated anti-I-Ab (AF6-120.1), FITC-conjugated anti-ICAM-1 (3E2), PEconjugated anti-Thy1.2 (30-H12), PE-conjugated anti-CD3 (145-2C11), FITC-conjugated anti-CD4 (L3T4), FITC-conjugated anti-CD8 (Lyt-2), and PE-conjugated anti-L-selectin (MEL-14) were purchased from PharMingen (San Diego, CA). Analyses of cell surface phenotypes were conducted by direct immunofluorescence staining of 0.5–1 3 106 cells with conjugated mAb. In each sample, 10,000 cells were analyzed by a FACScan flow microfluorometer (Becton Dickinson, Sunnyvale, CA).

Tumors

Recombinant cytokines

MCA102, MCA205, and MCA207 are antigenically distinct fibrosarcomas of B6 origin induced by intramuscular injection of 3-methylchoranthrene. LLC and B16F10 melanoma (B16) are also of B6 origin. These tumors were maintained in vivo in syngeneic mice by serial s.c. transplantation.

Recombinant hIL-2 was kindly supplied by Shionogi Pharmaceutical (Osaka, Japan). Purified material had a sp. act. of 1.1 3 107 IU/mg protein. Recombinant mIFN-g was kindly supplied by Otsuka Pharmaceutical (Tokyo, Japan). Purified material had a sp. act. of 1 3 106 IU/mg protein.

Materials and Methods Mice

Expression vector The eukaryotic cDNA expression vector, BCMGSNeo, conferring neomycin resistance, was kindly supplied by Dr. H. Karasuyama (Basel Institute for Immunology, Basel, Switzerland) (45). Mouse IL-2 (mIL-2), mIL-4, and human IL-6 (hIL-6) clones were kindly supplied by Dr. Y. Ohe (National Cancer Center Hospital, Tokyo, Japan). mIFN-g was kindly supplied by Dr. Y. Watanabe (Kyoto University, Kyoto, Japan). mB7-1 was kindly supplied by Dr. P. Linsley (Bristol-Myers Squibb, Seattle, WA). These cDNA clones were introduced into the XhoI or NotI site of BCMGSNeo. The biological activities of hIL-6 in mice have been described previously (22, 23).

Gene transfection BCMGSNeo(/Neo), mIL-2 cDNA-containing BCMGSNeo(/IL-2), mIL-4 cDNA-containing BCMGSNeo(/IL-4), hIL-6 cDNA-containing BCMGSNeo(/IL-6), mIFN-g cDNA-containing BCMGSNeo(/IFN-g), and/or mB71cDNA-containing BCMGSNeo (/B7-1) vectors were transfected into MCA102, LLC, B16, and MCA205 cells using a lipofectin reagent (Life Technologies, Gaithersburg, MD). An MCA102 clone with relatively high MHC class I expression (MCA102H) was produced by a limiting dilution method and transfected with B7-1 (MCA102H/B7-1). These tumor cells (1–2 3 105 cells) were plated in a 35-mm tissue culture dish in 2 ml RPMI 1640 medium supplemented with 10% heat-inactivated FCS and cultured until the cells were 40 – 60% confluent. The lipofectin-DNA complexes were overlaid onto the cells for a 12-h incubation period at 37°C in a CO2 incubator. After replacing the DNA-containing medium with RPMI 1640 medium containing 10% FCS, cells were incubated for an additional 48 h. The transfectants, MCA102Neo, MCA102/IL-2, MCA102/IL-4, MCA102/ IL-6, MCA102/IFN-g, MCA102/B7-1, MCA102/B7-1/IL-2, MCA102/B7-1/ IL-4, MCA102/B7-1/IL-6, MCA102/B7-1/IFN-g, MCA205/IFN-g, MCA 205/B7-1, MCA205/B7-1/IFN-g, LLC/IFN-g, LLC/B7-1, LLC/B7-1/IFN-g, B16/IFN-g, B16/B7-1, and B16/B7-1/IFN-g were selected by supplementation of the media for 14 days with 400-1000 mg/ml of the neomycin analogue, G418 (Life Technologies). These gene-modified tumor cells were maintained as monolayer cultures in complete medium (CM). CM consisted of RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM fresh L-glutamine, 100 mg/ml streptomycin sulfate, 10 U/ml penicillin, 50 mg/ml toburamicine (all from Life Technologies), 0.5 mg/ml amphotericin-B (Fungizon; Life Technologies), and 5 3 1025 M 2-ME (Sigma, St. Louis, MO).

Cytokine ELISA Medium (1 ml) conditioned by 5 3 105 tumor cells for 24 h was assayed for mIL-2, mIL-4, hIL-6, or murine IFN-g content by a quantitative “sandwich” enzyme immunoassay using a mIL-2 ELISA kit or a mIL-4 ELISA kit (Endogen, Boston, MA), a hIL-6 ELISA kit (Becton Dickinson Labware, Bedford, MA), or a mIFN-g ELISA kit (Genzyme, Cambridge, MA).

Lymphoid cell preparation and anti-CD3/IL-2 activation Tumor growth was initiated by inoculating syngeneic B6 mice s.c. in the bilateral flank with 107 viable tumor cells. Ten to 12 days later, tumordraining LNs were harvested and single-cell suspensions were prepared mechanically as described previously (35). The cells were stimulated in vitro by incubating ;108 cells in a 75-cm2 tissue culture flask containing 30 ml of CM with 2 mg/ml of anti-CD3 mAb. After 2 days of incubation at 37°C in a 5% CO2 atmosphere, activated cells were harvested, washed, and further cultured at a concentration of 6 3 105/ml in 30 ml of CM containing 40 U/ml of IL-2 for 3 days.

Adoptive immunotherapy B6 mice were injected i.v. with 8 3 105 MCA102, 4 3 105 MCA205, 106 MCA207, or 8 3 105 LLC tumor cells in 1 ml of HBSS to initiate pulmonary metastases. On day 3, effector cells were given i.v. to each mouse. On day 14, the metastatic foci in all mice were enumerated as described previously (46). Metastatic foci too numerous to count were assigned an arbitrary value of 250. The significance of differences in numbers of pulmonary metastases between groups was determined by the Wilcoxon’s rank sum test. Two-sided p values of , 0.05 were considered significantly different. Each treatment group consisted of at least five mice, and no animal was excluded from the statistical evaluation.

Fractionation of tumor-draining LN cells based on the expression of L-selectin T cells in the LN cell suspension were concentrated by passage through nylon wool columns (Wako Pure Chemical Industries, Osaka, Japan). After a 45-min incubation at 37°C, the nonadherent elution contained 90 –95% Thy1.21 T cells. Purified T cells were further fractionated into two subpopulations based on the expression of L-selectin. Cells were first incubated for 20 min at 4°C with the L-selectin hybridoma ascites fluid at a 1:1000 dilution. The cells were washed of unbound Ab. A total of 3– 4 3 107 cells in 4 ml CM were plated on a 25-cm2 flask that was precoated with goat anti-rat Ig Ab (American Qualex, San Clemente, CA). After 1 h incubation at 4°C, nonadherent (L-selectin2) cells were collected by gentle rocking. These cells were incubated on a new goat anti-rat Ig Ab-coated flask to yield highly purified (.90%) L-selectin2 cells. Adherent cells were collected from the first incubation flask with a cell scraper after rinsing twice with PBS. More than 95% of the recovered adherent cells were high L-selectin cells (L-selectin1).

Results Characteristics of gene-modified MCA102 tumor cells The morphology and in vitro proliferation of the transfectants were almost identical to those of parental MCA102 tumor cells (data not

3576

ADOPTIVE IMMUNOTHERAPY OF MURINE POORLY IMMUNOGENIC TUMOR

FIGURE 1. B7-1 expression on gene-transduced MCA102 tumor cells. Tumor cells were stained with FITC-labeled anti-B7-1 (16-10A) mAb. B7-1 expression was analyzed by flow cytometry. A total of 106 cells was analyzed for each sample. Each frame consists of 10,000 cells.

shown). The culture supernatants of MCA102 tumor cells transfected with mIL-2, mIL-4, hIL-6, or mIFN-g contained ;400 U/ml of mIL-2, 180 mg/ml of mIL-4, 240 ng/ml of hIL-6, or 20 IU/ml of mIFN-g, respectively. No cytokine was detected in the supernatants of parental or neomycin-resistant gene-transfected tumors. The expression of B7-1 in gene-modified MCA102 tumor cells was examined by flow cytometric analysis. B7-1 transfectants consistently expressed high levels of B7-1 (Fig. 1). Tumor growth of gene-modified tumor cells in C57BL/6 mice Confluent cultures of gene-modified and -unmodified tumor cells were harvested and inoculated s.c. in the right flank of B6 mice with 1 3 106 viable tumor cells in 0.05 ml of HBSS. In vivo tumor growth of gene-modified and -unmodified MCA102 tumors are shown in Fig. 2. MCA102/B7-1/IFN-g initially grew and then regressed. In contrast, other single or cotransfectants grew progressively (Fig. 2B). To assess the role of CD41 or CD81 T cells in the regression of MCA102/B7-1/IFN-g tumors,

mAb to the CD41 or CD81 determinant was administered 3 days before and 4 days after tumor inoculation. As shown in Fig. 3, the depletion of either CD41 or CD81 T cells abrogated the tumor regression, indicating that both CD41 and CD81 T cells were required for the regression of MCA102/B7-1/IFN-g tumors. The regression induced by the cotransfection of these two genes was confirmed further in other tumor models. LLC and B16, poorly immunogenic tumors, and MCA205, a weakly immunogenic tumor of B6 origin, were also transfected in vitro with B7-1 and/or IFN-g. In these tumor cells, cotransfection of B7-1 and IFN-g resulted in tumor regression when injected s.c. (Fig. 4). Because only cotransfection of B7-1 and IFN-g induced tumor regression, we examined whether vaccination with this gene-modified tumor could induce protective immunity against parental tumor challenge. Gene-modified or -unmodified MCA102 tumor cells were irradiated (4000 rad) and intradermally injected into B6 mice as a vaccination, and 2 wk after vaccination mice were challenged with parental tumors. Six of

FIGURE 2. In vivo tumor growth of genetransduced MCA102 tumor cells in B6 mice. Cells (106 in 0.05 ml of HBSS) were injected s.c. into the right flank of mice, and tumor size was measured serially. The results are expressed as mean diameter (in millimeters) of tumors from groups of five mice each.

The Journal of Immunology

3577 Table I. Challenge of mice with parental MCA102 or MCA205 tumor cells after immunization with B7-1- and/or IFN-g-transfected MCA102 tumor cells No. of Mice with Tumor/ No. of Mice Challenged withb Immunization witha

MCA102

MCA205

MCA102 MCA102/IFN-g MCA102/B7-1 MCA102/B7-1/IFN-g Control

10/10 10/10 10/10 4/10 10/10

10/10 10/10 10/10 10/10 10/10

a B6 mice were immunized intradermally in the abdomen with 2 3 107 irradiated (4000 rad) tumor cells. b Two weeks after immunization, these mice were challenged with 107 parental MCA102 or MCA205 tumor cells. The number of mice with tumor growth was counted.

FIGURE 3. Effect of in vivo T cell subset depletion on MCA102/B71/IFN-g tumor growth. Cells (106 in 0.05 ml of HBSS) were injected s.c. into the right flank of mice, and tumor size was measured serially. A 200-ml volume of ascites fluid containing mAb or 0.25 mg of rIg diluted to 1.0 ml of HBSS was administered i.v. 3 days before and 4 days after tumor inoculation. The results are expressed as mean diameter (in millimeters) of tumors from groups of five mice each.

10 mice that were vaccinated with MCA102/B7-1/IFN-g cells rejected parental tumor challenge in a tumor-specific manner (Table I). Although vaccination with MCA102/B7-1/IFN-g cells could efficiently induce protective immunity against s.c. challenge, it could not mediate the regression of pulmonary metastases (data not shown). Antitumor efficacy of effector cells generated from tumordraining LN of gene-modified MCA102 tumors We next examined the antitumor efficacy of cells generated from LN draining the gene-modified tumors by the anti-CD3/IL-2

FIGURE 4. In vivo tumor growth of B7-1and/or IFN-g-transfected tumor cells in B6 mice. Cells (106 in 0.05 ml of HBSS) were injected s.c. into the right flank of mice, and tumor size was measured serially. The results are expressed as mean diameter (in millimeters) of tumors from groups of five mice each.

method. We previously demonstrated that the activation of tumordraining but not normal or bacterial adjuvant Corynebacterium parvum-stimulated LN cells with anti-CD3/IL-2 resulted in the generation of specific antitumor effector cells (6). To investigate the advantage of gene-modified tumor cells for adoptive immunotherapy, we used this anti-CD3/IL-2 activation method to generate effector cells. Cells from tumor-draining LN of s.c. MCA102 parental, MCA102/IFN-g, MCA102/B7-1, or MCA102/B7-1/IFN-g tumors were harvested and activated in vitro with anti-CD3/IL-2. There was no significant difference in in vitro cell proliferation between these cells (approximately threefold). The antitumor efficacy of cells generated from different LNs was adoptively transferred to mice bearing 3-day established pulmonary metastases (Table II). The coexpression of B7-1 and IFN-g significantly enhanced the antitumor efficacy of anti-CD3/IL-2-activated cells when compared with that of cells generated from LNs draining parental MCA102, MCA102/IFN-g, or MCA102/B7-1 tumors. In addition, the enhancing effect of IFN-g was augmented further by the coexpression of B7-1. The adoptive transfer of fresh noncultured cells from MCA102/B7-1/IFN-g tumor-draining LN failed to demonstrate antitumor efficacy (data not shown), indicating that the enhancing effect could be induced by promoting the precursor

3578


Table II. Adoptive immunotherapy with anti-CD3/IL-2-activated cells generated from LN-draining B7-1- and/or IFN-g-transfected MCA102 tumor cells No. of Cells Transferreda 3 107

3 1 0.3 0

Table IV. Specificity of adoptive immunotherapy mediated by antiCD3/IL-2-activated cells generated from LN-draining MCA102/B7-1/ IFN-g tumors

Mean No. of Pulmonary Metastases (SEM)b MCA102

199 (41) 214 (36) 250 250

Mean No. of Pulmonary Metastases (SEM)b

MCA102/B7-1 MCA102/IFN-g MCA102/B7-1/IFN-g

183 (36) 250 240 (10)

†‡

106 (22)* 250 216 (26)

†‡§

14 (6)* 73 (15)*†‡§ 184 (28)

a Inguinal LN cells obtained from mice bearing s.c. MCA102, MCA102/B7-1, MCA102/IFN-g, or MCA102/B7-1/IFN-g tumors for 12 days were activated by the anti-CD3/IL-2 method. These cells were given i.v. to mice with 3-day established pulmonary MCA102 metastases. b Lungs were harvested and metastases were counted 14 days after tumor i.v. inoculation. Significant differences were detected from groups receiving: p, no treatment; †, activated MCA102 tumor-draining LN cells; ‡, activated MCA102/B7-1 tumor-draining LN cells; or §, activated MCA102/IFN-g tumor-draining LN cells.

response. To investigate the T cell subset populations that participated in tumor eradication, we depleted a CD41 or CD81 T cell subset population in vivo with mAb. As shown in Table III, depletion of either a CD41 or CD81 T cell subset resulted in abrogation of the antitumor effect, indicating that both populations of T cells participated in mediating the tumor eradication. Specificity of adoptive immunotherapy mediated by anti-CD3/ IL2-activated cells generated from LN-draining MCA102 tumors transfected with B7-1 and IFN-g We examined the specificity of adoptive immunotherapy mediated by anti-CD3/IL-2-activated cells generated from LN-draining MCA102/B7-1/IFN-g tumors. Cells from MCA102/B7-1/IFN-g tumor-draining LN had significant therapeutic efficacy against MCA102, but not MCA205, MCA207, or LLC pulmonary metastases (Table IV). These results demonstrate that cells generated from MCA102/B7-1/IFN-g tumor-draining LN cells mediated specific tumor eradication. Expression of MHC class I molecule on B7-1 and/or IFN-g gene-modified MCA102 tumor cells To clarify the mechanisms of enhanced antitumor response induced by MCA102/B7-1/IFN-g cells, we analyzed the expression of cell surface molecules on the gene-modified tumors by flow cytometry. As shown in Fig. 5, up-regulation of MHC class I molecules (Kb and Db) was observed in both MCA102/IFN-g and MCA102/B7-1/IFN-g tumors, but not in MCA102/B7-1 or parental MCA102 tumors. No expression of MHC class II molecule

Table III. Effect of in vivo T cell subset depletion on therapeutic efficacy of anti-CD3/IL-2-activated cells generated from LN-draining MCA102/B7-1/ IFN-g tumors No. of Cells mAb for Mean No. of Transferred (3 107)a In Vivo Depletionb Pulmonary Metastases (SEM)c

0 3 3 3

rIg Anti-CD4 Anti-CD8

168 (15) 16 (7)d 150 (26) 141 (25)

a Cells from LN-draining s.c. MCA102/B7-1/IFN-g tumors for 12 days were activated by the anti-CD3/IL-2 method. These cells were given i.v. to mice with 3-day established pulmonary MCA102 metastases. b A 200-ml volume of ascites fluid containing mAb or 0.25 mg of rIg diluted to 1.0 ml of HBSS was administered i.v. within 1 h of cell transfer. c Lungs were harvested and metastases were counted 14 days after tumor i.v. inoculation. d Significantly different from group receiving no treatment.

Adoptive Immunotherapya

MCA102

MCA205

MCA207

LLC

2 1

250 23 (10)c

235 (8) 234 (10)

117 (20) 124 (19)

101 (11) 100 (6)

a Cells from LN-draining s.c. MCA102/B7-1/IFN-g tumors for 12 days were activated by the anti-CD3/IL-2 method. B6 mice were sublethally irradiated (500 rad) and injected with 8 3 105 MCA102, 4 3 105 MCA205, 106 MCA207, or 8 3 105 LLC tumor cells to initiate pulmonary metastases. On day 3, activated LN cells (3 3 107/mouse) were given i.v. to each mouse. b Lungs were harvested and metastases were counted 14 days after tumor i.v. inoculation. c Significantly different from group receiving no treatment.

(I-Ab) or ICAM-1 (CD54) was observed in these transfectants. To elucidate the role of MHC class I Ag on tumor cells in induction of host antitumor immunity, MCA102H, a clone with relatively high MHC class I expression, was produced by limiting dilution method and transfected in vitro with B7-1 (MCA102H/B7-1) (Fig. 5). In vivo tumor growth of MCA102H and MCA102H/B7-1 cells are shown in Fig. 6. The transfection of B7-1 into MCA102H induced tumor regression, indicating that intensity of MHC class I expression on tumor cells had important implications for the outcome of antitumor response by B7 costimulation. Although the culture of parental MCA102 or MCA102/B7-1 cells with 500 IU/ml of recombinant mIFN-g for 2 days resulted in transient upregulation of MHC class I expression on tumor cells, these cultured cells did not regress when injected s.c. (data not shown). These results suggest that persistent up-regulation of MHC class I is essential for tumor regression induced by B7-1 expression. Comparison of antitumor efficacy of cells generated from LN-draining MCA102H/B7-1 and MCA102/B7-1/IFN-g tumors To examine whether up-regulation of MHC class I could be the sole cause of the enhanced precursor response by IFN-g, the antitumor efficacy of cells generated from MCA102H/B7-1 or MCA102/B7-1/IFN-g tumors was compared in adoptive immunotherapy for established pulmonary metastases. Cells from LNdraining s.c. MCA102H/B7-1 or MCA102/B7-1/IFN-g tumors were harvested and activated in vitro with anti-CD3/IL-2. As shown in Table V, the antitumor efficacy of activated MCA102/ B7-1/IFN-g tumor-draining LN cells was significantly superior to that of activated MCA102H/B7-1 tumor-draining LN cells. These results suggest that the enhancement effect of IFN-g from tumor cells could not be explained solely by the up-regulation of MHC class I expression. Phenotypic analysis of gene-modified or -unmodified tumorprimed LN cells before and after anti-CD3/IL-2 activation Phenotypic analysis of cells from gene-modified or -unmodified tumor-draining LN for CD3, CD4, CD8, or the homing molecule L-selectin are shown in Fig. 7. Although LN draining these tumors consisted of almost identical fractions of CD41 and CD81 T cells, LN-draining MCA102/B7-1/IFN-g tumors had an increased proportion of cells with down-regulation of L-selectin (L-selectin2). Approximately 25% of cells from LN-draining MCA102/B7-1/ IFN-g tumors were L-selectin2 cells. These L-selectin2 cells consisted of almost identical fractions of CD41 and CD81 T cells (data not shown).

The Journal of Immunology

3579

FIGURE 5. MHC class I, MHC class II, or ICAM-1 expression of IFN-g-transfected MCA102 tumor cells. Tumor cells were stained with FITC-labeled anti-Kb, FITC-labeled anti-Db mAb, PE-conjugated anti-I-Ab (AF6-120.1), or FITC-conjugated antiICAM-1 (3E2). A total of 106 cells was analyzed for each sample. Each frame consists of 10,000 cells. Values indicate the mean channel number (MCN).

Antitumor efficacy of adoptive immunotherapy mediated by activated L-selectin2 cells derived from LN-draining MCA102/ B7-1/IFN-g tumors To determine whether the increased proportion of cells with downregulated L-selectin may reflect the efficient precursor response in tumor-draining LN, cells purified according to the expression of L-selectin were activated by the anti-CD3/IL-2 method, and the antitumor efficacy of these activated cells were analyzed in adoptive immunotherapy. On activation by the anti-CD3/IL-2 method, L-selectin2 cells proliferated more vigorously than L-selectin1

cells and unfractionated LN cells. L-selectin2 cells increased approximately eightfold, compared with a threefold increase observed in L-selectin1 cells and unfractionated LN cells. These activated cells were adoptively transferred to mice bearing 3-day established pulmonary metastases. As shown in Table VI, the transfer of 0.3 3 107 L-selectin2 cells were therapeutically equally effective as 1 3 107 unfractionated cells, whereas the transfer of 1 3 107 L-selectin1 cells did not demonstrate any antitumor efficacy. Because activated L-selectin2 cells generated from LN-draining MCA102/B7-1/IFN-g tumors efficiently eliminated microscopic 3-day established pulmonary metastases, we further investigated whether adoptive transfer of the cells could prolong the survival. Mice that received 2 3 107 L-selectin1 cells had an equivalent median survival time to untreated mice. However, the transfer of 2 3 107 L-selectin2 cells resulted in long-term survival and indicated more efficient tumor eradication than unfractionated cells (Fig. 8). These results indicate that the antitumor efficacy of activated L-selectin2 cells is far greater than that of activated L-selectin1 or unfractionated cells and that the precursor response in LN-draining MCA102/B7-1/IFN-g is exclusively augmented.

Table V. Adoptive immunotherapy with anti-CD3/IL-2-activated cells generated from LN cells draining MCA102H/B7-1 or MCA102/B7-1/ IFN-g tumors

FIGURE 6. In vivo tumor growth of MCA102 tumor cells with a high expression of MHC class I. Cells (106 in 0.05 ml of HBSS) were injected s.c. into the right flank of mice, and tumor size was measured serially. The results are expressed as mean diameter (in millimeters) of tumors from groups of five mice each.


No. of Cells Transferreda (3 107)

MCA102H/B7-1

MCA102/B7-1/IFN-g

3 1 0.3 0

75 (13)* 115 (9)* 187 (17) 213 (14)

3 (2)*† 58 (15)*† 181 (14)

a Cells from LN-draining s.c. MCA102H/B7-1 or MCA102/B7-1/IFN-g tumors for 12 days were activated by the anti-CD3/IL-2 method. These cells were given i.v. to mice with 3-day established pulmonary MCA102 metastases. b Lungs were harvested and metastases were counted 14 days after tumor i.v. inoculation. Significant differences were detected from groups receiving: p, no treatment; or †, activated MCA102H/B7-1 tumor-draining LN cells.

3580


FIGURE 7. Phenotypic analysis of tumor-draining LN cells. Cells were stained with PE-conjugated anti-CD3 mAb, FITCconjugated anti-CD4 mAb, FITC-conjugated anti-CD8 mAb, or PE-conjugated antiL-selectin mAb. A total of 106 cells was analyzed for each sample. Each frame consists of 10,000 cells. Values indicate the percentage of CD3-positive, CD4-positive, CD8positive, or L-selectin-negative (L-selectin2) cells in each preparation.

Discussion In many animal models, tumors transduced with genes for cytokine such as IL-2 (17–19), IL-4 (20, 21), IL-6 (22), IFN-g (24 –28), TNF (29 –31), granulocyte-CSF (32), or granulocyte-macrophageCSF (33, 34) may have reduced tumorigenicity with an augmented host-immune response. Although vaccination with the gene-modified tumors resulted in the rejection of parental tumor challenge, this strategy is not thought to be always directly applicable for treatment of human cancers, which often lack apparent immunogenicity. We previously reported that vaccine therapy with gene-modified tumor cells could be used to augment the antitumor efficacy of adoptive immunotherapy (35). Although vaccination therapy with tumor cells modified to secrete cytokines alone did not mediate the tumor regression of established visceral metastases, vaccination could enhance the in vivo priming of antitumor precursor T cells in tumor-draining LN and subsequently augment the antitumor efficacy of adoptive immunotherapy. Cells derived from LN draining these gene-modified tumors showed potent antitumor efficacy in the eradication of pulmonary metastases. We further demonstrated that the suppressed function of CD41 T cells by tumor burden was restored by the local secretion of the Th cytokines from tumor cells and subsequently facilitated the priming of precursor lymphocytes of effector cells (35).

In the current study, we first investigated whether such genetic modifications could enhance the host-immune reaction against tumors that lacked apparent immunogenicity. Although transfection of cytokines successfully enhanced the generation of antitumor effector T cells when relatively immunogenic tumors were used, our results indicated that transfection of cytokines alone failed to induce a host-immune response, which led to the eradication of poorly immunogenic tumors and priming of tumor- reactive T cells (Fig. 2 and Tables I and II). Transfection of costimulatory molecules such as B7-1 have been demonstrated to reduce or abrogate tumorigenicity by eliciting a specific antitumor response against parental tumors (38 – 44). The transfection of B7-1 enhanced the host-immune reaction, and the expression on weakly immunogenic MCA205 tumor cells reduced tumorigenicity of these cells when injected s.c. (Fig. 4). In contrast, B7-1 transfection into poorly immunogenic tumors did not induce tumor regression or precursor

Table VI. Adoptive immunotherapy with anti-CD3/IL-2-activated cells generated from LN cells draining MCA102/B7-1/ IFN-g tumors separated based on L-selectin expression

Cellsa

No. of Cells Transferred (3 107)


Unfractionated L-selectin1 L-selectin2

0 1 1 0.3

217 (20) 56 (8)c 210 (26) 62 (10)c

a Cells from LN-draining s.c. MCA102/B7-1/IFN-g tumors for 12 days were separated based on L-selectin expression and activated by the anti-CD3/IL-2 method. These cells were given i.v. to mice with 3-day established pulmonary MCA102 metastases. b Lungs were harvested and metastases were counted 14 days after tumor i.v. inoculation. c Significantly different from group receiving no treatment.

FIGURE 8. Antitumor efficacy of unfractionated, L-selectin2, and L-selectin1 cells derived from MCA102/B7-1/IFN-g tumor-draining LNs in adoptive immunotherapy of established MCA102 pulmonary metastases. Cells from LN-draining s.c. MCA102/B7-1/IFN-g tumors for 12 days were separated based on L-selectin expression and were activated by the anti-CD3/IL-2 method. These cells were given i.v. to mice with 3-day established pulmonary MCA102 metastases. Each group consisted of at least 10 mice. The group of mice that received 2 3 107 L-selectin cells had a significantly longer survival time than mice in all the other groups (p 5 0.005).

The Journal of Immunology response in the tumor-draining LN (Fig. 4 and Table II). These data are consistent with the past observation that tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity (41). Considering these facts, we speculated that dysfunction of CD41 T cells, which is observed in a weakly immunogenic tumor system, may preclude the generation of effector T cells facilitated by B7-1 expression on poorly immunogenic tumor cells. Therefore, we investigated whether cotransfection of the Th cytokines and B7-1 could enhance the host antitumor immunity. The coexpression of IFN-g and B7-1 induced the regression of s.c. tumors, but combinations of other cytokines and B7-1 had no effect (Fig. 2B). This particular combination also induced tumor regression of two other poorly immunogenic tumors (Fig. 4). The regression was mediated by a host-immune reaction, because mice that were given sublethal irradiation were incapable of rejecting the cotransfected tumor cells (data not shown). Mice vaccinated with MCA102/B7-1/IFN-g cells rejected parental tumor challenge, indicating that vaccination could induce systemic tumor immunity (Table I). Furthermore, cells from MCA102/B7-1/IFN-g tumordraining LN acquired potent antitumor efficacy after in vitro activation with anti-CD3 and IL-2. Adoptive transfer of fresh tumordraining LN cells did not mediate antitumor efficacy (data not shown), indicating that the vaccination could enhance priming of antitumor precursor T cells in tumor-draining LN and subsequently augment the antitumor efficacy of adoptive immunotherapy. These effector cells were tumor-specific because the cells derived from LN-draining MCA102/B7-1/IFN-g tumors had no antitumor reactivity against MCA207, LLC, or B16 tumors (Table IV). A deficiency in the presentation of endogenous Ag may be one reason for the lack of tumor immunogenicity (47), and the upregulation of MHC class I expression by IFN-g enables weakly immunogenic tumors to express endogenous Ag (27). Tumor cells modified to secrete IFN-g had reduced tumorigenicity, and this phenomenon was explained by the up-regulation of MHC class I Ag expression on tumor cells (24 –28). Because IFN-g is a potent inducer of MHC class I, class II Ag, and other molecules such as ICAM-1, we analyzed the expression of these molecules on IFNg-transfected MCA102 tumor cells. MHC class I expression was up-regulated on IFN-g-transfected MCA102 tumor cells, but neither MHC class II nor ICAM-1 was expressed on IFN-g-transfected tumor cells (Fig. 5). To examine this correlation between the up-regulation of MHC class I expression on tumor cells and the augmented antitumor reactivity, we selected one clone from the parental tumor cells that had a high level of MHC class I Ag expression (MCA102H). B7-1 transfection of MCA102H resulted in tumor regression, indicating that MHC class I expression plays an important role in the regression of tumors (Fig. 6). However, interestingly the antitumor efficacy of activated MCA102/B7-1/IFN-g tumor-draining LN cells was far superior to that of activated MCA102H/B7-1 tumor-draining LN cells (Table V). This significant difference was not attributed to the difference in mean intensity of B7-1 and MHC class I Ag on tumor cells, indicating that enhanced priming of precursor lymphocytes of antitumor effector T cells in MCA102/B7-1/IFN-g tumor-draining LN could not be explained solely by the up-regulation of MHC class I expression. Our data is consistent with previous studies that demonstrated that IFN-g-transfected tumor cells were more effective in the immunotherapy of tumor-bearing mice than MHC class I Ag gene-transfected tumor cells (28) and that up-regulation of MHC class I expression by low IFN-g secretors was insufficient to decrease tumorigenicity (26).

3581 IFN-g may influence the outcome of an immune response in several distinct ways, and its importance in tumor immunology has been demonstrated by numerous reports (24 –28). We and others previously demonstrated that the function of the CD41 T cell subset, especially CD41 Th cells, is depressed in a tumor-bearing host, and the intensity of the suppressive effect is correlated to the tumor burden (35, 48, 49). Among the many cytokines expressed by CD41 Th cells, a recent study revealed down-regulation of IFN-g but not IL-2 levels of CD41 T cells in a particular tumor model (50). This selective down-regulation of cytokine expression may also occur in our tumor model, because coexpression of IFN-g but not IL-2 abrogated tumorigenicity and enhanced the precursor response. In our tumor model, there was no expression of MHC class II or ICAM-1 on IFN-g-transfected tumor cells (Fig. 5). Nevertheless, both CD41 and CD81 T cells appeared to be required for antitumor efficacy, because in vivo depletion of either CD41 or CD81 T cell subsets by mAb abrogated the tumor regression (Fig. 3). This result may suggest an important role for host APC as an MHC class II Ag presenter to prime CD41 T cells. IFN-g from tumor cells could act primarily on macrophages and dendritic cells through up-regulation of MHC class II expression, thereby enhancing the Ag presentation to CD41 T cells. This could be an alternative explanation for the enhancing effect by IFN-g. We have confirmed the enhanced generation of precursor cells of antitumor effector T lymphocytes by analyzing the antitumor efficacy of in vitro activated tumor-draining LN cells in adoptive immunotherapy. Recently, it was demonstrated that the down-regulation of the homing molecule L-selectin could serve as a surrogate marker for identifying specific tumor-sensitized T cells (51, 52). Using this method, we further confirmed the generation of precursor T cells in the tumor-draining LN. Phenotypic analysis of tumor-draining LN cells revealed an increased population of cells with down-regulated L-selectin expression in MCA102/B7-1/ IFN-g but not in MCA102H/B7-1 tumor-draining LN (Fig. 7). Cells with down-regulated L-selectin expression constituted all the antitumor reactivity, indicating that the coexpression of B7-1 and IFN-g of tumor cells induced an efficient precursor response in the tumor-draining LN (Table VI, Fig. 8). In conclusion, the cotransfection of poorly immunogenic murine tumors with B7-1 and IFN-g could enhance the in vivo priming of precursor cells of tumor-specific T cells in the tumor-draining LN. MHC class I up-regulation on tumor cells by IFN-g alone could not account for the enhanced antitumor immunity, indicating that this particular combination of transfection could facilitate a suitable microenvironment for the priming of tumor-reactive T cells. Our observation implies the therapeutic utility of gene-modification of tumors for the treatment of tumors that lack apparent immunogenicity. The strategy presented here will help us to understand the mechanisms of induction of host antitumor immunity as well as to establish more effective immunotherapy for human cancer.

Acknowledgments We thank Dr. Toshiyuki Koya for his technical assistance.

References 1. Rosenberg, S. A., and W. D. Terry. 1977. Passive immunotherapy of cancer in animals and man. Adv. Cancer Res. 25:323. 2. Smith, H. G., R. P. Harmel, M. G. Hanna, Jr., B. S. Zwilling, B. Zbar, and H. J. Rapp. 1977. Regression of established intradermal tumors and lymph node metastases in guinea pigs after systemic transfer of immune lymphoid cells. J. Natl. Cancer Inst. 58:1315. 3. Fernandez-Cruz, E., B. Halliburton, and J. D. Feldman. 1979. In vivo elimination by specific effector cells of an established syngeneic rat moloney virus-induced sarcoma. J. Immunol. 123:1772.

3582


4. Cheever, M. A., P. D. Greenberg, and A. Fefer. 1984. Potential for specific cancer therapy with immune T lymphocytes. J. Biol. Response Mod. 3:113. 5. Shu, S., and S. A. Rosenberg. 1985. Adoptive immunotherapy of a newly induced sarcoma: immunologic characteristics of effector cells. J. Immunol. 135:2895. 6. Yoshizawa, H., K. Sakai, A.E. Chang, and S. Shu. 1991. Activation by anti-CD3 of tumor-draining lymph node cells for specific adoptive immunotherapy. Cell. Immunol. 134:473. 7. Yoshizawa, H., A. E. Chang, and S. Shu. 1991. Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with antiCD3 and IL-2. J. Immunol. 147:729. 8. Yoshizawa, H., K. Sakai, A. E. Chang, and S. Shu. 1992. Cellular interactions in effector generation and tumor regression mediated by anti-CD3/interleukin 2 activated tumor-draining lymph node cells. Cancer Res. 52:1129. 9. Mitsuma, S., H. Yoshizawa, K. Ito, H. Moriyama, M. Wakabayashi, T. Chou, M. Arakawa, and S. Shu. 1994. Adoptive immunotherapy mediated by anti-TCR/ IL-2-activated tumour-draining lymph node cells. Immunology 83:45. 10. Miyao, H., T. Chou, K. Ito, H. Moriyama, S. Mitsuma, M. Wakabayashi, H. Yoshizawa, and M. Arakawa. 1994. Treatment of advanced primary lung cancer associated with malignant pleural effusion by the combination of immunotherapy and chemotherapy. Oncology 51:87. 11. Ito, K., T. Chou, S. Mitsuma, H. Moriyama, M. Wakabayashi, A. Mizusawa, H. Miyao, H. Yoshizawa, T. Tsuchiya, T. Ebe, L. Yuejing, and M. Arakawa. 1993. A novel chemo-immunotherapy for advanced non-small cell lung cancer. J. Jpn. Soc. Cancer Ther. 29:36. 12. Rosenberg, S. A., B. S. Packard, P. M. Aebersold, D. Solomon, S. L. Topalian, S. T. Toy, P. Simon, M. T. Lotze, J. C. Yang, C. A. Seipp, et al. 1988. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma: a preliminary report. N. Engl. J. Med. 319:1676. 13. Rosenberg, S. A., P. Spiess, and R. Lafreniere. 1986. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233:1318. 14. Topalian, S. L., D. Solomon, F. P. Avis, A. E. Chang, D. L. Freerksen, W. M. Linehan, M. T. Lotze, C. N. Robertson, C. A. Seipp, P. Simon, et al. 1988. Immunotherapy of patients with advanced cancer using tumor-infiltrating lymphocytes and recombinant interleukin-2: a pilot study. J. Clin. Oncol. 6:839. 15. Chang, A. E., H. Yoshizawa, K. Sakai, M. J. Cameron, V. K. Sondak, and S. Shu. 1993. Clinical observations on adoptive immunotherapy with vaccine-primed Tlymphocytes secondarily sensitized to tumor in vitro. Cancer Res. 53:1043. 16. Chang, A. E., A. Aruga, M. J. Cameron, V. K. Sondak, D. P. Normolle, B. A. Fox, and S. Shu. 1997. Adoptive immunotherapy with vaccine-primed lymph node cells secondarily activated with anti-CD3 and interleukin-2. J. Clin. Oncol. 15:796. 17. Fearon, E. R., D. M. Pardoll, T. Itaya, P. Golumbek, H. I. Levitsky, J. W. Simons, H. Karasuyama, B. Vogelstein, and P. Frost. 1990. Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response. Cell 60:397. 18. Tsai, S. C., B. Gansbacher, L. Tait, F. R. Miller, and G. H. Heppner. 1993. Induction of antitumor immunity by interleukin-2 gene-transduced mouse mammary tumor cells versus transduced mammary stromal fibroblasts. J. Natl. Cancer Inst. 85:546. 19. Ohe, Y., E. R. Podack, K. J. Olsen, Y. Miyahara, T. Ohira, K. Miura, K. Nishio, and N. Saijo. 1993. Combination effect of vaccination with IL2 and IL4 cDNA transfected cells on the induction of a therapeutic immune response against Lewis lung carcinoma cells. Int. J. Cancer 53:432. 20. Tepper, R. I., P. K. Pattengale, and P. Leder. 1989. Murine interleukin-4 displays potent anti-tumor activity in vivo. Cell 57:503. 21. Golumbek, P. T., A. J. Lazenby, H. I. Levitsky, L. M. Jaffee, H. Karasuyama, M. Baker, and D. M. Pardoll. 1991. Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4. Science 254:713. 22. Okada, M., M. Kitahara, S. Kishimoto, T. Matsuda, T. Hirano, and T. Kishimoto. 1988. IL-6/BSF-2 functions as a killer helper factor in the in vitro induction of cytotoxic T cells. J. Immunol. 141:1543. 23. Porgador, A., E. Tzehoval, A. Katz, E. Vadai, M. Revel, M. Feldman, and L. Eisenbach. 1992. Interleukin 6 gene transfection into Lewis lung carcinoma tumor cells suppresses the malignant phenotype and confers immunotherapeutic competence against parental metastatic cells. Cancer Res. 52:3679. 24. Gansbacher, B., R. Bannerji, B. Daniels, K. Zier, K. Cronin, and E. Gilboa. 1990. Retroviral vector-mediated g-interferon gene transfer into tumor cells generates potent and long lasting antitumor immunity. Cancer Res. 50:7820. 25. Nishihara, K., S. Miyatake, T. Sakata, J. Yamashita, H. Kikuchi, Y. Kawade, Y. Zu, Y. Namba, M. Hanaoka, and Y. Watanabe. 1988. Augmentation of tumor targeting in a line of glioma-specific mouse cytotoxic T-lymphocytes by retroviral expression of mouse g-interferon complementary DNA. Cancer Res. 48: 4730. 26. Watanabe, Y., K. Kuribayashi, S. Miyatake, K. Nishihara, E. Nakayama, T. Taniyama, and T. Sakata. 1989. Exogenous expression of mouse interferon-g cDNA in mouse neuroblastoma C1300 cells results in reduced tumorigenicity by augmented anti-tumor immunity. Proc. Natl. Acad. Sci. USA 86:9456. 27. Restifo, N. P., P. J. Spiess, S. E. Karp, J. J. Mule, and S. A. Rosenberg. 1992. A poorly immunogenic sarcoma transduced with the cDNA for interferon-g elicits CD81 T cells against the wild-type tumor: correlation with antigen presentation capability. J. Exp. Med. 175:1423. 28. Porgador, A., R. Bannerji, Y. Watanabe, M. Feldman, E. Gilboa, and L. Eisenbach. 1993. Antimetastatic vaccination of tumor-bearing mice with two types of IFN-g gene-inserted tumor cells. J. Immunol. 150:1458.

29. Asher, A. L., J. J. Mule, A. Kasid, N. P. Restifo, J. C. Salo, C. M. Reichert, G. Jaffe, B. Fendly, M. Kriegler, and S. A. Rosenberg. 1991. Murine tumor cells transduced with the gene for tumor necrosis factor-a: evidence for paracrine immune effects of tumor necrosis factor against tumors. J. Immunol. 146:3227. 30. Karp, S. E., A. Farber, J. C. Salo, P. Hwu, G. Jaffe, A. L. Asher, E. Shiloni, N. P. Restifo, J. J. Mule, and S. A. Rosenberg. 1993. Cytokine secretion by genetically modified poorly immunogenic murine fibrosarcoma: tumor inhibition by IL-2 but not tumor necrosis factor. J. Immunol. 150:896. 31. Vanhaesebroeck, B., M. Mareel, F. Van Roy, J. Grooten, and W. Fiers. 1991. Expression of the tumor necrosis factor gene in tumor cells correlates with reduced tumorigenicity and reduced invasiveness in vivo. Cancer Res. 51:2229. 32. Colombo, M. P., G. Ferrari, A. Stoppacciaro, M. Parenza, M. Rodolfo, F. Mavilio, and G. Parmiani. 1991. Granulocyte colony-stimulating factor gene transfer suppresses tumorigenicity of a murine adenocarcinoma in vivo. J. Exp. Med. 173:889. 33. Dranoff, G., E. Jaffee, A. Lazenby, P. Golumbek, H. Levitsky, K. Brose, V. Jackson, H. Hamada, D. Pardoll, and R. C. Mulligan. 1993. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc. Natl. Acad. Sci. USA 90:3539. 34. Arca, M. J., J. C. Krauss, A. Aruga, M. J. Cameron, S. Shu, and A. E. Chang. 1996. Therapeutic efficacy of T cells derived from lymph nodes draining a poorly immunogenic tumor transduced to secrete granulocyte-macrophage colony-stimulating factor. Cancer Gene Ther. 3:39. 35. Ohno, K., H. Yoshizawa, H. Tsukada, T. Takeda, Y. Yamaguchi, K. Ichikawa, Y. Maruyama, Y. Suzuki, E. Suzuki, and M. Arakawa. 1996. Adoptive immunotherapy with tumor-specific T lymphocytes generated from cytokine genemodified tumor-primed lymph node cells. J. Immunol. 156:3875. 36. Linsley, P. S., and J. A. Ledbetter. 1993. The role of the CD28 receptor during T cell responses to antigen. Annu. Rev. Immunol. 11:191. 37. Schwartz, R. H. 1992. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 71: 1065. 38. Hathcock, K. S., G. Laszlo, C. Pucillo, P. Linsley, and R. J. Hodes. 1994. Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function. J. Exp. Med. 180:631. 39. Townsend, S. E., and J. P. Allison. 1993. Tumor rejection after direct costimulation of CD81 T cells by B7-transfected melanoma cells. Science 259:368. 40. Chen, L., S. Ashe, W. A. Brady, I. Hellstrom, K. E. Hellstrom, J. A. Ledbetter, P. McGowan, and P. S. Linsley. 1992. Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell 71:1093. 41. Chen, L., P. McGowan, S. Ashe, J. Johnston, Y. Li, I. Hellstrom, and K. E. Hellstrom. 1994. Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity. J. Exp. Med. 179:523. 42. Baskar, S., Rosenberg S. Ostrand, N. Nabavi, L. M. Nadler, G. J. Freeman, and L. H. Glimcher. 1993. Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major histocompatibility complex class II molecules. Proc. Natl. Acad. Sci. USA 90:5687. 43. Li, Y., P. McGowan, I. Hellstrom, K. E. Hellstrom, and L. Chen. 1994. Costimulation of tumor-reactive CD41 and CD81 T lymphocytes by B7, a natural ligand for CD28, can be used to treat established mouse melanoma. J. Immunol. 153:421. 44. Ramarathinam, L., M. Castle, Y. Wu, and Y. Liu. 1994. T cell costimulation by B7/BB1 induces CD8 T cell-dependent tumor rejection: an important role of B7/BB1 in the induction, recruitment, and effector function of antitumor T cells. J. Exp. Med. 179:1205. 45. Karasuyama, H., A. Kudo, and F. Melchers. 1990. The proteins encoded by the VpreB and l5 pre-B cell-specific genes can associate with each other and with m heavy chain. J. Exp. Med. 172:969. 46. Wexler, H. 1966. Accurate identification of experimental pulmonary metastases. J. Natl. Cancer Inst. 36:641. 47. Restifo, N. P., F. Esquivel, A. L. Asher, H. Stotter, R. J. Barth, J. R. Bennink, J. J. Mule, J. W. Yewdell, and S. A. Rosenberg. 1991. Defective presentation of endogenous antigens by a murine sarcoma: implications for the failure of an anti-tumor immune response. J. Immunol. 147:1453. 48. Zou, J. P., J. Shimizu, K. Ikegame, N. Yamamoto, S. Ono, H. Fujiwara, and T. Hamaoka. 1992. Tumor-bearing mice exhibit a progressive increase in tumor antigen-presenting cell function and a reciprocal decrease in tumor antigen-responsive CD41 T cell activity. J. Immunol. 148:648. 49. Li, X. F., H. Takiuchi, J. P. Zou, T. Katagiri, N. Yamamoto, T. Nagata, S. Ono, H. Fujiwara, and T. Hamaoka. 1993. Transforming growth factor-b (TGF-b)mediated immunosuppression in the tumor-bearing state: enhanced production of TGF-b and a progressive increase in TGF-b susceptibility of anti-tumor CD41 T cell function. Jpn. J. Cancer Res. 84:315. 50. Cheng, X., and D. M. Lopez. 1998. CD41, but not CD81, T cells from mammary tumor-bearing mice have a down-regulated production of IFN-g: role of phosphatidyl serine. J. Immunol. 160:2735. 51. Kagamu, H., J. E. Touhalisky, G. E. Plautz, J. C. Krauss, and S. Shu. 1996. Isolation based on L-selectin expression of immune effector T cells derived from tumor-draining lymph nodes. Cancer Res. 56:4338. 52. Kagamu, H., and S. Shu. 1998. Purification of L-selectin (low) cells promotes the generation of highly potent CD4 antitumor effector T lymphocytes. J. Immunol. 160:3444.