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[Cancer Research 64, 1490-1495, February 15, 2004]
© 2004 American Association for Cancer Research

Immunology

Generation and Targeting of Human Tumor-Specific Tc1 and Th1 Cells Transduced with a Lentivirus Containing a Chimeric Immunoglobulin T-Cell Receptor

Hiroshi Gyobu1,2, Takemasa Tsuji1, Yoshinori Suzuki1,2, Takayuki Ohkuri1, Kenji Chamoto1, Masahide Kuroki3, Hiroyuki Miyoshi4, You Kawarada2, Hiroyuki Katoh2, Tsuguhide Takeshima1 and Takashi Nishimura1

1 Division of Immunoregulation, Institute for Genetic Medicine and2 Surgical Oncology, Cancer Medicine, Division of Cancer Medicine, Hokkaido University, Sapporo;3 First Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka; and4 Subteam for Manipulation of Cell Fate, BioResource Center, RIKEN Tsukuba Institute, Ibaraki Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD4+ Th cells, in particular IFN-{gamma}-producing Th1 cells, play a critical role in the activation and maintenance of Tc1 cells that are essential for tumor eradication. Here, we report the generation of artificial tumor-specific Th1 and Tc1 cells from nonspecifically activated T cells using a lentiviral transduction system. Anti-CD3-activated T cells from healthy human donors were transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor gene composed of single-chain variable fragments derived from an anticarcinoembryonic antigen (CEA)-specific monoclonal antibody fused to an intracellular signaling domain derived from the cytoplasmic portions of membrane-bound CD28 and CD3{zeta}. These artificial tumor-specific Tc1 and Th1 cells, termed Tc1- and Th1-T bodies, respectively, could be targeted to CEA+ tumor cells independently of MHC restriction. Specifically, Tc1-T bodies demonstrated high cytotoxicity and produced IFN-{gamma} in response to CEA+ tumor cell lines but not CEA- tumors. Although Th1-T bodies exhibited low cytotoxicity, they secreted high levels of IFN-{gamma} and interleukin-2 in response to CEA+ tumor cells. Such CEA+ tumor-specific activation was not observed in mock gene-transduced nonspecific Tc1 and Th1 cells. Moreover, Tc1- and Th1-T bodies exhibited strong antitumor activities against CEA+ human lung cancer cells implanted into RAG2-/- mice. Furthermore, combined therapy with Tc1- and Th1-T bodies resulted in enhanced antitumor activities in vivo. Taken together, our findings demonstrate that Tc1- and Th1-T bodies represent a promising alternative to current methods for the development of effective adoptive immunotherapies.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been well accepted that T cells, most notably CD4+ Th and CD8+ Tc cells, play an important role in cancer immunotherapy (1) . T cells with antitumor activities recognize peptides derived from tumor rejection antigens (TRAs) bound by MHC molecules. Because genes encoding TRA were isolated, many investigators have tried to identify MHC class I- or class II-binding TRA peptides that can be used to develop tumor vaccines for treatment of cancer patients (2, 3, 4, 5) . However, although clinical studies have indicated that TRA peptides can elicit antigen-specific, tetramer-positive CTLs in vivo, the increased frequency of CTLs did not lead to tumor rejection in these patients (6) . The low effectiveness of antitumor CTLs may be caused by a number of factors: (a) CTLs may be ineffective in reacting with tumor cells because such cells lack or down-regulate MHC and/or costimulatory molecules that are essential for inducing the full activation of T cells (7 , 8) ; and (b) CTLs may fail to be fully activated in tumor-bearing hosts because such individuals often exhibit significant immunosuppression and defective helper T-cell function (9 , 10) .

To overcome these problems, we have developed an efficient method to prepare tumor-specific artificial Tc1 and Th1 cells from nonspecifically activated human CD8+ and CD4+ T cells using a lentiviral transduction system. We infected anti-CD3-stimulated CD4+ and CD8+ T cells isolated from human peripheral blood mononuclear cells (PBMCs) with a lentivirus containing a chimeric immunoglobulin T-cell receptor (cIgTCR) composed of single-chain variable fragments (scFv) derived from an anticarcinoembryonic antigen (CEA) monoclonal antibody (mAb) and an intracellular signaling domain derived from the cytoplasmic part of membrane-bound CD28 and CD3{zeta}. Such artificially generated antigen-specific T cells, which we refer to as T bodies, thereby possess the advantage of binding with Ag in an MHC-independent manner, while maintaining the capacity to efficiently activate the effector functions of T cells.

Since T body technology was reported (11 , 12) , most investigators have focused on the cytotoxic function and targeting of these cells. The functional consequences of preparing T bodies with distinct effector functions but with the identical Ag specificity have not been evaluated. It is now well accepted that Th1-dominant immunity is important for successful induction of antitumor immunity in tumor-bearing hosts (13 , 14) . Moreover, the Th1/Tc1 circuit in tumor-bearing hosts is crucial for complete tumor eradication (15 , 16) . Therefore, we reasoned that an efficient method to prepare tumor-specific Th1- and Tc1-T bodies with the same antigenic specificity will be helpful for the development of novel adoptive tumor immunotherapies.

In the present study, we document the generation and targeting of human tumor-specific artificial Tc1- and Th1-T bodies. Tc1-T bodies derived from anti-CD3 mAb-activated CD8+ T cells specifically lysed tumor cells and produced IFN-{gamma}. Th1-T bodies produced high levels of interleukin (IL)-2 and IFN-{gamma} in response to CEA+ tumor cells but showed limited cytotoxicity. Moreover, we demonstrate that Tc1- and/or Th1-T bodies exhibit strong antitumor activities against CEA+ human lung cancer cells implanted into recombination activating gene (RAG)2-/- mice. Thus, our novel and effective protocol for the preparation of Tc1- and Th1-T bodies may provide a new strategy for adoptive tumor immunotherapy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The human tumor cell lines HLC-1 (lung cancer), Daudi (lymphoma), KATO-III (gastric cancer), SH10 (gastric cancer), and AZ521 (gastric cancer) were cultured in RPMI 1640 (Sigma, St. Louis, MO) containing 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.) and supplemented with 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol (Sigma), HEPES, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate (RPMI-10S). 293T cells were maintained in DMEM (Sigma) containing 10% fetal bovine serum and supplemented with 2 mM L-glutamine, HEPES, penicillin, and streptomycin (DMEM-10S).

Animals.
BALB/c background RAG-2-/- mice were donated by Dr. M. Ito (Central Institute for Experimental Animals, Kanagawa, Japan). All of the mice were male and used at 10–12 weeks of age.

cIgTCR Gene and Lentiviral Vector.
The cDNA F39scFv/CIR-2, containing the scFv derived from F11–39 mAb specific for CEA, the CD8{alpha} hinge lesion, the CD28 transmembrane and cytoplasmic domain, and the CD3{zeta} cytoplasmic domain, has been described (Refs. 17 and 18 ; Fig. 1Citation ). The third generation lentivirus vector system used in the experiments was developed by one of us (H. M). Plasmid vector CSII-EF-MCS-IRES-hrGFP (CSII-GFP) contains a multiple cloning site (MCS) and the gene encoding green fluorescent protein (GFP; Ref. 19 ). F39scFv/CIR-2 cDNA was inserted into the EcoRI and NotI restriction enzyme sites within the multiple cloning site of the CSII-GFP vector (CSII-CIR-GFP). As a control vector, CSII-GFP was used.



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  		Fig. 1. Schematic representation of the chimeric immunoglobulin T-cell receptor cDNA: EF, human elongation factor 1{alpha} subunit gene promoter; L, leader peptide gene; V{kappa}, light chain variable region gene of F11–39 anticarcinoembryonic antigen monoclonal antibody; VH, heavy chain variable region of F11–39 monoclonal antibody; TM, transmembrane domain; Cyto, cytoplasmic domain; IRES, internal ribosomal entry site; GFP, green fluorescent protein.

 
Preparation of Lentiviral Vectors.
293T cells (5 x 106) were plated on 24 10-cm dishes precoated with 0.002% poly-L-lysine (Sigma). Later (20–24 h), CS II, pMD.G, pMDLg/pRRE, and pRSV-Rev were cotransfected by calcium phosphate transfection (20) . After 12–16 h, medium was aspirated, and 7.5 ml DMEM-10S containing 10 µM forskolin (Sigma F3917) were added to each dish. Later (48 h), virus vector-containing supernatant was collected and passed through a 0.45-µm filter. Then, supernatant was concentrated by ultracentrifugation at 68,000 x g and stored at -80°C until use.

Generation of T Bodies.
PBMCs (2 x 105 cells) were isolated from healthy donors by density gradient centrifugation using Ficoll-Paque PLUS (Amersham Bioscience, Sweden). Purified cells were then cultured with immobilized anti-CD3 mAb (5 µg/ml; PharMingen, San Diego, CA) and Retronectin (25 µg/ml; Takara Biomedicals, Shiga, Japan) for 2 h at 37°C. After incubation, PBMCs were infected twice with lentivirus vectors at a 24-h interval by adding viral supernatants. The infected cells were expanded by culture with RPMI-10S medium containing IL-2 (100 units/ml), IL-12 (50 units/ml), IFN-{gamma} (20 ng/ml), and anti-IL-4 mAb (2 µg/ml; PharMingen) for the generation of Th1- or Tc1-T bodies. Lentivirus supernatants were added at multiplicities of infection of 100–150.

Purification of T Bodies.
To determine the transduction efficacy, T cells expressing cIgTCR were detected by GFP fluorescence using flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA). The GFP gene is expressed under the control of the internal ribosomal entry site sequence, which is inserted downstream of the cIgTCR gene in the lentiviral vectors (Fig. 1)Citation . CD4+ or CD8+ cells were first separated by using immunomagnetic bead systems (MACS; Miltenyi Biotec, Glodbach, Germany) according to the manufacturer’s instructions. CD4+GFP+ or CD8+GFP+ cells were then separated by using a FACSVantage instrument (BD Biosciences). Isolated T bodies used in the following experiments were >95% pure.

Detection of Cytokine Activity.
T bodies (5 x 104 cells) and tumor cell lines (5 x 104 cells) were cocultured in 96-well round-bottomed plates in 200 µl of RPMI-10S medium. After 24-h incubation, supernatants were collected, and IFN-{gamma} and IL-2 levels were determined by ELISA using the OptEIA Human IFN-{gamma} set (PharMingen) and Quantikine Human IL-2 Immunoassay kit (R&D Systems, Minneapolis, MN).

Determination of Cytotoxicity.
The cytotoxicity mediated by T bodies was measured by 4-h-51Cr-release assays as described previously (21) . Tumor-specific cytotoxicity was determined using human CEA+ cell lines HLC-1 and KATO-III as target cells. As a control, CEA- human cell lines AZ521, SH10, and Daudi were used. The percentage of cytotoxicity was calculated as described previously (21) .

Winn’s Assay.
A mixture of HLC-1 with or without T bodies was injected intradermally into the abdominal skin of RAG-2-/--BALB/c mice. The tumor growth inhibitory effect of T bodies was determined by measuring change over time in the means of two perpendicular diameters of the tumor mass, as described previously (16) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Preparation of T Bodies.
We first examined the efficacy of lentiviral transduction. PBMCs obtained from healthy donors were stimulated with immobilized anti-CD3 mAb and then infected with lentiviral vectors. To polarize T cells into IFN-{gamma}-producing Th1- or Tc1-T bodies, the transduced T cells were expanded in the presence of IL-2 (100 units/ml), IL-12 (50 units/ml), IFN-{gamma} (20 ng/ml), and anti-IL-4 mAb (2 µg/ml) for 7–10 days. The transduction efficacy of lentivirus vectors containing cIgTCR or a mock gene into CD4+ Th or CD8+ Tc cells was determined by measuring the percentage of GFP+ cells using flow cytometry. As shown in Fig. 2Citation , ~30% of CD4+ Th cells and ~20% of CD8+ Tc cells were successfully transduced with the cIgTCR gene in this experiment. We repeatedly tried this experiment using PBMCs obtained from six different donors, and the average of transduction efficiency in 16 separate experiments was 42% for CD4+ T cells and 23% for CD8+ T cells. The efficacy of transduction of the control CSII-GFP vector into CD4+ Th or CD8+ Tc cells was ~90 and 80%, respectively. The cIgTCR gene-transduced CD4+- and CD8+-T bodies were highly enriched by cell sorting at >95% purity and used for the experiments. The prepared Th1- or Tc1-T bodies were easily expanded by stimulation with immobilized anti-CD3 mAb.



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  		Fig. 2. Green fluorescent protein (GFP) expression of Th1 and Tc1 cells transduced with a lentiviurs containing chimeric immunoglobulin T-cell receptor-GFP or mock-GFP gene. Anti-CD3 monoclonal antibody-activated peripheral blood mononuclear cells were transduced with lentiviral vectors and expanded for 7–10 days under Th1-polarizing conditions as described in "Materials and Methods." Cells were stained with phycoerythrin-conjugated anti-CD4 or -CD8 monoclonal antibody, and GFP expression on Th1 and Tc1 cells was investigated by flow cytometry (A–D). GFP-expressing Th1 or Tc1 cells were isolated by cell sorting and reanalyzed (E–H). GFP expression on mock-GFP gene-transduced Th1 (A and E) and Tc1 (C and G) cells and chimeric immunoglobulin T-cell receptor-GFP gene-transduced Th1 (B and F) and Tc1 (D and H) is shown. The typical data in 16 different experiments were shown.

 
Cytokine Production by Th1- and Tc1-T Bodies in Response to CEA-Positive Human Tumor Cell Lines.
The capacity of T bodies to produce cytokines in response to CEA-expressing tumor cells was examined by measuring cytokine levels in the culture supernatant. T bodies or mock gene-transduced T cells were cocultured with CEA+ or CEA- tumor cells for 24 h. Culture of CD4+- or CD8+-T bodies by themselves caused no significant release of IFN-{gamma} (data not shown). In contrast, CD4+- or CD8+-T bodies produced high levels of IFN-{gamma} when they were cocultured with CEA+ tumor cells (Fig. 3)Citation . These CD4+ or CD8+ T bodies did not produce IL-4, IL-5, or IL-13 in response to stimulation with CEA+ tumor cells (data not shown), indicating that Th1- and Tc1-T bodies were successfully induced from nonspecifically activated T cells in Th1-polarizing conditions. Th1- and Tc1-T bodies did not produce IFN-{gamma} in response to CEA- tumor cells, indicating that IFN-{gamma} production from T bodies was induced in an antigen-specific manner (Fig. 3)Citation . No significant IFN-{gamma} production was detected in supernatants of control T cells cocultured with CEA+ or CEA- tumor cells. We also evaluated the amount of IL-2 secretion from Th1- or Tc1-T bodies stimulated with tumor cells. As shown in Fig. 4Citation , Th1-T bodies produced high amounts of IL-2 in response to CEA+ HLC-1 cells but not CEA- Daudi cells, whereas Tc1-T bodies produced no significant IL-2. It was also demonstrated that Th1-T bodies produced high levels of cytokines independently on Class II and B7-signaling molecules, because HCL-1 cells expressed neither class II, B7–1, nor B7–2 on their cell surface (Fig. 3)Citation .



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  		Fig. 3. Antigen-specific IFN-{gamma} production of T bodies after 24-h coincubation with tumor cells. A, carcinoembryonic antigen (CEA) expression of target tumor cell lines. Tumor cell lines were stained with F11–39 anti-CEA monoclonal antibody or mouse myeloma IgG as control antibody. After extensive washing, cells were stained with FITC-conjugated rabbit antimouse IgG antibody. CEA expression of HLC-1 (a), KATO-III (b), AZ521 (c), SH10 (d), and Daudi (e) cells is shown in histograms. In B, expression of MHC class I (a), class II (b), B7–1 (c), and B7–2 (d) on HLC-1 was examined by flow cytometry. Dotted lines, unstained control curve; solid lines, stained curve. In C, T bodies or control T cells (5 x 104) and target tumor cells (5 x 104) were cocultured in 96-well round-bottomed plates for 24 h, and the IFN-{gamma} levels in the supernatants were measured by ELISA. ND, not detectable. The similar results were obtained in three separate experiments.

 


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  		Fig. 4. Production of interleukin (IL)-2 by Th1-T bodies activated by carcinoembryonic antigen-expressing tumor cells. T bodies or control cells (5 x 104) were cocultured with carcinoembryonic antigen+ HLC-1 tumor cells or carcinoembryonic antigen- Daudi tumor cells (5 x 104) in 96-well round-bottomed plates for 24 h. IL-2 levels in supernatants were measured by ELISA. The similar results were obtained in three separate experiments.

 
Anti-CEA Antibody Blocks Cytokine Production by T Bodies Stimulated with CEA-Expressing Tumor Cells.
To evaluate whether T bodies specifically recognize CEA antigen expressed on tumor cells, we examined the capacity of anti-CEA mAb to block cytokine production by Th1- or Tc1-T bodies cocultured with CEA+ tumor cells. As clearly shown in Fig. 5Citation , IFN-{gamma} production of T bodies was almost completely blocked by adding anti-CEA mAb, whereas the addition of control mAb caused no significant changes in IFN-{gamma} production.



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  		Fig. 5. Blocking effect of parental F11–39 anticarcinoembryonic antigen (CEA) monoclonal antibody (mAb) on IFN-{gamma} production by T bodies. T bodies or control cells (5 x 104cells) were cocultured with CEA+ HLC-1 tumor cells (5 x 104) in 96-well round-bottomed plates with or without anti-CEA mAb or with control mouse myeloma IgG at a concentration of 20 µg/ml. After 20 h, supernatants were collected, and the IFN-{gamma} levels were measured by ELISA. ND, not detectable. The similar results were obtained in three separate experiments.

 
Cytotoxicity Mediated by T Bodies against CEA+ Tumor Cells.
The cytotoxicity mediated by Th1- or Tc1-T bodies was examined by 4-h-51Cr-release assays. As shown in Fig. 6Citation , Tc1-T bodies exhibited a strong cytotoxicity against CEA+ tumor cells but not against CEA- tumor cells. In contrast, Th1-T bodies showed marginal cytotoxicity against CEA+ tumor cells. In some experiments, Th1-T bodies exhibited low but significant cytotoxicity, but their cytotoxicity was always lower than that of Tc1-T bodies. Control Th1 or Tc1 cells lysed neither CEA+ nor CEA- tumor cells. We further demonstrated that the cytotoxicity as well as cytokine production capacity of Tc1- and Th1 T bodies was strongly blocked by anti-CEA mAb (Fig. 7)Citation .



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  		Fig. 6. Antigen-specific cytotoxic activities of Th1- and Tc1-T bodies. Cytotoxicities of T bodies or control T cells against carcinoembryonic antigen+ tumor cells (a, HLC-1; b, KATO-III) or carcinoembryonic antigen- tumor cells (c, AZ521; d, SH10; e, Daudi) were determined by 4-h 51Cr-release assay. The similar results were obtained in three separate experiments.

 


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  		Fig. 7. Cytotoxic activities of T bodies are blocked by parental F11–39 anticarcinoembryonic antigen (CEA) monoclonal antibody (mAb). Cytotoxicities against the CEA+ tumor cell line HLC-1 were measured by 4-h 51Cr-release assay. Blocking effect of F11–39 mAb on cytotoxicities was evaluated by adding anti-CEA mAb (20 µg). Mouse myeloma IgG (20 µg/ml) was used as a control mAb. The similar results were obtained in three separate experiments.

 
Thus, these findings clearly demonstrate that T bodies artificially generated from nonspecifically activated T cells exhibit cytokine production and cytotoxicity in an antigen-specific manner.

Antitumor Activities of T Bodies in Vivo.
We next evaluated the antitumor activity of T bodies against human CEA+ tumor cells in vivo. Th1-T bodies, Tc1-T bodies, or both were mixed with CEA+ tumors and intradermally injected into RAG2-/- mice (Fig. 8)Citation . Th1-T bodies alone were unable to completely eradicate tumors, despite the fact that these cells strongly inhibited tumor growth in vivo. Tc1-T bodies by themselves initially reduced tumors to a size that was not palpable or visible, but tumors recurred. However, when both Th1- and Tc1-T bodies were injected together with CEA+ tumor cells, RAG2-/- mice completely rejected the tumors. Such antitumor activity was not induced when control T cells were mixed with tumors and transferred into RAG2-/- mice. Neither Th1- nor Tc1-T bodies, either individually or in combination, exhibited antitumor activity in vivo against CEA- tumor cells implanted into RAG2-/- mice (data not shown). Thus, these results demonstrate that artificial human Tc1-T bodies have profound antitumor activity in vivo if they were combined with Th1-T bodies.



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  		Fig. 8. Antitumor activities mediated by T bodies in vivo. A mixture of HLC-1 (2 x 106 cells) and T bodies or control T cells was injected intradermally into RAG2-/--BALB/c mice. The mean of two perpendicular measurements of the tumor diameter was determined thereafter. {circ}, tumor only; {triangleup}, mock gene-transduced Th1 and Tc1 cells (2 x 106 cells each); {blacksquare}, Th1-T body (2 x 106 cells); {blacktriangleup}, Tc1-T body (2 x 106cells); {bullet}, Th1-T and Tc1-T bodies (2 x 106 cells each). Five mice per each group were used for experiments. The similar results were obtained in two separate experiments.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have established an efficient method for the preparation of Th1- and Tc1-T bodies by lentiviral transduction of a cIgTCR containing the scFv derived from an anti-CEA mAb fused with the intracellular signaling domains of membrane-bound CD28 and CD3{zeta} molecules. These artificial tumor-specific Tc1- and Th1-T bodies, as compared with physiologically induced TRA-specific Tc1 and Th1 cells, have several advantages for tumor immunotherapy.

First, T bodies can be easily induced from anti-CD3 mAb-activated PBMCs (Fig. 2)Citation , and this technology is applicable to all tumor patients. Since the discovery of TRA peptides, it has become possible to expand tumor-specific CTLs in vivo using TRA-bound DC cells (2 , 3) . However, it still takes a long time to expand enough numbers of tumor-specific CTLs from PBMCs or tumor-infiltrating lymphocytes isolated from patients. Although some MHC class II-binding tumor rejection peptides were discovered, expansion of tumor-specific Th1 cells in vitro has proven very challenging (4) . These difficulties in inducing and expanding tumor-specific Th1 and Tc1 cells have hampered their application to adoptive tumor immunotherapy. Our established method provides a rapid, easy, and general protocol for inducing and expanding tumor-specific IFN-{gamma}-producing Tc1 and Th1 cells. This methodology should prove useful for adoptive immunotherapy of patients with tumors or infectious diseases.

Secondly, T bodies can recognize tumor-specific antigens via the Ag-binding domain of an scFv in an MHC-nonrestricted fashion. Therefore, T bodies can exhibit their functions in response to tumor cells even when tumor cells express low levels of MHC. It has been reported that MHC expression is down-modulated in tumor-bearing hosts (7 , 22) . Such down-modulation of MHC may results in the escape of tumor cells from immunosurveillance mechanisms mediated by T-cell immunity. In addition to MHC molecules, the expression of costimulatory molecules, such as B7 and CD40, play a crucial role for long-lasting and effective T-cell activation (23, 24, 25, 26) . It has been demonstrated that FcR{gamma}- or CD3{zeta}-mediated signaling is sufficient to induce cytotoxicity against tumor cells. However, cytokine production by T bodies equipped with T-cell receptor-mediated signaling was greatly enhanced by CD28-mediated signaling (27) . We therefore prepared T bodies using cIgTCR gene containing signal transduction units of CD28 and CD3{zeta} molecules, which are essential for induction of cytokine production. Consistent with previous results (28 , 29) , T bodies transduced with cIgTCR gene exhibit a strong tumor-specific response to CEA+ tumor cells and show high levels of cytokine production and cytotoxicity, although HCL-1 cells expressed neither B7–1 nor B7–2. Moreover, HCL-1 tumor cells expressed class I but not class II molecules on their cell surface. However, Th1-T bodies produced high levels of IFN-{gamma} in response to CEA+ HLC tumor cells, indicating that T bodies recognize tumor antigen in MHC-independent manner, and trasduction units of CD28 in addition to CD3 {zeta} may contribute to transduce activation signaling in response to B7-negative tumor cells (Fig. 3)Citation .

Thirdly, it is anticipated that coinjection of Th1-T bodies with Tc1-T bodies may overcome immunosuppression in tumor-bearing hosts and facilitate the induction of antitumor immunity in vivo. It has been reported that T cells derived from tumor-bearing hosts show reduced immune responses as compared with healthy controls. Several factors may contribute to this immunosuppression, including defective Th cell function, overactivation of suppressive Treg cells, defective T-cell receptor{zeta} signaling, and transforming growth factor-ß production (9 , 10 , 30) . Using an animal model of tumor adoptive immunotherapy, we have proposed that introduction of local help to CTL with Th cells, most importantly Th1 cells, is crucial for overcoming immunosuppression. This hypothesis is supported by a number of recent findings: (a) DC/Th1 cell–cell interactions are critically important for inducing the complete activation of CTLs and long-term maintenance of CTL memory in vivo (31, 32, 33, 34) ; and (b) Th1/Tc1 circuits in tumor-bearing hosts are critical for inducing complete tumor eradication in vivo (14 , 15 , 19) . As shown in Fig. 8Citation , combined therapy with Th1- and Tc1-T bodies induced complete rejection of tumor cells, although Th1- or Tc1-T bodies alone were unable to completely reject coinjected tumor cells. Th1-T bodies showed the same levels of growth inhibition of tumor in vivo, although they exhibited lower cytotoxicity than Tc1-T bodies in vitro. This might be because of the potentiation of mouse natural effector cells (natural killer, M{phi}, etc.) involved in antitumor immunity by cytokines produced by Th1-T bodies. Although Tc1-T bodies exhibit higher levels of cytotoxicity against tumor cells in vitro, they failed to reject tumor cells by themselves. This may be caused by the failure of Tc1-T bodies to produce IL-2, which is essential for long-lasting CTL functions in vivo. In contrast, Th1-T bodies produced both IFN-{gamma} and IL-2 but showed low levels of cytotoxicity. Therefore, coinjection of Tc1-T and Th1-T bodies might be able to overcome the deficiency of Tc1-T bodies to produce IL-2 and exhibited the most effective antitumor activity in vivo.

The final goal of tumor immunotherapy is the development of an efficient strategy to enhance the concentration of tumor-specific Tc1 and Th1 cells at the local tumor site, either by tumor vaccine therapy or adoptive tumor cell therapy. To overcome the difficulties in inducing physiological tumor-specific Th1 and Tc1 cells, the TRA-specific immunoreceptor viral transduction strategy should prove highly effective. However, thus far, most investigators have focused on the cytotoxic function of T bodies or natural killer-T bodies. Moreover, nobody has tried to establish general protocol for preparing and expanding IFN-{gamma}-producing Th1- or Tc1-T bodies, although there were some reports that CD4+- or CD8+-T bodies were accidentally induced from PBMCs (35) . However, in the present study, we have first succeeded in establishment of a general protocol to prepare pure IFN-{gamma}-producing Th1-T and Tc1-T bodies, and we demonstrate that T bodies induce their effector functions in response to receptor engagement, in an antigen-specific but MHC-independent manner. The finding that combined therapy with Th1- and Tc1-T bodies induces complete rejection of tumor cells in vivo indicates that the efficient and effective protocol described here provides a novel strategy for adoptive tumor immunotherapy.


   ACKNOWLEDGMENTS
 
We thank Dr. Luc Van Kaer (Vanderbilt University School of Medicine, Nashville, TN) for reviewing this manuscript. We also thank Dr. Michiko Kobayashi (Genetics Institute, Cambridge, MA) and Takuko Sawada (Shionogi Pharmaceutical Institute Co., Osaka, Japan) for their kind donations of IL-12 and IL-2, respectively.


   FOOTNOTES
 
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.

Requests for reprints: Takashi Nishimura, at Division of Immunoregulation, Institute for Genetic Medicine, Hokkaido University, N-15 W-7, Kita-ku, Sapporo 060-0815, Japan. Phone and Fax: 81-11-706-6835; E-mail: tak24{at}imm.hokudai.ac.jp

Received 9/ 4/03. Revised 12/11/03. Accepted 12/12/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Van der Bruggen P., Traversari C., Chomoez P., Lurquin C., De Plaen E., Van den Eynde B., Knuth A., Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science (Wash. DC), 254: 1643-1647, 1991.[Abstract/Free Full Text]
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