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[Cancer Research 66, 1809-1817, February 1, 2006]
© 2006 American Association for Cancer Research

Immunology

An Essential Role of Antigen-Presenting Cell/T-Helper Type 1 Cell-Cell Interactions in Draining Lymph Node during Complete Eradication of Class II–Negative Tumor Tissue by T-Helper Type 1 Cell Therapy

Kenji Chamoto, Daiko Wakita, Yoshinori Narita, Yue Zhang, Daisuke Noguchi, Hideaki Ohnishi, Takeshi Iguchi, Tomoaki Sakai, Hiroaki Ikeda and Takashi Nishimura

Division of Immunoregulation, Section of Disease Control, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

Requests for reprints: Takashi Nishimura, Division of Immunoregulation, Section of Disease Control, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan. Phone: 81-81-706-7546; Fax: 81-11-706-7546; E-mail: tak24{at}igm.hokudai.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prior studies have shown that transfer of ovalbumin (OVA)-specific T helper type 1 (Th1) cells into mice bearing MHC class II+ OVA–expressing tumor cells (A20-OVA) causes complete tumor rejection. Here we show that, although Th1 cell therapy alone was not effective against MHC class II– OVA–expressing tumor cells (EG-7), treatment of mice bearing established EG-7 tumors by i.v. transfer of Th1 cells combined with i.t. injection of the model tumor antigen OVA induced complete tumor rejection. Transferred Th1 cells enhanced the migration of tumor-infiltrating antigen-presenting cells (APC) that had processed OVA into the draining lymph node (DLN). Although transferred Th1 cells were randomly distributed in DLN, distal LN, spleen, and tumor tissue, active proliferation of Th1 cells always initiated in DLN, where Th1 cells efficiently interacted with APC that presented OVA. In parallel, OVA-tetramer+ CTLs, showing EG-7-specific cytotoxicity, were highly induced in DLN and the local tumor site. The OVA-tetramer+ CTL functioned systemically because two bilateral tumor masses were both completely rejected on treatment of one tumor. Furthermore, either active proliferation of transferred Th1 cells or generation of tetramer+ CTL was not induced in MHC class II–deficient mice and LN-deficient Aly/Aly mice. These results indicate that DLN is an indispensable organ for initiating active APC/Th1 cell interactions, which is critical for inducing complete eradication of tumor mass by tumor-specific CTL. (Cancer Res 2006; 66(3): 1809-17)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ultimate goal of tumor immunotherapy is to develop an efficient strategy to induce tumor-specific CTLs, which eradicate tumors in vivo. Although many investigators approached this problem using MHC class I–binding peptides, this approach has been hampered by strong immunosuppression and unknown immune-escape mechanisms in tumor-bearing hosts (1–3). Recently, it has been reported that CD4+ T cells are crucial for the induction of effective antitumor immunity (4–12). In particular, introducing T helper type 1 (Th1)-dominant immunity in tumor-bearing hosts is critically important to overcome the immunosuppression and to induce fully activated tumor-specific CTL (4–7, 13). We have previously shown the distinct role of tumor-specific Th1 and Th2 cells in tumor immunity and provided evidence that adoptively transferred tumor-specific Th1 cells exhibited strong antitumor activity in vivo. These antitumor responses were able to induce complete rejection of a well-established MHC class II+ tumor mass by promoting the generation of tumor-specific CTL in vivo (4–7). However, because most human tumors do not express MHC class II molecules, it is necessary to develop an alternative therapeutic model to induce complete eradication of MHC class II– tumor mass by tumor-specific Th1 cells.

In some mouse models, MHC class II– tumor cells were rejected by treatment with tumor-specific CD4+ T cells alone or Th1 cells alone. However, these experiments investigated the capacity of adoptively transferred cells to prevent the development of tumors injected into mice after adoptive transfer or to inhibit the growth of small tumor mass (14–16). To develop a novel therapeutic strategy that is applicable to tumor patients with a strong immunosuppressive state, it is important to establish a method to cure mice bearing established MHC class II– tumors. Here, we show that ovalbumin (OVA)-specific Th1 cells can completely eradicate established OVA-expressing EG-7 tumor tissue by a combination therapy involving i.t. injection of a model tumor antigen, OVA. Using this model, we show that adoptively transferred Th1 cells, combined with i.t. injected of OVA, promote the generation of OVA-tetramer+ tumor-specific CD8+ CTL, which can function as final effector T cells against MHC class II– tumor cells. Moreover, we provide evidence for an indispensable role of draining lymph node (DLN), rather than of distal LN and spleen, for inducing efficient antigen-presenting cell (APC)/Th1 cell-cell interactions essential for the generation of tetramer+ tumor-specific CTL.

It has been shown that LN plays a central role in immune responses (17). Moreover, recent studies with chemokines and improved imaging technology have shown the dynamics of T-cell/dendritic cell interactions in LN (18–24). However, the precise role of LN in antitumor immunity has not been definitely elucidated yet and several contradictory results have been reported (25–28).

To resolve this issue, we have examined the role of DLN, distal LN, and spleen during the generation of tumor-specific CTL. Using LN-deficient Aly/Aly mice and MHC class II–deficient mice, we show that the initial MHC class II–mediated APC/Th1 interaction in DLN is critically important for inducing active proliferation and subsequent generation of tetramer+ tumor-specific CTL that can completely eradicate established tumor tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. C57BL/6 and BALB/c mice were obtained from Charles River Japan (Yokohama, Japan). OVA323-339-specific I-Ab-restricted T-cell receptor-transgenic mice (OT-2) maintained on the C57BL/6 background were kindly provided by F.R. Carbone (University of Melbourne, Victoria, Australia; ref. 29). OVA323-339-specific I-Ad-restricted T-cell receptor-transgenic mice (DO11.10) maintained on the BALB/c mice background were kindly donated by Dr. K.M. Murphy (Washington University School of Medicine, St. Louis, MO; ref. 30). MHC class II–deficient mice (Abb–/– mice, C57BL/6 background) were purchased from Takonic (Germantown, NY). Alymphoplasia mutant mice (Aly/Aly mice, C57BL/6 background), which are characterized by the systemic absence of lymph nodes and Peyer's patches (31), were purchased from CLEA Japan (Tokyo, Japan). All mice were female and were used at 6 to 8 weeks of age.

Cytokines, monoclonal antibodies, antigens, and tetramers. Interleukin (IL)-12 was kindly donated by Wyeth Research (Cambridge, MA). IL-2 was supplied by Takuko Sawada (Shionogi Pharmaceutical Institute Co. Ltd., Osaka, Japan). IFN-{gamma} was purchased from PeproTech EC Ltd. (London, United Kingdom). Anti-IL-4 monoclonal antibody (mAb; 11B11) was purchased from American Type Culture Collection (Rockville, MD). Phycoerythrin (PE)-anti-CD4 mAb, FITC-anti-CD45RB mAb, FITC-anti-CD8 mAb, FITC-anti-CD69 mAb, cychrome-anti-T-cell receptor ß mAb, PE-anti-CD8 mAb, PE-anti-NK1.1 mAb, PE-anti-CD11c mAb, and PE-anti-CD11b mAb were purchased from PharMingen (San Diego, CA). Anti-CD8 mAb-conjugated microbeads for the magnetic cell sorting system were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Anti-CD8 mAb for CD8+ T cell depletion was purchased from Meiji Nyugyo (Kanagawa, Japan). H-2Kb OVA tetramer-SIINFEKL-PE (OVA Tetramer) and H-2Kb tetramer-SIYRYYGL-PE (Control Tetramer) were purchased from MBL (Nagoya, Japan). Recombinant OVA323-339 peptide was kindly supplied by Fujiya Co. Ltd. (Hadano, Japan). OVA protein was purchased from Sigma-Aldrich Japan (Tokyo, Japan).

Generation of OVA-specific Th1 cells from DO11.10 or OT-2 T-cell receptor-transgenic mice. CD4+ CD45RB+ naïve T cells were isolated from nylon-passed spleen cells of DO11.10 or OT-2 T-cell receptor-transgenic mice using FACSVantage (Becton Dickinson, San Jose, CA) as previously reported (4). Purified CD4+ CD45RB+ cells were stimulated with 10 µg/mL OVA323-339 peptide in the presence of mitomycin C (MMC)–treated spleen cells, 20 units/mL IL-12, 1 ng/mL IFN-{gamma}, 50 µg/mL anti-IL-4 mAb, and 100 units/mL IL-2 for Th1 development. At 48 hours, cells were restimulated with OVA323-339 under the same conditions and used at 9 to 12 days of culture.

Analysis of cell surface phenotype. The phenotypic characterization of each cell subpopulation was carried out using a FACSCalibur flow cytometer (Becton Dickinson) and Cell Quest software. Detail procedures for staining have been described in a previous article (32). Fluorescence data were collected with logarithmic amplification. Mean fluorescence intensity was calculated using Cell Quest program.

Detection of carboxyfluorescein diacetate succinimidyl ester–labeled Th1 cells in vivo. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Molecular Probes, Inc. (Eugene, OR) and used for monitoring daughter cell generation in vivo as indicated in the instructions of the manufacturer. In brief, 2 µL of a CFSE stock solution (5 mmol/L in DMSO) were incubated with 10 mL of Th1 cells (1 x 107/mL) in PBS for 5 minutes at room temperature. Cells were washed thrice with 10% FCS-containing medium. Thirty-six hours after the transfer of CFSE-labeled Th1 cells (2 x 107 per mouse), the generation of daughter cells by the labeled Th1 cells in DLN, distant LN, spleen, or tumor tissue was analyzed using FACSCalibur. The fluorescence intensity was analyzed by ModFit LT software, which provides a robust, statistical method for identifying peaks in histograms (33). Fluorescence data were collected with logarithmic amplification. Mean fluorescence intensity was calculated using Cell Quest program.

Tumor immunotherapy model. MHC class II– EG-7 cells (2 x 106) were i.d. inoculated into C57BL/6 mice. When the tumor mass became large (8-9 mm), the tumor-bearing mice were treated with saline, OVA protein (200 µg/mouse), OVA-specific Th1 cells, or Th1 cells plus OVA. Th1 cells (2 x 107 per mouse) were i.v. injected into tumor-bearing mice combined with i.t. injection of OVA antigen thrice at intervals of 2 days. In the experiments testing bilateral established-tumor therapy, EG-7 cells (2 x 106) were inoculated on both flanks of the animals. When the tumor size reached 8 to 9 mm, OVA protein (200 µg/mouse) was injected into the tumor mass at the left side only, combined with i.v. transfer of Th1 cells (2 x 107 per mouse). The therapeutic model for MHC class II+ tumors was established using A20-OVA-bearing BALB/c mice and DO11.10-derived Th1 cells as previously described (4). The antitumor activity mediated by the transferred cells was determined by measuring tumor size in perpendicular diameters. Tumor volume was calculated by the following formula: tumor volume = 0.4 x length (mm) x [width (mm)] 2 (4). Tumor-bearing mice that survived for >60 days after therapy were considered completely cured. The mean of five mice per group is indicated in the graphs.

Monitoring of APC migration into DLN from tumor tissue. For analyzing cell migration of OVA antigen-presenting dendritic cells or M{phi} into DLN, OVA protein labeled with Alexa F488 (Molecular Probes) was i.t. injected into tumor-bearing mice. Thirty-six hours after the first round of therapy, DLN cells or tumor-infiltrating cells were stained with PE-conjugated anti-CD11c or anti-CD11b mAb to measure the percentage of Alexa F488–labeled OVA-presenting CD11c+ dendritic cells or CD11b+ M{phi} by FACSCalibur. Then, the absolute numbers of Alexa F488–labeled CD11c+ dendritic cells or Alexa F488–labeled CD11b+ M{phi} were calculated by the following formula: absolute number of the cells = percentage of Alexa F488–labeled dendritic cells (or M{phi}) in the tissue x total cell number in the tissue x 1/100. For analyzing APC/Th1 cell interactions in DLN by immunohistochemical analysis, CFSE–labeled Th1 cells were i.v. transferred into tumor-bearing mice and Alexa F594 (red)–labeled OVA was injected into the tumor tissue. Thirty-six hours after the treatment, DLNs were removed, frozen in optimum cutting temperature compound, and 4-µm cryostat sections were prepared. After washing with PBS, the samples were fixed in acetone for 10 minutes and APC/Th1 cell interactions in the section were examined under fluorescence microscopy equipped with FLUOVIEW FV500 software (Olympus, Tokyo, Japan).

Cytotoxicity assay. The cytotoxicity mediated by tumor-specific CTL was measured by 6-hour 51Cr-release assay as previously described (34). Tumor-specific cytotoxicity was determined using EG-7 cells (OVA gene–transfected EL-4 cells) as target cells. Parental EL-4 cells were used as control target cells. To confirm the antigen specificity of H-2Kb-restricted CTL, 51Cr-labeled target cells were incubated with CD8+ CTL pretreated with OVA-tetramer, which can block the recognition of H-2Kb-restricted target peptide (OVA257-264) by CTL. CD8+ T cells were enriched by magnetic cell sorting system according to the protocol of the manufacturer. The percent cytotoxicity was calculated as previously described (34).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adoptive transfer of tumor-specific Th1 cells, combined with i.t. injection of antigen, induces complete eradication of a well-established MHC class II– tumor mass. As previously reported (4), i.v. transfer of OVA-specific Th1 cells (2 x 107), differentiated in vitro from T-cell receptor-transgenic mice, into mice bearing MHC class II+ A20 tumor cells expressing OVA caused complete regression of the tumor mass (Fig. 1A). In contrast, Th1 cell transfer alone did not completely cure mice bearing MHC class II– OVA–expressing EG-7 tumors (Fig. 1B). However, treatment of mice bearing a large EG-7 tumor mass (8-9 mm) with three rounds of i.v. transfer of Th1 cells and i.t. injection of the model tumor antigen protein, OVA (200 µg), resulted in profound inhibition of tumor growth and all mice treated were completely cured (Fig. 1C-H). Neither Th1 cells alone (Fig. 1F) nor i.t. injection of OVA alone (Fig. 1G) induced a significant therapeutic effect against established EG-7 tumors. We further showed that no significant antitumor activity was induced by cell transfer of naïve OT-2-derived CD4+ T cells, indicating that in vitro activated Th1 cells are essential for inducing tumor cell rejection (data not shown).


Figure 1
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  		Figure 1. Transfer of tumor-specific Th1 cells combined with i.t. injection of tumor-related antigen causes complete eradication of a well-established MHC class II– tumor mass. A, MHC class II+ tumor A20-OVA cells were i.d. inoculated into BALB/c mice. When the tumor mass became palpable (8-9 mm), OVA-specific Th1 cells (2 x 107; bullet) or saline ({circ}) was i.v. injected once. B, MHC class II–/– tumor EG-7 cells were i.d. inoculated into C57BL/6 mice. When the tumor mass became palpable (8-9 mm), OVA-specific Th1 cells (2 x 107; {blacktriangleup}) or saline ({triangleup}) was i.v. transferred once. C to H, EG-7-bearing mice were treated thrice with saline (C and D, {triangleup}; photo E), Th1 cells (2 x 107; C and D, {blacktriangleup}; photo F), OVA protein (200 µg/mouse; C and D, {circ}; photo G), or Th1 cells + OVA protein (C and D, bullet; photo H). In each group, Th1 cells (2 x 107 per mouse) were i.v. transferred and OVA protein was i.t. injected thrice at 2-day intervals. The volume of the tumor mass was calculated as described in Materials and Methods. The fractional numbers in (A-C) indicate dead mice per total number of mice 60 days after tumor inoculation. Points, mean of five mice in each experimental group; bars, SE. Similar results were obtained in three separate experiments.

 

Figure 2
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  		Figure 2. Systemic Th1 cell therapy promotes the migration of tumor-infiltrating APC into DLN. A, EG-7-bearing mice were treated with i.v. transfer of Th1 cells plus i.t. injection of Alexa F488 (green)–conjugated OVA as described in Materials and Methods. Thirty-six hours after the first round of therapy, Alexa F488–labeled OVA-presenting CD11c+ dendritic cells or CD11b+ M{phi} in DLN were monitored by flow cytometry. B to D, the absolute numbers of OVA-presenting CD11c+ dendritic cells (Alexa F488+ CD11c+ dendritic cells) or CD11b+ M{phi} (Alexa F488+ CD11b+ M{phi}) in DLN (B and C) or in the tumor (D) were examined 36 hours after the first (B and D) or second round of therapy (C) as described in Materials and Methods. {square}, tumor-bearing mice treated with i.t. injection of model tumor antigen, OVA; {blacksquare}, tumor-bearing mice treated with Th1 cell transfer combined with i.t. injection of OVA. Columns, mean of five mice in each experimental group; bars, SE. Similar results were obtained in three separate experiments.

 
Systemic Th1 cell therapy promotes migration of APC that have taken up tumor antigens into DLN. To monitor the migration of APC that had taken up the tumor antigen (OVA) from the tumor tissue to DLN, we injected OVA conjugated with Alexa Fluor 488 carboxylic acid, succinimidyl ester (Alexa F488; green) i.t. into the tumor mass and we monitored the migration of Alexa F488+ APC, which processed Alexa F488–labeled OVA by flow cytometry in DLN. Thirty-six hours after the first or second round of Th1 cell therapy combined with i.t. injection of Alexa F488–labeled OVA, the percentage of Alexa F488+ CD11c+ and Alexa F488+ CD11b+ APC in DLN was monitored by fluorescence-activated cell sorting (FACS) after staining with PE-conjugated anti-CD11c or anti-CD11b mAbs (Fig. 2A). Then, using this staining strategy, the absolute numbers of migrated CD11c+ or CD11b+ APC in DLN or nonmigrated APC in the tumor mass were calculated (Fig. 2B-D). Interestingly, the absolute numbers of Alexa F488+ CD11c+ or Alexa F488+ CD11b+ APC in the tumor were significantly reduced 36 hours after the first round of Th1 cell therapy although this treatment did not exhibit any effect on tumor size at this time point (Fig. 2D). Conversely, the absolute numbers of labeled CD11c+ and CD11b+ APC, which migrated into DLN from the tumor tissue, increased with Th1 cell therapy (Fig. 2B and C). In particular, the increase of CD11c+ or CD11b+ APC in DLN became prominent 36 hours after the second round of Th1 cell therapy (Fig. 2C). Moreover, when bovine serum albumin was injected instead of OVA as a control, such enhancement of APC migration into DLN was not observed (data not shown). These results indicate that systemically transferred Th1 cells enhance the migration of dendritic cells and M{phi} that have taken up antigens from the tumor mass into DLN although we cannot completely deny the possibility that some of injected OVA protein moved to DLN directly.

Proliferation of transferred Th1 cells is mediated by interaction with tumor APC in the DLN. To monitor both migration and activation of transferred Th1 cells in lymphoid tissues (DLN, distal LN, and spleen) and the tumor site, Th1 cells were labeled with CFSE, which made it possible to monitor cellular proliferation in vivo by measuring the decrease in green fluorescence intensity. Mice were treated with CFSE-labeled Th1 cells and/or i.t. injection of Alexa F594–labeled OVA, and 36 hours later, cellular division of Th1 cells was evaluated by flow cytometry. As shown in Fig. 3B to D, active daughter cell generation was observed in DLN where transferred Th1 cells (green labeled) interacted with OVA APC (red labeled; Fig. 3A). The most extensive proliferation of transferred Th1 cells was induced when EG-7-bearing mice were treated with Th1 cell transfer combined with i.t. injection of OVA, but not with Th1 cells or i.t. OVA injection alone (Fig. 3B and C). Although CFSE-labeled Th1 cells were randomly distributed in lymphoid tissues and the tumor tissue, the most extensive Th1 cell proliferation was always observed within DLN 36 hours after the first or second round of Th1 cell therapy. It is also noteworthy that Th1 cell proliferation in DLN occurred earlier than in other tissues such as distal LN, spleen, and tumor mass (Fig. 3D). These results indicate that tumor antigen (OVA)-presenting APCs, which migrated into DLN from tumor tissue, play a pivotal role in inducing the initial active proliferation of transferred Th1 cells within DLN.


Figure 3
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  		Figure 3. Proliferation of transferred Th1 cells by interaction with APC in DLN. A, tumor-bearing mice were treated with i.v. transfer of CFSE-labeled Th1 cells (green) combined with i.t. injection of Alexa F594-labeled OVA (red). Thirty-six hours after treatment, tissue sections of DLN were examined by fluorescence microscopy as described in Materials and Methods. Typical APC/Th1 cell interactions are shown in photo A. B and C, the rate of proliferation of CFSE-labeled Th1 cells was analyzed using DLN of tumor-bearing mice 36 hours after the first (B) or second (C) round of therapy with saline (Control), OVA, Th1 cells, or Th1 cells + OVA. Hatched columns, rate of parental generation and first to fourth daughter cell generations in CFSE-labeled Th1 cells. D, tumor-bearing mice were treated with Th1 cell transfer combined with i.t. injection of OVA. As a control, mice were treated with saline (Control). Then, the proliferation of CFSE-labeled Th1 cells in DLN, distal LN, spleen, or tumor tissue (TIL) was measured by FACS. Hatched columns, rate of parental generation and first to fourth daughter cell generations in CFSE-labeled Th1 cells. Similar results were obtained in three separate experiments.

 
Th1 cell therapy combined with i.t. injection of OVA promotes the migration and activation of both nonspecific innate and tumor-specific acquired effector cells within DLN. Next, we investigated the expression levels of the early activation marker CD69 on natural killer (NK), NK T cells, CD8, dendritic cells, and M{phi} in DLN of EG-7-bearing mice, which were treated with Th1 cell adoptive transfer and/or i.t. injection of OVA. As shown in Fig. 4, the expression levels of CD69 were greatly increased on both innate effector cells (dendritic cells, M{phi}, NK and NK T cells) and acquired CD8+ T cells in tumor-bearing mice that were treated with Th1 cells and injected with OVA. In addition, higher numbers of NK, NK T cells, dendritic cells, M{phi}, and T cells were present in DLN of these animals (data not shown). In sharp contrast, neither enhanced cell infiltration nor activation was observed in tumor-bearing mice treated with Th1 cells or injected with OVA alone. Thus, Th1 cells are beneficial not only for enhancing the migration of antigen presenting APC into DLN but also for promoting migration and activation of innate and acquired effector cells into DLN.


Figure 4
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  		Figure 4. Th1 cell therapy combined with i.t. injection of OVA promotes the activation of both innate and acquired effector cells in DLN. DLNs were prepared from tumor-bearing mice 36 hours after treatment with saline (Control), OVA, Th1 cells, or Th1 cells + OVA. Then, the expression of the early activation marker CD69 on NK1.1+ TCR– NK cells, NK1.1+ TCR+ NK T cells, CD8+ T cells, CD11c+ dendritic cells (DC), and CD11b+ M{phi} was examined by FACS analysis. The number attached in each box means the percentage of CD69+ cell population among the upper cell subsets indicated as NK, NK T cells, CD8, dendritic cells, and M{phi}. Similar results were obtained in three separate experiments.

 
Concomitant with nonspecific activation of innate effector cells, CD69 expression levels of CD8+ T cells were 3-fold increased by treatment with Th1 cells and OVA injection. Therefore, we wanted to investigate whether our treatment protocol induced the development of OVA-tetramer+ CD8+ CTL. As shown in Fig. 5A(a-j), increased numbers of tetramer+ CD8+ T cells were induced in tumor-bearing mice 24 hours after treatment with Th1 cells and OVA antigen injection. The increase in tetramer+ CD8+ T cells was not due to nonspecific binding of OVA-tetramers because <0.1% of CD8+ T cells stained with control tetramer reagent (Fig. 5A, e and j). A higher percentage of tetramer+ CD8+ T cells was induced in DLN (4.6%) and these cells also accumulated in the tumor [17.9% of CD8+ tumor-infiltrating lymphocytes (TIL)] as compared with the distal LN (1.4%) (Fig. 5A, k-n). These tetramer+ CD8+ T cells exhibited specific cytotoxicity against EG-7 but not against parental EL-4 tumor cells (Fig. 5B). The differential susceptibility of EG-7 and EL-4 was not due to the distinct levels of MHC expression (data not shown). CD8+ T cells purified from DLN and TIL exhibited strong cytotoxicity against EG-7 cells and their cytotoxicity was strongly blocked by H-2b-OVA-tetramer (Fig. 5B, c-f). Thus, Th1 cell therapy combined with i.t. injection of OVA induces tetramer+ CD8+ CTLs that specifically lyse EG-7 tumor cells by recognition of H-2b-binding OVA257-264 peptide antigen.


Figure 5
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  		Figure 5. Th1 cell therapy combined with i.t. injection of OVA antigen induces systemic antitumor immunity mediated by tumor-specific tetramer+ CD8+ T cells generated in DLN and the local tumor site. A, induction of OVA-tetramer+ CTL was examined by flow cytometry as described in Materials and Methods. The percentage of OVA-tetramer+ CD8+ T cells in DLN (a-e) and among TIL (f-j) of tumor-bearing mice was examined 24 hours after the second round of therapy with saline (Control; a and f), OVA (b and g), Th1 cells (c and h), or Th1 cells + OVA (d and i). As a control staining, control tetramer was used in both DLN (e) and TIL (j). The distribution of OVA-tetramer+ CD8+ T cells was examined in DLN (k), distal LN (l), spleen (m), or TIL (n) obtained from tumor-bearing mice treated with Th1 cells + OVA. B, mice bearing EG-7 tumors were treated twice with saline ({circ}), OVA antigen ({triangleup}), Th1 cells ({blacktriangleup}), or Th1 cells + OVA antigen (bullet) as described in the legend of Fig. 1. Unfractionated DLN cells in each treated group were prepared 24 hours after the second round of therapy and their cytotoxicity against EG-7 (a) and EL-4 (b) cells was measured by 6-hour 51Cr-release assay. CD8+ T cells (c-f, bullet) were isolated from DLN (c and d) or TIL (e and f) of EG-7-bearing mice, which were treated with Th1 cell transfer plus i.t. injection of OVA. Then, their cytotoxicity against EG-7 (c and e) or EL-4 (d and f) cells was examined by 51Cr-release assay. To determine H-2Kb-restricted antigenic peptide specificity, CD8+ CTLs were pretreated with H-2Kb-OVA-tetramer-SIINFKL and examined for cytotoxicity (c-f, {circ}) against EG-7 (c and e) or EL-4 (d and f) cells. Points, mean of triplicate samples; bars, SE. C, a, mice bearing EG-7 tumors were treated thrice with saline ({circ}) or Th1 cells + OVA ({blacktriangleup}, bullet) at 10, 12, and 14 days after tumor inoculation. Mice were also treated with i.v. injection of saline ({circ}), rat immunoglobulin M (IgM; {blacktriangleup}), or anti-CD8 mAb (bullet) at 9, 10, 15, 20, and 25 days. b and c, EG-7 cells (2 x 106) were inoculated into both ventral sides. When the tumor size reached 8 to 9 mm, saline ({circ}) or Th1 cells + OVA antigen (bullet) were injected only into the tumor mass at the left side of the mice (b) and the growth of the inoculated tumors at both the left (b) and right (c) side was measured as described in Materials and Methods. Points, mean of five mice in each experimental group; bars, SE. A to C, similar results were obtained in three separate experiments.

 
Th1 cell therapy combined with i.t. injection of OVA antigen induces systemic antitumor immunity mediated by CTL. To define the final effector T cells in our therapeutic protocol, CD8+ T cells were depleted by injecting anti-CD8 mAb. As shown in Fig. 5C(a), the therapeutic effect was completely abolished in CD8+ T cell–depleted tumor-bearing mice. Taking together this finding with the results in Fig. 5B, we conclude that OVA-tetramer+ CD8+ CTLs are the major effector cells that eradicate the EG-7 tumor mass in vivo. We further evaluated whether these tumor-specific CTL exhibited systemic antitumor activity against EG-7 tumor cells. EG-7 (2 x 106) cells were i.d. inoculated into both ventral sides of mice. When tumor masses became palpable (8-9 mm), OVA protein was injected into the tumor mass at the left side of the mice and Th1 cells were i.v. transferred. As shown in Fig. 5C(b,c), the untreated tumor mass on the right side of the animals was also eradicated, concomitant with the regression of the tumor mass at the left side. These results indicate that introduction of Th1-mediated immunity at the local tumor site induces systemic antitumor immunity, which is mediated by tumor-specific CD8+ tetramer+ CTL.

APC/Th1 interactions in DLN are crucial for the induction of OVA-tetramer+ CTL in tumor tissue. To investigate the role of APC/Th1 cell interactions in DLN for the subsequent induction of OVA-tetramer+ CTL into the tumor site, we used MHC class II–deficient mice and LN-deficient Aly/Aly mice. EG-7-bearing MHC class II–deficient mice were treated with CFSE-labeled Th1 cells as described in Fig. 3. In contrast to tumor-bearing wild-type mice (Fig. 6A, b, e, and h), MHC class II–deficient tumor-bearing mice showed neither active proliferation of transferred Th1 cells nor augmented CD69 expression on CD8+ T cells in DLN (Fig. 6A, c and f). Moreover, tetramer+ CTL generation in the tumor tissue was not induced by treatment with Th1 cells plus OVA injection in MHC class II–deficient tumor-bearing mice (Fig. 6A, g, h, and i). Likewise, OVA-tetramer+ CTLs failed to be generated at the tumor site of LN-deficient Aly/Aly mice even after two rounds of therapy with Th1 cells plus OVA injection (Fig. 6A, j, k, and l). These findings indicate that MHC class II–mediated APC/Th1 cell interactions are crucial for the induction of OVA-tetramer+ CTL into the tumor tissue and that DLN is an indispensable lymphoid organ to induce complete eradication of established tumors in our therapeutic protocol.


Figure 6
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  		Figure 6. MHC class II–dependent APC/Th1 cell interactions in DLN is crucial for inducing the activation and proliferation of tetramer+ CTL in the local tumor site. A, EG-7 tumor cells were inoculated into wild-type mice (a, b, d, e, g, h, j, and k), MHC class II–deficient mice (c, f, and i), or LN-deficient Aly/Aly mice (l). After the tumor became palpable, mice were treated with Th1 cell transfer (a, d, g, and j) or Th1 cell transfer + OVA injection (b, c, e, f, h, i, k, and l). The frequency of parental cells and daughter cells (1st-5th generation) in DLN was monitored using CFSE-labeled Th1 cells (a-c). Activation of DLN CD8+ T cells was determined by measuring expression levels of CD69 antigen (d-f). Generation of tumor-specific CTL in the local tumor site (TIL) was determined by staining with PE-conjugated OVA-tetramer 24 hours after the second round of therapy (g-l). Similar results were obtained in three separate experiments. B, the possible mechanisms of Th1-dependent antitumor immunity in tumor-bearing mice are illustrated. a, APCs such as dendritic cells and M{phi} are activated by i.t. injection of tumor-related antigen protein and these cells migrate from the tumor tissue into DLN; b, APC/Th1 cell interactions in DLN promote the activation and proliferation of transferred Th1 cells, which induce the subsequent cell infiltration and activation of antitumor effector cells including innate effector cells and acquired CD8+ tumor-specific tetramer+ CTL; c, OVA-tetramer+ CTL in DLN migrate into the tumor tissue to eradicate the tumor cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In previous works (4–7, 35–37), we have shown a critical role of Th1 immunity to induce tumor-specific CTL in tumor-bearing mice. We showed that adoptive transfer of antigen-specific Th1 cells into mice bearing established tumors causes the induction of tumor-specific CTL and induces the complete eradication of MHC class II+ A20-OVA tumor mass (4). In contrast to MHC class II+ tumor, it has been shown that MHC class II– tumors are highly refractory to immunotherapy. We also showed that, in contrast to MHC class II+ A20-OVA, it is hard to completely cure mice of established MHC class II– EG-7 tumors by Th1 cell transfer alone (Fig. 1). However, in the present article, we succeed in establishing a novel therapeutic protocol for inducing the complete eradication of established MHC class II– tumors from mice by cell transfer of antigen-specific Th1 cells combined with i.t. injection of a model tumor antigen protein (Fig. 1).

The resistance of MHC class II– EG-7 tumors against Th1 cell therapy may be because Th1 cells cannot initiate active immune responses at the local tumor site by interacting with class II– OVA–expressing EG-7 cells. To overcome this problem, the model tumor-antigen protein, OVA, was i.t. injected into EG-7 tumor tissue with or without Th1 cell transfer. A modest inhibition of tumor growth was observed in mice treated with OVA alone or with Th1 cells alone, and none of the treated mice were cured. In sharp contrast, Th1 cell transfer with OVA injection resulted in complete rejection of established MHC class II– EG-7 tumors and all treated mice were completely cured. Although several reports have shown that cell transfer of tumor-specific naïve OT-2 CD4+ T cells combined with naïve CD8+ T cells prevented or rejected MHC class II– tumor cells (12, 38), the same therapeutic protocol was unable to eradicate large EG-7 tumor masses in our model (data not shown). Moreover, transfer of tumor-specific naïve CD4+ T cells, instead of tumor-specific Th1 cells combined with OVA injection, did not completely cure mice from tumors (data not shown). These findings indicate that in vitro activated Th1 cells can overcome the strong immunosuppression in tumor-bearing hosts.

In the early events of tumor eradication by Th1 cells plus OVA injection, the migration of APC (CD11c+ dendritic cells and CD11b+ M{phi}) into DLN is a critical step for inducing active proliferation of transferred Th1 cells (Figs. 2 and 3). Th1 cell transfer also augments the migration of OVA-presenting APC from tumor tissue into DLN (Fig. 2). Thus, transferred Th1 cells synergistically act with i.t. injected OVA for initiating Th1-dependent immune responses in DLN and the local tumor site. Introducing such Th1-dominant immunity in DLN also induces up-regulation of the CD69 early activation marker on NK, NK T cells, dendritic cells, M{phi}, and CD8 T cells (Fig. 4) and enhances infiltration of these cells into DLN (data not shown). Thus, both innate and acquired immunity are efficiently activated within DLN in tumor-bearing host in our combined treatment protocol. This enhanced migration of APC and immunoregulatory cells into DLN might be due to the production of certain chemokines up-regulated by Th1-dominant immune responses. Because it has been reported that the SLC chemokine plays a crucial role in the migration of dendritic cells into LN (18, 19, 39), we did our established therapeutic protocol using SLC-deficient mice (Plt/Plt mice). However, the frequency of cell migration and activation was not changed between wild-type and Plt/Plt mice (data not shown), suggesting that chemokines other than SLC are involved in this phenomenon.

In the second step, APCs encounter i.v. transferred Th1 cells in DLN (Fig. 3A), which results in active proliferation of Th1 cells in DLN, which is less pronounced in distal LN, spleen, and tumor tissue (Fig. 3D). In this step, antitumor immunoregulatory cells, including CD8+ T cells and APCs, are already activated to express the CD69 early activation molecule (Fig. 4). We further showed that tumor-specific OVA-tetramer+ CTLs with specific cytotoxicity against EG-7 are induced in DLN and TIL at 24 hours after the second round of Th1 cell therapy (Fig. 5A). Thus, Th1-dominant immunity induced by Th1 cell transfer and local OVA injection is sufficient to overcome immunosuppression at the local tumor site and induces tumor-specific CTL, which can completely cure mice of established MHC class II– tumors. The efficient induction of tetramer+ CTL by our combination therapy may be due to augmented interaction of Th1 cells and antigen-presenting MHC class II+ APC within DLN. Indeed, neither active proliferation of transferred Th1 cells nor CD69 expression on infiltrated host-derived cells including CD8+ T cells is induced in DLN in MHC class II–deficient mice (Fig. 6A, a-f). Moreover, CD8+ tetramer+ CTL generation at the local tumor site is not observed in MHC class II–deficient mice (Fig. 6A, g-i). We also showed that tetramer+ tumor-specific CTL are not induced in LN-deficient Aly/Aly mice after treatment with Th1 cells and OVA injection (Fig. 6A, j-l). Aly/Aly mice were used to investigate the role of LN in tumor immunity (40). However, we cannot deny the possibility that the failure of the generation of antitumor immunity in Aly/Aly mice was due to some other immune disfunction because it has been reported that Aly/Aly mice have defects of reduced serum levels of immunoglobulins and there was no class switch to immunoglobulin A (41). Taken together, it seems that OVA-tetramer+ CTLs are induced in DLN soon after Th1-cell activation by interacting with APC in DLN. Subsequently, CTLs migrate from DLN into the tumor tissue. As shown in Fig. 5C, once tumor-specific CTLs are generated in our treatment protocol, the locally induced CTL are redistributed in the whole body to eradicate tumor tissue at a distant site from the original tumor mass. These results indicate that APC/Th1 cell interactions within DLN play a critically important role in combination therapy with Th1 cells and local injection of tumor antigen protein.

The possible mechanisms involved in the eradication of MHC class II– tumors by Th1 cell therapy and local OVA injection are summarized in Fig. 6B. The following steps may be involved: (a) APCs such as dendritic cells and M{phi} are activated by i.t. injection of tumor-antigen protein and migrate into DLN; (b) APC/Th1 cell interactions promote the activation and proliferation of transferred Th1 cells, leading to infiltration, activation, and generation of antitumor effector cells, including innate effector cells and acquired CD8+ tumor-specific tetramer+ CTL; and (c) OVA-tetramer+ CTLs in DLN migrate into the tumor tissue to eradicate the tumor cells. Thus, tumor-specific protective immunity will spread to the whole body to prevent tumor recurrence or metastasis.

Whereas we show a critical role of LN for tumor rejection in our therapy model, Mullins et al. (27) reported that the requirement of LN during dendritic cells-vaccine therapy depends on the location of tumors and the route of dendritic cells administration. Therefore, it may be possible to induce tetramer+ CTL specific to MHC class II– tumor cells by treatment with Th1 cell therapy combined with injection of antigen at another site (e.g., i.d. injection of antigen protein near DLN of tumor site instead of i.t. injection of antigen). If effective, this method would expand the range of application of our therapeutic protocol. We are currently trying this possibility.

In this tumor therapy model, we used xenogeneic antigen (OVA) as a tumor model antigen. It would be better to use a weak self antigen instead of OVA to establish this model because most tumor-antigens used in clinical study are likely weak self antigens. However, it has been recently shown that self natural tumor antigen protein (carcinoembryonic antigen), but not peptide, induced tumor-specific CTL in tumor-bearing mice by combination with flt3L-activated dendritic cells (42). Thus, natural tumor antigen was shown to have a capability of inducing tumor-specific CTL in vivo although it is a weak self antigen. Therefore, we believe that our established protocol may be applicable to clinical trial although there are still unresolved problems.

In contrast to MHC class I–restricted CTL, the generation of MHC class II–restricted tumor-specific Th1 cells has been difficult because of limited information about the tumor-specific peptide epitopes that are recognized by MHC class II–restricted T cells. However, recently, we have established a culture system for inducing tumor-specific Th1 cells by stimulation with tumor-antigen protein and monocyte-derived dendritic cells (data not shown). Moreover, it is now possible to induce tumor-specific Th1 cells from nonspecifically activated Th cells by T-cell receptor gene modification (35–37). Thus, it is now feasible to prepare tumor-specific Th1 cells that are suitable for clinical trials. We believe that Th1 cell therapy using tumor-specific Th1 cells and tumor antigen protein will become a novel strategy for therapy of human tumors in the near future.


   Acknowledgments
 
Grant support: A Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, a Grant-in-Aid for Scientific Research on priority Areas, a Grant-in-Aid for Immunological Surveillance and Its Regulation and a Grant-Aid for Ministry of Education, Culture, Sports, Science and Technology Cancer Translational Research Project.

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.

We thank Dr. Luc Van Kaer (Vanderbilt University School of Medicine, Nashville, TN) and Dr. Micallef Mark (TORAY, Tokyo, Japan) for helpful and critical comments during the preparation of this manuscript, and Dr. Steve Herrmann (Wyeth Research, Cambridge, MA) and Takuko Sawada (Shionogi Pharmaceutical Institute Co., Osaka, Japan) for their kind donations of IL-12 and IL-2, respectively.


   Footnotes
 
Note: K. Chamoto and D. Wakita contributed equally to this work.

Received 6/29/05. Revised 9/ 6/05. Accepted 11/22/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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