Cancer Research Cancer Health Disparities Conference 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Y.
Right arrow Articles by Xiang, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Y.
Right arrow Articles by Xiang, J.
[Cancer Research 65, 4955-4962, June 1, 2005]
© 2005 American Association for Cancer Research

Immunology

Tumor-Infiltrating Dendritic Cell Subsets of Progressive or Regressive Tumors Induce Suppressive or Protective Immune Responses

Yongqing Liu, Xuguang Bi, Shulin Xu and Jim Xiang

Research Unit, Division of Health Research, Saskatchewan Cancer Agency, Departments of Oncology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Requests for reprints: Jim Xiang, Research Unit, Division of Health Research, Saskatchewan Cancer Agency, Departments of Oncology and Immunology, College of Medicine, University of Saskatchewan, 20 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 4H4. Phone: 306-655-2917; Fax: 306-655-2910; E-mail: JXiang{at}scf.sk.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor-infiltrating dendritic cells (TID) have an ambivalent role in regulation of tumor regression or growth. However, their precise natures and molecular mechanisms have not been elucidated. In this study, we studied TIDs recruited in progressive P815 and regressive P198 tumors of the same origin. Our data showed that P815 tumors contained CD4+8+ and CD4–8– TID815 subsets, whereas P198 tumors contained CD4+8+ and CD4+8– TID198 subsets. They similarly stimulate allogeneic T cell proliferation and have nitric oxide–mediated cytotoxicity to tumor cells with an exception of CD4–8– TID815 with less efficiency. The newly identified fourth CD4+8+ TID815 or TID198 subset and the CD4+8– TID198 all express high levels of IFN-{gamma} and interleukin (IL)-6, whereas CD4–8– TID815 secrete a marked level of transforming growth factor-ß. Vaccination of mice with P815 tumor lysate–pulsed CD4+8+ TID815 or TID198 and CD4+8– TID198 induced IFN-{gamma}–secreting Th1 and effective CTL responses leading to protective immunity against P815 tumor, whereas CD4–8– TID815 stimulated IL-10–expressing Tr1 responses leading to immune suppression. Transfer of CD4+ Tr1 cells obtained from CD4–8– TID815-immunized wild-type, but not IL-10–/– mice, into CD4+8+ TID815 immunized mice abolished otherwise inevitable development of antitumor immunity. Taken together, our findings provide an important insight into immunologic alterations in progressive and regressive tumors and an implication for dendritic cell–based approaches in the design of cancer vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Murine splenic CD11c+ dendritic cells could be subdivided into three distinct subsets, with CD4+8– cells representing about 50% of splenic dendritic cells and CD4–8+ and CD4–8– dendritic cells with about 25% each of the total population (1). The three populations are derived from discrete developmental streams (2), are located in the T cell areas as well as the marginal zones of lymph nodes, and can stimulate allogeneic T cell responses in vitro (3, 4). In vivo, however, dendritic cell subsets reportedly induced distinctive immune responses. CD4–8+ and CD4–8– dendritic cells can efficiently prime male antigen-specific CTL responses, whereas CD4+8– dendritic cells do so only weakly (3). CD4+8– dendritic cells reportedly mediate tolerance or bystander suppression against diverse T cell specificities (5), whereas CD4–8+ dendritic cells can induce tolerance to tissue-associated antigens (6) and prolong the survival of heart allografts (7). The reasons for the different (i.e., immunogenic versus tolerogenic) results observed using the different dendritic cell subsets are at present unclear.

The repertoire of chemokines and cytokines at tumor sites is a critical determinant for successful immune responses against tumors. In general, tumor-derived chemokines are able to chemoattract various leukocytes including dendritic cells, lymphocytes, and granulocytes infiltrating into tumors (8, 9). Among them, the tumor-infiltrating dendritic cells (TID) represent a major component of the lymphoreticular infiltrates of tumors (8). Originally, TIDs were hypothesized to be the cells responsible for mediating tumor destruction. Many studies have associated the infiltration of tumors by dendritic cells with a favorable prognosis for cancer patients (10, 11). TIDs activated by cytokines or chemicals facilitated tumor regression in animal tumor models (12–14). However, others showed that TIDs were associated with poor prognosis (15), promoted tumor growth (3, 16), and had suppressive effects leading to tumor progression (17, 18). Therefore, the role of TIDs in tumor immunity still remains controversial with the possibility that distinct subsets of TIDs may either aid tumor destruction or foster tumor growth.

The experimental progressive/regressive tumor cell system has been extensively used to determine the precise nature of the cells involved in progressive and regressive rat tumors (19, 20). In this study, we selected two mouse tumor cell lines, a progressive tumor cell line, P815, and a regressive tumor cell line, P198, which is a derivative of P815 line through in vitro drug-induced mutagenesis (21, 22). These two cell lines expressing tumor-associated antigen P1A constitutively differ in immunogenicity and tumorigenicity in syngeneic hosts. The progressive line P815 gives rise to progressive and lethal tumors, whereas the regressive line P198 yields spontaneously regressive tumors due to CTL-mediated immune responses. Therefore, this experimental P815/P198 cell system provides a well-suited model to determine the precise nature of TIDs recruited in progressive and regressive tumors. Based upon this mouse tumor model, TIDs purified from P815 and P198 tumors were phenotypically and functionally characterized. We then further investigated their functional effects in stimulation of T cells in vitro and antitumor immunity in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, antibodies, cytokines, and animals. P815 is a mastocytoma cell line derived from a DBA/2 mouse (21). P198 is an immunogenic variant obtained after mutagenic treatment of P815 (22). The leukemia cell line L1210 was derived from a DBA/2 mouse. All these three tumor cell lines obtained from American Tissue Culture Collection (Rockville, MD) were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS. The biotin-conjugated anti-mouse H-2Kd (SF1.1), I-Ad (AF6-120.1), CD4 (GK1.5), CD8 (53-6.7), CD11b, CD11c (HL3), CD40 (IC10), CD54 (3E2), CD80 (16-10A1), B220 (RA3-6B2), and Gr-1 (RB6-8C5) antibodies, and the FITC-conjugated anti-CD4 and R-phycoerythrin-conjugated anti-CD8 antibodies were all purchased from BD Biosciences (Mississauga, Ontario, Canada). The anti-P1A antibodies were obtained from Dr. Yang Liu, Ohio State University Medical Center, Columbus, OH. The FITC-conjugated avidin was purchased from Caltag (Burlingame, CA). The rabbit anti-mouse TNF-{alpha} antibodies were obtained from Sigma (Oakville, Ontario, Canada). Recombinant mouse interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor were purchased from R&D Systems (Minneapolis, MN). Female DBA/2 mice (H-2Kd) and C57BL/6 mice (H-2Kb) were obtained from Charles River Laboratories (St. Laurent, Quebec, Canada). Homozygous IL-10–/– knock-out mice on H-2Kd background were obtained from Jackson Laboratories (Bar Harbor, ME). All mice were housed in the animal facility of the Saskatoon Cancer Center; all animal experiments were carried out according to the guidelines of the Canadian Council for Animal Care.

Tumor-infiltrating dendritic cells and tumor-infiltrating dendritic cell subset preparation. DBA/2 mice were s.c. injected in their right thighs with 0.5 x 106 P815 and P198 tumor cells, respectively. Tumors were removed when they reached ~6 mm in diameter. Tumor tissues were cut into small fragments and incubated in DMEM containing 1 mg/mL collagenase (4 mL per gram of tumor tissue; Worthington Biochemical Corp., Lakewood, NJ) at 37°C for 45 to 60 minutes. Single cell suspension was prepared by pressing the digested tumor tissues through a stainless mesh. Dead cells were removed by Ficoll-Paque density gradients. The tumor cells were first removed by incubation of cell samples in anti-P1A antibody-coated plate at room temperature for 1 hour (23). TIDs were then purified from the nonadherent cells by using anti-CD11c microbeads (Miltenyi Biotech, Auburn, CA) according to the manufacturer's instructions. After overnight culture in DMEM plus 10% FCS, IL-4 and granulocyte macrophage colony-stimulating factor (15-20 ng/mL), these purified CD11c+ TIDs were harvested. For TID subset purification, these TIDs were first stained with FITC-anti-CD4 and phycoerythrin-anti-CD8 antibodies and then analyzed by flow cytometry. P198 CD4+8+ and CD4+8– TID subsets (CD4+8+ TID198 and CD4+8– TID198) as well as P815 CD4+8+ and CD4–8– TID subsets (CD4+8+ TID815 and CD4–8– TID815) were then purified by flow cytometric cell sorting, respectively.

Tumor lysate pulsing. P185 tumor lysates were prepared as previously described (24). Briefly, P815 cells were resuspended in extraction buffer containing 0.01 mol/L Tris-HCl (pH 7.2) and 0.2 mmol/L CaCl2 (10 mL per gram of tumor cells) and homogenized for 2 minutes on ice using a Silverson homogenizer. Cell extracts were harvested by centrifugation at 1,000 x g for 5 minutes to remove cellular debris. For tumor lysate pulsing, 5 to 10 x 106 TID subsets were pulsed with P815 tumor lysate at a ratio of 3:1 (three tumor cell lysates per dendritic cell) in AIM-V medium (Life Technologies) plus granulocyte macrophage colony-stimulating factor (15-20 ng/mL) at 37°C for overnight.

Regional lymph node T cell preparation. To examine the CD4+ T cell response, regional lymph nodes of mice s.c. immunized with 1 x 106 tumor lysate–pulsed TID subsets were harvested 5 days after the immunization for cytokine secretion analysis (25). To block the transforming growth factor-ß (TGF-ß) effect of CD4–8– TID815, mice immunized with tumor lysate–pulsed CD4–8– TID815 were i.p. injected with anti-TGF-ß antibody (0.5 mg per mouse) 1 day before and 2 days after the immunization for a total of two times. Briefly, cell suspensions were prepared from regional lymph nodes of immunized mice 5 days after the immunization. T cells were obtained by passage of lymph node cell suspensions through nylon wool columns. The CD4+ lymph node T cells were further purified by using anti-CD4 microbeads (Miltenyi Biotech).

Immunophenotypic analysis. For phenotypic analysis of tumor cells and TIDs, these cells were stained with a panel of biotin-conjugated antibodies (each, 5 µg/mL), respectively, followed with FITC-conjugated avidin (1:100). Isotype-matched biotin-conjugated antibodies were used as controls. For phenotypic analysis of dendritic cell subsets, the purified TID subsets were cultured overnight and then stained with FITC-conjugated anti-CD11c, CD54, CD80, B220, and Gr1 antibodies, respectively, and analyzed by flow cytometry.

Cytokine secretion. For assessment of cytokine production, TID subsets were cultured in the presence of granulocyte macrophage colony-stimulating factors (20 ng/mL) with or without the commonly used stimulus reagent lipopolysaccharide (1 µg/mL; ref. 26), whereas CD4+ T cells harvested from mice immunized with tumor lysate–pulsed TID subsets were restimulated with anti-CD3 antibodies (1 µg/mL; ref. 27). After 1 day, the supernatants were assayed for IFN-{gamma}, IL-4, IL-6, IL-10, IL-12, TNF-{alpha}, and TGF-ß using ELISA kits (R&D Systems; ref. 28).

Mixed lymphocyte reaction. Primary mixed lymphocyte reactions were done as previously described (26). Briefly, graded doses of irradiated (2,000 rad) TIDs or TID subsets starting at 1 x 105 cells per well were cocultured in 96-well plates with constant numbers (2 x 105 per well) of allogeneic T cells of C57BL/6 mice. After 2 days, T cell proliferation was measured using an overnight [3H]thymidine (1 mCi/mL, Amersham Canada, Ltd., Baie D'Urfe, Quebec) uptake assay (1 µCi per well). The levels of [3H]thymidine incorporation into the cellular DNA were determined by liquid scintillation counting.

Cytotoxicity assays. To test TID cytotoxicity, TID815 and TID198 were used as effector cells, whereas 51Cr-labeled P815, P198 and L1210 tumor cells were used as target cells. For investigating the killing mechanisms, TID subsets were assayed for their capacities to kill the tumor target cells in presence of the nitric oxide production inhibitor NG-methyl-L-arginine (1 mmol/L) or anti-TNF-{alpha} antibodies (5 µg/mL; ref. 29). To test CTL cytotoxicity, splenic lymphocytes from mice immunized with tumor lysate–pulsed TID subsets were cocultured with irradiated (2,000 rad) P815 tumor cells for 3 days (24). CTLs were harvested and used as effector cells, whereas 51Cr-labeled P815, P198, and L1210 tumor cells were used as target cells. Ten thousand labeled target cells per well were mixed with effector cells at various effector/target ratios in triplicate and incubated for 24 and 6 hours for TID and CTL cytotoxicity, respectively. Percentage of specific lysis was calculated as: 100 x [(experimental cpm – spontaneous cpm) / (maximal cpm – spontaneous cpm)]. Spontaneous counts per minute (cpm) released in the absence of effector cells was <10% of specific lysis. The maximal cpm was released by adding 1% Triton X-100 to wells in the experiment.

Animal studies. DBA/2 mice (eight each group) were s.c. injected in their right thighs with 1 x 106 viable P815 and P198 tumor cells, respectively. Mice were then monitored daily for tumor progression or regression. For evaluation of TID vaccination, mice were s.c. vaccinated with 1 x 106 TIDs with or without tumor lysate pulsing and TID subsets with tumor lysate pulsing, respectively. Ten days later, the mice (n = 8 per group) were challenged by s.c injection of 1 x 104 P815 tumor cells. To confirm that the regulatory T cells were functional, these CD4+ Tr1 cells (2 x 106 per mouse) harvested from lymph nodes of CD4–8– TID815-immunized wild-type or IL-10–/– knock-out mice were i.v. injected into the mice s.c. vaccinated with tumor lysate–pulsed CD4+8+ TID815 subset (1 x 106 per mouse). Ten days later, mice (n = 8 per group) were challenged by s.c injection of 1 x 104 P815 tumor cells. Animal mortality and tumor growth were monitored daily for up to 10 weeks; for humanitarian reasons, all mice with tumors that achieved a size of 1.5 cm in diameter were sacrificed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P815 and P198 are progressive and regressive tumor cell lines. Both P815 and P198 cell lines express MHC class I and P1A, but not MHC class II and CD11c (Fig. 1A). However, P198 cell line expresses less MHC class I molecule than P815. When these two cell lines are inoculated into mice, they both initially grew into visible tumor masses (~6 mm in diameter) within 10 days. Interestingly, P815 tumors continuously grew, whereas P198 tumors started to gradually regress, leading to complete disappearance of most P198 tumors in another 2 weeks (Fig. 1B).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 1. Tumor cell flow cytometric analysis and growth in mice. A, flow cytometric analysis. Tumor cells were stained with the biotin-labeled anti-H-2Kd, I-Ad, P1A, and CD11c antibodies, followed with the FITC-conjugated avidin and then analyzed by flow cytometry (solid lines). Isotype-matched irrelevant antibodies were used as controls (dotted thin lines). B, tumor cell growth in DBA/2 mice. Tumor cells (1 x 106) were s.c. injected into each mouse. Tumor growth was monitored daily. One of two representative experiments is shown.

 
Distinct tumor-infiltrating dendritic cell subsets recruited in P815 and P198 tumors. Both tumors of hematologic origin contained significant amount of TIDs. Purified TID815 accounted for 12 ± 3% of the whole cell population from P815 tumors, which is much more than ≤0.5% of TIDs seen in tumors of epithelial cell origin (30, 31). This is probably because most tumors of hematologic origin tend to secrete chemokines. TID198 from regressive P198 tumors accounted for 19 ± 5% of the total cell population, reflecting the significant cellular immune responses seen in P198 tumors, which is much higher than that seen in aggressive P815 tumors.

To study the potential difference in dendritic cells recruited in tumors, TIDs were purified from P815 and P198 tumors, respectively, using anti-CD11c MACS microbeads, and then subjected to flow cytometric analysis. As shown in Fig. 2A, freshly isolated TID815 and TID198 all expressed similar amounts of CD11b, CD11c, CD40, CD54, CD80, B220, and Gr-1, but not I-Ad. In addition, all or a part of TID198 expressed CD4 or CD8 marker, whereas a part of TID815 expressed CD4 and CD8, respectively. TIDs pulsed with P815 tumor lysate in overnight culture expressed similar phenotypes as above, except for an up-regulation of I-Ad (data not shown). Interestingly, when stained with both FITC-anti-CD4 and phycoerythrin-anti-CD8 antibodies, TID815 were divided into two distinct populations, 37% CD4+8+ and 63% CD4–8– dendritic cell subsets, whereas TID198 were divided into another two distinct populations, 48% CD4+8+ and 52% CD4+8– dendritic cell subsets (Fig. 2B). For further characterization, the above four TID subsets including CD4+8+ and CD4–8– TID815 and CD4+8+ and CD4+8– TID198 were then separated and purified by flow cytometric cell sorting. As shown in Fig. 2C, all four TID subsets expressed similar amounts of CD11c, CD54, and CD80, except that CD4–8– TID815 expressed lower level of CD54 and CD80, indicating that CD4–8– TID815 are relatively less mature dendritic cells. In addition, these CD4–8– TID815 expressed CD11b, low amount of B220 and Gr-1 (Fig. 2D), which is consistent with the previous characterization of CD4–8– dendritic cells reported by MeLellan et al. (3).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 2. Flow cytometric analysis of TID. A, P815 and P198 TIDs were stained with biotin-labeled anti-CD4, CD8, CD11b, CD11c, CD40, CD54, CD80, B220, and Gr-1 antibodies, followed with the FITC-conjugated avidin (solid lines) and then analyzed by flow cytometry. Isotype-matched irrelevant antibodies were used as controls (dotted thin lines). B, TIDs and flow cytometrically sorted TID subsets were stained with both FITC anti-CD4 and phycoerythrin anti-CD8 antibodies. C, purified TID subsets were cultured overnight and stained with FITC-conjugated anti-CD11c, CD54, and CD80 antibodies, respectively, and analyzed by flow cytometry. D, CD4–8– TID815 were stained with FITC-conjugated anti-CD11b, B220, and Gr1 antibodies, respectively, and analyzed by flow cytometry. FITC-conjugated isotype-matched irrelevant antibodies were used as controls. One of two representative experiments is shown.

 
Distinct cytokine expression profiles of tumor-infiltrating dendritic cell subsets. The cytokine expression profiles of these distinct TID subsets were very different (Fig. 3). All three TID subsets including CD4+8+ TID815 and CD4+8+ and CD4+8– TID198 were quite similar in high-level expression (600-1,700 pg/mL) of IFN-{gamma} and IL-6 and moderate level expression (~300 pg/mL) of TNF-{alpha}, but little or no TGF-ß expression. However, the cytokine expression profiles of CD4–8– TID815 did differ markedly. The CD4–8– TID815 secreted a marked level of TGF-ß (500 pg/mL), but little other cytokines. All four TID subsets under lipopolysaccharide stimulation did not secrete IL-10 (Fig. 3) and IL-12 (data not shown), which is consistent with the liver dendritic cells reported by Pillarisetty et al. (32). In addition, all four TID subsets expressed relatively low levels of cytokines without lipopolysaccharide stimulation (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Cytokine secretion assay. The TID subset supernatants were measured for IFN-{gamma}, IL-6, IL-10, TNF-{alpha}, and TGF-ß secretion by ELISA. One of two representative experiments is depicted.

 
Mixed lymphocyte reaction of tumor-infiltrating dendritic cell subsets. In general, dendritic cells are excellent antigen-presenting cells, and thus potently induce antigen-specific T cell responses against allogeneic or other antigens in vitro (28). We found that TID198 more efficiently activated allogeneic T cell cycling than TID815 (Student's t test, P < 0.05) in a conventional mixed leukocyte culture (Fig. 4A). When the four subsets were subjected to assessment, we found that all three TID subsets including CD4+8+ TID815 and CD4+8+ and CD4+8– TID198 effectively stimulated allogeneic T cell responses (Fig. 4B and C), whereas the CD4–8– TID815 were less effective in stimulation of allogeneic T cells (Student's t test, P < 0.01), probably due to the lower expression of CD54 and CD80 (Fig. 2C).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. In vitro allogeneic T cell proliferation assays. Irradiated (A) P815 and P198 TIDs, (B) P815 TID subsets, and (C) P198 TID subsets and their 2-fold dilutions were cultured with a constant number (2 x 105 per well) of allogeneic C57BL/6 T cells, respectively. After 48 hours, thymidine incorporation was determined by liquid scintillation counting. *, P < 0.05 versus cohorts of (A) TID815, (B) CD4–8– TID815, and (C) CD4+8– TID198 (Student's t test). One of three representative experiments is depicted.

 
Tumor-infiltrating dendritic cell cytotoxicity. Cytotoxicity assays were done in vitro to determine the direct effects of TIDs on tumor cells. Both P815 and P198 TIDs isolated 10 days after the graft in mice were strongly cytotoxic to both P815 and P198 tumor cells. The latter is more sensitive to TID killing than the former (Student's t test, P < 0.05; Fig. 5A, i and iii). In addition, TID198 displayed stronger cytotoxicity on both P815 and P198 tumor cells than TID815 as TID198 induced nearly 60% killing activity at effector/target of 50:1 on P198 cells, compared with 38% killing activity of TID815. Both TID815 and TID198 also showed killing activity on P1A-negative L1210 tumor cells, but in less extent, indicating that TID's killing activity is not P1A-specific. The killing activities of P815 and P198 TID subsets were similar to their unfractionated P815 and P198 TIDs (data not shown). In addition, we also investigated the killing mechanisms of TIDs. Addition of 1 mmol/L NG-methyl-L-arginine, an inhibitor of nitric oxide generation from arginine, strongly inhibited both P815 and P198 killing by P815 and P198 TIDs (Student's t test, P < 0.05), whereas TID-induced cytotoxicity was not significantly influenced by the anti-TNF-{alpha} antibodies (Student's t test, P > 0.05; Fig. 5A, ii and iv), indicating that TID's killing activity was mainly mediated by nitric oxide.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 5. Cytotoxicity assays. A, TID cytotoxicity assay. To test TID cytotoxicity, (i) TID815 and (iii) TID198 were used as effector cells (E), whereas 51Cr-labeled P815, P198, and L1210 tumor cells were used as target cells (T). Ten thousand labeled target cells per well were mixed with effector cells at various effector/target ratios in triplicate and incubated for 24 hours in chromium release assay. *, P < 0.05 versus cohorts of P815 and L1210 (Student's t test). For investigating the killing mechanisms, TID subsets were assayed for their capacities to kill the tumor target cells in the presence of NG-methyl-L-arginine [(ii) 1 mmol/L] or anti-TNF-{alpha} antibodies [(iv) 5 µg/mL]. *, P < 0.05 versus cohorts of tumor cell killing without addition of reagents. B, CTL cytotoxicity assay. (i) CTLs from CD4+8+ TID815 [T (CD4+8+ TID815)] and CD4–8– TID815 [T (CD4–8– TID815)] as well as (ii) CTLs from CD4+8+ TID198 [T (CD4+8+ TID198)], and CD4+8– TID198 [T (CD4+8– TID198)] immunized mice were used as effector cells, whereas 51Cr-labeled P815, P198, and L1210 tumor cells were used as target cells. Ten thousand labeled target cells per well were mixed with effector cells at various effector/target ratios in triplicate and incubated for 6 hours in chromium release assay. *, P < 0.05 versus cohorts of (i) the CTLs [T (CD4–8– TID815)] and (ii) the CTLs [T (CD4+8– TID198)] (Student's t test).

 
CD4+8+ or CD4+8– and CD4–8– tumor-infiltrating dendritic cells stimulate Th1 and Tr1 cell response, respectively. In general, CD4+ T helper cells are divided into two types. Type 1 T helper cells (Th1) producing IL-2 and IFN-{gamma} and type 2 T helper cells (Th2) secreting IL-4, IL-5, and IL-10 are responsible for cellular and humoral immunity, respectively (33). In addition, a type 1 regulatory CD4+ T cell (Tr1) population was defined, which produces high level of IL-10, but low level of IL-4, and suppresses Th1 response (33). To study the type of CD4+ T cell immune response induced by TID subsets, mice were s.c. immunized with tumor lysate–pulsed TID subsets. Three days after immunization, CD4+ T cells were purified from regional lymph nodes and restimulated with anti-CD3 antibodies. After 1 day, the supernatants were assayed for different cytokine expressions. As shown in Fig. 6, CD4+ T cells derived from CD4+8+ TID815 and CD4+8+ TID198 or CD4+8– TID198 subsets secreted high and moderate levels of IFN-{gamma} (~1,300 to 2,000 pg/mL) and TNF-{alpha} (~800 to 1,200 pg/mL), but low levels of IL-4 (~100 to 200 pg/mL) and little IL-10, which is consistent with a Th1 cell response (34). Importantly, CD4+ T cells derived from CD4–8– TID815 subset secreted moderate levels of IFN-{gamma} (~800 pg/mL) and IL-4 (~500 pg/mL), but high levels of IL-10 (~1,300 pg/mL), which is consistent with a Tr1 cell response (34). Interestingly, in vivo injection of anti-TGF-ß antibodies into mice immunized with CD4–8– TID815 subset resulted in more than 8-fold decrease of IL-10 secretion (~150 pg/mL) by CD4–8– TID815 subset-stimulated CD4+ T cells, indicating TGF-ß secretion of CD4–8– TID815 subset is responsible for induction of IL-10 secreting Tr1 cells.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 6. The supernatants of CD4+ T cells derived from regional lymph nodes of mice immunized with P815 tumor lysate–pulsed CD4+8+ TID198 [T (CD4+8+ TID198)], CD4+8– TID198 [T (CD4+8– TID198)], CD4+8+ TID815 [T (CD4+8+ TID815)], and CD4–8– TID815 [T (CD4–8– TID815)] were measured for IFN-{gamma}, IL-10, TNF-{alpha}, and IL-4 secretion by ELISA. In addition, the supernatants of CD4+ T cells derived from regional lymph nodes of another group of mice immunized with CD4–8– TID815 as well as treated with anti-TGF-ß antibody [T (CD4–8– TID815) + anti-TGF] were also measured for cytokine secretion. One of three representative experiments is shown.

 
CD4+8+ or CD4+8– and CD4–8– tumor-infiltrating dendritic cells have and lack the capacity to stimulate antigen-specific CTL responses, respectively. We next examined the capacity of each TID subpopulation to induce antigen-specific CTL responses in mice, using splenic lymphocytes from mice immunized with these tumor lysate–pulsed TID subsets as effector cells against 51Cr-labeled P815 tumor target cells. CTLs from mice immunized with CD4–8– TID815 did not show any cytotoxic activity against P815 tumor cells (Fig. 5B). On the other hand, CTLs from mice immunized with CD4+8+ TID815 and TID198 or CD4+8– TID198 subsets were all cytotoxic for these P1A-positive target cells. At an effector/target ratio of 50, CTLs from CD4+8+ TID815 and TID198, as well as CD4+8– TID198 immunized mice killed 38%, 45%, and 36% of their P815 targets, but essentially none of the P1A-negative L1210 tumor cells, indicating that the killing activities are P1A tumor–specific.

CD4+8+or CD4+8– and CD4–8– tumor-infiltrating dendritic cells induce in vivo protective and suppressive immune responses against tumors, respectively. To study the antitumor immune responses of TIDs, we vaccinated groups of mice with purified TID815 and TID198, and then 10 days later, challenged them with P815 tumor cells. As shown in Fig. 7A, TID815-immune and PBS-treated (i.e., control) mice invariably died within 5 weeks of P815 cell implantation, whereas two of eight TID198-immunized mice were protected against P815 tumor challenge for at least 10 weeks, indicating that TID815 and TID198 induce in vivo suppressive and protective immune responses against tumors, respectively. To enhance antitumor immunity, we then immunized the mice with tumor lysate–pulsed TID815 and TID198, and then 10 days later challenged them with P815 tumor cells. This time, most (six of eight) TID198-immunized mice were protected against P815 tumor challenge (Fig. 7B). To further study the potentially distinct effects of different TID subsets, we vaccinated groups of mice with the different tumor lysate–pulsed TID subsets, and then 10 days later, challenged them with P815 tumor cells. Interestingly, all three TID subsets including CD4+8+ TID815 and TID198 as well as CD4+8– TID198 induced protective immunity resulting in one or two out of eight mice protected against P815 tumor challenge (Fig. 7C), indicating protective immune responses against P815 tumor. On the other hand, however, CD4–8– TID815-immunized mice all died within 4 weeks of P815 cell inoculation, indicating a suppressive immune response.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 7. Animal studies. For evaluation of TID vaccination, mice (n = 8 per group) were s.c. vaccinated with 1 x 106 (A) purified TID815 ({circ}) and TID198 ({blacksquare}), and (B) tumor cell lysate-pulsed TID815 ({circ}) and TID198 ({blacksquare}), and (C) CD4+8+ TID815 ({circ}), CD4–8– TID815 ({blacksquare}), CD4+8+ TID198 ({blacktriangledown}), CD4+8– TID198 ({bullet}) or PBS ({blacktriangleup}), respectively. D, mice were immunized with CD4+8+ TID815 ({circ}) alone or combined with CD4+8+ TID815-induced CD4+ Th1 cells (*), or CD4–8– TID815-induced CD4+ Tr1 cells ({square}), or CD4–8– TID815-induced CD4+ Tr1 cells with IL-10–/– ({square}). Ten days after TID immunization, these mice were then challenged s.c. with 1 x 104 P815 tumor cells. Animal mortality was monitored daily for up to 10 weeks. The data are representative of two experiments with similar results.

 
CD4–8– tumor-infiltrating dendritic cell–induced Tr1 cells deliver immune suppression through IL-10 secretion. Given our data showing that the tumor lysate–pulsed CD4–8– TID815 stimulated a Tr1 response in wild-type mice and induced suppressive immune response against tumor, we next characterized the abilities of these in vivo-generated Tr1 cells to induce functional tolerance in vivo. We transferred the CD4–8– TID815-induced Tr1 cells into tumor lysate–pulsed CD4+8+ TID815-immunized mice, then 10 days later, challenged the mice with P815 tumor cells. Transfer of these putative wild-type Tr1 cells obtained from CD4–8– TID815-immunized wild-type mice abolished antitumor immunity, such that all of the mice rapidly succumbed to their tumors (Fig. 7D). To study the role of IL-10 in this Tr1-dependent immune suppression, CD4+ "Tr1" cells obtained from CD4–8– TID815-immunized IL-10–/– BALB/c mice were injected into CD4+8+ TID815-immunized mice. Unlike the above wild-type Tr1 cells, these IL-10–/– Tr1 cells had little impact on CD4+8+ TID815-driven antitumor immunity, such that most (seven of eight) of the recipients remained tumor-free at 10 weeks after P815 challenge. As expected, when we transferred the control CD4+8+ TID815-induced Th1 cells into CD4+8+ TID815-immunized mice, we observed an augmented tumor protection, such that all animals remained P815 tumor–free for the duration of the experiments. Therefore, our data clearly implicated IL-10-producing Tr1 cells as central to the tolerance observed in this model system.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
More and more evidence has now indicated that TIDs have an ambivalent role in regulation of tumor growth. The presence of TIDs in tumors with regression suggests that TIDs may stimulate protective immune responses against tumors. On the other hand, the presence of TIDs in the tumors with aggressive growth suggests that the TID functions may be altered. Alterations in functional activities of TIDs in aggressive tumors have been reported to include (a) the defective production of reactive oxygen and nitrogen intermediates (35) and important cytokines (36), and (b) the defective functions in antigen presentation (37), costimulation (38), and T cell stimulation (39). However, the precise nature of TIDs and its molecular mechanisms in induction of either suppressive or protective immune responses in aggressive or regressive tumors have not been elucidated.

Our data showed that TIDs freshly isolated from aggressive P815 and regressive P198 tumors all expressed similar amounts of CD11b, CD11c, CD40, CD54, CD80, B220, and Gr-1, but not Iad. Interestingly, for the first time, we discovered that distinct dendritic cell subsets including CD4+8– and CD4–8– dendritic cell subsets, as well as the newly identified fourth CD4+8+ dendritic cell subset (40), were recruited in the progressive and regressive tumors, respectively. Miller et al. (40) first reported a fourth CD4+8+ dendritic cell subset accounting for 9% of the total spleen dendritic cell populations. However, no further characterization was conducted on this dendritic cell subset in their report. In this study, our data showed that TID815 contained 37% CD4+8+ and 63% CD4–8– dendritic cell subsets, whereas TID198 contained 48% CD4+8+ and 52% CD4+8– dendritic cell subsets.

Furthermore, the cytokine expression profiles of these distinct TID subsets were very different. We found that CD4+8+ TID815 and TID198 all express high levels of IFN-{gamma} and IL-6 and moderate level of TNF-{alpha}, but little or no TGF-ß. The cytokine expression profiles of CD4+8– TID198 were similar to the above CD4+8+ TIDs. Recently, it has been reported that IL-6 secreted by dendritic cells is able to overcome CD4+25+ Tr-mediated suppression (41). Therefore, CD4+8+ TID815 and TID198 and CD4+8– TID198 expressing high levels of IL-6 are also expected to be immune active. However, the cytokine expression profiles of CD4–8– TID815 differed markedly. Lipopolysaccharide-stimulated B cells expressing TGF-ß have recently been shown to induce T cell anergy (42) via activation of Tr cells (43). Interestingly, the CD4–8– TID815 observed in this study also secreted a marked level of TGF-ß, but little expression of other cytokines, suggesting an immune tolerogenic type of dendritic cells (44).

The Th1/Th2 balance has been shown to be critically important in various immune responses including antitumor immunity (45). Our data showed that CD4+ T cells derived from CD4+8+ TID815 and TID198 or CD4+8– TID198 subsets secreted high levels of IFN-{gamma}, but low levels of IL-4 and little IL-10, indicating a Th1 response (25) essential for priming antitumor responses (46). Interestingly, as predicted, CD4+ T cells derived from TGF-ß-secreting CD4–8– TID815 subset immunized mice secreted moderate levels of IFN-{gamma} and IL-4, but high levels of IL-10, indicating a Tr1 cell response (34). Tr1 cells can also be induced by addition of exogenous IL-10 to primary murine T cell cultures or by coculturing T cells with TGF-ß/IL-10-expressing "tolerogenic" dendritic cells (44). These Tr1 cells are distinct from Th1 or Th2 cells in that they produce high levels of IL-10, and proliferate poorly upon TCR ligation suppress immune responses in vitro and in vivo through secreted IL-10 (44, 47).

We next examined the capacity of each TID subpopulation to induce antigen-specific CTL responses in mice. CTLs from mice immunized with CD4–8– TID815 did not show any cytotoxic activity against P815 tumor cells. On the other hand, CTLs from mice immunized with CD4+8+ TID815 and TID198 or CD4+8– TID198 subsets were all cytotoxic for these P1A-positive target cells. We again vaccinated groups of mice with the tumor lysate–pulsed P815 and P198 TID subsets. We found that CD4+8+ TID815 and TID198 as well as CD4+8– TID198 all induced protective immunity, whereas CD4–8– TID815-immunized mice all died within 4 weeks of P815 cell inoculation, indicating a suppressive immune response.

It has previously been shown that macrophages with surface expression of CD11b+GR-1+ MHC II+ and CD11b+CD45+ MHC II+ had immune suppressive effects leading to tumor progression (18). The mechanism of macrophage-induced immune suppression was later linked to dysfunctional natural killer cell activity mediated via prostaglandin-E2 released by the macrophages (48) and the nitric oxide released by suppressor macrophages, which was cytotoxic to CTLs (49). However, no studies on TID subsets and their molecular immune mechanisms were conducted in these reports. Given our data showing that the tumor lysate–pulsed CD4–8– TID815 stimulated a Tr1 response and induced suppressive immune response against tumor, we next addressed the question of whether the above suppressive immune response is mediated by tolerogenic CD4–8– TID815-induced Tr1 cells expressing high levels of immune suppressive IL-10. In this study, our data clearly showed that transfer of these putative wild-type Tr1 cells abolished antitumor immunity. Unlike the wild-type Tr1 cells, the IL-10–/– Tr1 cells had little impact on CD4–8+ dendritic cellOVA-driven antitumor immunity. Therefore, our results clearly implicated IL-10-producing Tr1 cells as central to the tolerance observed in this model system, and this is consistent with some other previous reports (43, 44). Interestingly, it has recently been shown that the IL-10-stimulated CD11clow CD45RBhigh tolerogenic dendritic cells having a phenotype similar to our CD4–8– dendritic cells as described in this study also induced Tr1 cell differentiation and immune tolerance in vivo (50).

Taken together, we showed that different dendritic cell subsets were recruited into tumors. The newly identified fourth CD4+8+ dendritic cell subset as well as the CD4+8– dendritic cell subset recruited in regressive tumors all induce Th1 and active immune response, whereas the CD4+8– TIDs expressing TGF-ß, which are recruited in progressive tumors, stimulate Tr1-mediated immune suppression through Tr1-secreted IL-10. Therefore, our findings will be very important in understanding the immunologic alterations in progressive and regressive tumors as well as in implications for dendritic cell–based approaches to the design of cancer vaccines.


   Acknowledgments
 
Grant support: Research grant MOP 63259 from the Canadian Institute of Health Research. Y. Liu was supported by a Postdoctoral Fellowship from the Canadian Institute of Health Research.

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.

Received 11/ 4/04. Revised 3/11/05. Accepted 3/24/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Moser M, Murphy KM. Dendritic cell regulation of TH1-TH2 development. Nat Immunol 2000;1:199–205.[CrossRef][Medline]
  2. Kamath AT, Pooley J, O'Keeffe MA, et al. The development, maturation, and turnover rate of mouse spleen dendritic cell populations. J Immunol 2000;165:6762–70.[Abstract/Free Full Text]
  3. McLellan AD, Kapp M, Eggert A, et al. Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression. Blood 2002;99:2084–93.[Abstract/Free Full Text]
  4. Pooley JL, Heath WR, Shortman K. Cutting edge: intravenous soluble antigen is presented to CD4 T cells by CD8– dendritic cells, but cross-presented to CD8 T cells by CD8+ dendritic cells. J Immunol 2001;166:5327–30.[Abstract/Free Full Text]
  5. Legge KL, Gregg RK, Maldonado-Lopez R, et al. On the role of dendritic cells in peripheral T cell tolerance and modulation of autoimmunity. J Exp Med 2002;196:217–27.[Abstract/Free Full Text]
  6. Belz GT, Behrens GM, Smith CM, et al. The CD8{alpha}(+) dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens. J Exp Med 2002;196:1099–104.[Abstract/Free Full Text]
  7. O'Connell PJ, Li W, Wang Z, Specht SM, Logar AJ, Thomson AW. Immature and mature CD8{alpha}+ dendritic cells prolong the survival of vascularized heart allografts. J Immunol 2002;168:143–54.[Abstract/Free Full Text]
  8. Mantovani A. Tumor-associated macrophages in neoplastic progression: a paradigm for the in vivo function of chemokines. Lab Invest 1994;71:5–16.[Medline]
  9. Giovarelli M, Cappello P, Forni G, et al. Tumor rejection and immune memory elicited by locally released LEC chemokine are associated with an impressive recruitment of APCs, lymphocytes, and granulocytes. J Immunol 2000;164:3200–6.[Abstract/Free Full Text]
  10. Hillenbrand EE, Neville AM, Coventry BJ. Immunohistochemical localization of CD1a-positive putative dendritic cells in human breast tumours. Br J Cancer 1999;79:940–4.[CrossRef][Medline]
  11. Ishigami S, Aikou T, Natsugoe S, et al. Prognostic value of HLA-DR expression and dendritic cell infiltration in gastric cancer. Oncology 1998;55:65–9.[CrossRef][Medline]
  12. Ha SJ, Lee SB, Kim CM, Shin HS, Sung YC. Rapid recruitment of macrophages in interleukin-12-mediated tumour regression. Immunology 1998;95:156–63.[CrossRef][Medline]
  13. Key M, Haskill JS. Immunohistologic evidence for the role of antibody and macrophages in regression of the murine T1699 mammary adenocarcinoma. Int J Cancer 1981;28:225–36.[Medline]
  14. Weinberg JB, Hibbs JB Jr. Enhanced macrophage tumoricidal activity and tumor suppression or regression caused by heat-killed Candida albicans. J Natl Cancer Inst 1979;63:1273–8.
  15. Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J, Harris AL. Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res 1996;56:4625–9.[Abstract/Free Full Text]
  16. Garcia-Hernandez ML, Hernandez-Pando R, Gariglio P, Berumen J. Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation. Immunology 2002;105:231–43.[CrossRef][Medline]
  17. Sluyter R, Halliday GM. Enhanced tumor growth in UV-irradiated skin is associated with an influx of inflammatory cells into the epidermis. Carcinogenesis 2000;21:1801–7.[Abstract/Free Full Text]
  18. Robinson MK, Truitt GA, Okayasu T, Wheelock EF. Enhanced suppressor macrophage activity associated with termination of the L5178Y cell tumor-dormant state in DBA/2 mice. Cancer Res 1983;43:5831–6.
  19. Martin F, Caignard A, Jeannin JF, Leclerc A, Martin M. Selection by trypsin of two sublines of rat colon cancer cells forming progressive or regressive tumors. Int J Cancer 1983;32:623–7.[Medline]
  20. Caignard A, Martin MS, Michel MF, Martin F. Interaction between two cellular subpopulations of a rat colonic carcinoma when inoculated to the syngeneic host. Int J Cancer 1985;36:273–9.[Medline]
  21. Dunn TB, Potter M. A transplantable mast-cell neoplasm in the mouse. J Natl Cancer Inst 1957;18:587–601.
  22. Uyttenhove C, Van Snick J, Boon T. Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. I. Rejection by syngeneic mice. J Exp Med 1980;152:1175–83.[Abstract/Free Full Text]
  23. Maric M, Liu Y. Strong cytotoxic T lymphocyte responses to a macrophage inflammatory protein 1{alpha}-expressing tumor: linkage between inflammation and specific immunity. Cancer Res 1999;59:5549–53.[Abstract/Free Full Text]
  24. Xiang J, Moyana T. Regression of engineered tumor cells secreting cytokines is related to a shift in host cytokine profile from type 2 to type 1. J Interferon Cytokine Res 2000;20:349–54.[CrossRef][Medline]
  25. Maldonado-Lopez R, Maliszewski C, Urbain J, Moser M. Cytokines regulate the capacity of CD8{alpha}(+) and CD8{alpha}(–) dendritic cells to prime Th1/Th2 cells in vivo. J Immunol 2001;167:4345–50.[Abstract/Free Full Text]
  26. Yasumi T, Katamura K, Yoshioka T, et al. Differential requirement for the CD40-CD154 costimulatory pathway during Th cell priming by CD8{alpha}+ and CD8{alpha}– murine dendritic cell subsets. J Immunol 2004;172:4826–33.[Abstract/Free Full Text]
  27. Carbonneil C, Saidi H, Donkova-Petrini V, Weiss L. Dendritic cells generated in the presence of interferon-{alpha} stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells. Int Immunol 2004;16:1037–52.[Abstract/Free Full Text]
  28. Chen Z, Moyana T, Saxena A, Warrington R, Jia Z, Xiang J. Efficient antitumor immunity derived from maturation of dendritic cells that had phagocytosed apoptotic/necrotic tumor cells. Int J Cancer 2001;93:539–48.[CrossRef][Medline]
  29. Bonnotte B, Larmonier N, Favre N, et al. Identification of tumor-infiltrating macrophages as the killers of tumor cells after immunization in a rat model system. J Immunol 2001;167:5077–83.[Abstract/Free Full Text]
  30. Chiodoni C, Paglia P, Stoppacciaro A, Rodolfo M, Parenza M, Colombo MP. Dendritic cells infiltrating tumors cotransduced with granulocyte/macrophage colony-stimulating factor (GM-CSF) and CD40 ligand genes take up and present endogenous tumor-associated antigens, and prime naive mice for a cytotoxic T lymphocyte response. J Exp Med 1999;190:125–33.[Abstract/Free Full Text]
  31. Hartmann E, Wollenberg B, Rothenfusser S, et al. Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer. Cancer Res 2003;63:6478–87.[Abstract/Free Full Text]
  32. Pillarisetty VG, Shah AB, Miller G, Bleier JI, DeMatteo RP. Liver dendritic cells are less immunogenic than spleen dendritic cells because of differences in subtype composition. J Immunol 2004;172:1009–17.[Abstract/Free Full Text]
  33. Jonuleit H, Schmitt E. The regulatory T cell family: distinct subsets and their interrelations. J Immunol 2003;171:6323–7.[Free Full Text]
  34. Mills KH. Regulatory T cells: friend or foe in immunity to infection? Nat Rev Immunol 2004;4:841–55.[CrossRef][Medline]
  35. Dinapoli MR, Calderon CL, Lopez DM. The altered tumoricidal capacity of macrophages isolated from tumor-bearing mice is related to reduce expression of the inducible nitric oxide synthase gene. J Exp Med 1996;183:1323–9.[Abstract/Free Full Text]
  36. Sica A, Saccani A, Bottazzi B, et al. Autocrine production of IL-10 mediates defective IL-12 production and NF-{kappa}B activation in tumor-associated macrophages. J Immunol 2000;164:762–7.[Abstract/Free Full Text]
  37. Chaux P, Favre N, Martin M, Martin F. Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats. Int J Cancer 1997;72:619–24.[CrossRef][Medline]
  38. Chaux P, Martin MS, Martin F. Defect of the CTLA4-Ig ligands on tumor-infiltrating dendritic cells. Adv Exp Med Biol 1995;378:389–92.[Medline]
  39. Yang AS, Lattime EC. Tumor-induced interleukin 10 suppresses the ability of splenic dendritic cells to stimulate CD4 and CD8 T-cell responses. Cancer Res 2003;63:2150–7.[Abstract/Free Full Text]
  40. Miller G, Pillarisetty VG, Shah AB, Lahrs S, Xing Z, DeMatteo RP. Endogenous granulocyte-macrophage colony-stimulating factor overexpression in vivo results in the long-term recruitment of a distinct dendritic cell population with enhanced immunostimulatory function. J Immunol 2002;169:2875–85.[Abstract/Free Full Text]
  41. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 2003;299:1033–6.[Abstract/Free Full Text]
  42. Parekh VV, Prasad DV, Banerjee PP, Joshi BN, Kumar A, Mishra GC. B cells activated by lipopolysaccharide, but not by anti-Ig and anti-CD40 antibody, induce anergy in CD8+ T cells: role of TGF-ß1. J Immunol 2003;170:5897–911.[Abstract/Free Full Text]
  43. Jooss K, Gjata B, Danos O, von Boehmer H, Sarukhan A. Regulatory function of in vivo anergized CD4(+) T cells. Proc Natl Acad Sci U S A 2001;98:8738–43.[Abstract/Free Full Text]
  44. Groux H, O'Garra A, Bigler M, et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997;389:737–42.[CrossRef][Medline]
  45. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:2348–57.[Abstract]
  46. Goedegebuure PS, Eberlein TJ. The role of CD4+ tumor-infiltrating lymphocytes in human solid tumors. Immunol Res 1995;14:119–31.[Medline]
  47. Cottrez F, Hurst SD, Coffman RL, Groux H. T regulatory cells 1 inhibit a Th2-specific response in vivo. J Immunol 2000;165:4848–53.[Abstract/Free Full Text]
  48. Young MR, Wheeler E, Newby M. Macrophage-mediated suppression of natural killer cell activity in mice bearing Lewis lung carcinoma. J Natl Cancer Inst 1986;76:745–50.
  49. Medot-Pirenne M, Heilman MJ, Saxena M, McDermott PE, Mills CD. Augmentation of an antitumor CTL response In vivo by inhibition of suppressor macrophage nitric oxide. J Immunol 1999;163:5877–82.[Abstract/Free Full Text]
  50. Wakkach A, Fournier N, Brun V, Breittmayer JP, Cottrez F, Groux H. Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity 2003;18:605–17.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 GutHome page
S Le Gouvello, S Bastuji-Garin, N Aloulou, H Mansour, M-T Chaumette, F Berrehar, A Seikour, A Charachon, M Karoui, K Leroy, et al.
High prevalence of Foxp3 and IL17 in MMR-proficient colorectal carcinomas
Gut, June 1, 2008; 57(6): 772 - 779.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
J. H. Aberle, S. W. Aberle, R. M. Kofler, and C. W. Mandl
Humoral and Cellular Immune Response to RNA Immunization with Flavivirus Replicons Derived from Tick-Borne Encephalitis Virus
J. Virol., December 15, 2005; 79(24): 15107 - 15113.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Y.
Right arrow Articles by Xiang, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Y.
Right arrow Articles by Xiang, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online