Cancer Research Cancer Medicine 8 2010 Workshops
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
Cancer Research 68, 5432, July 1, 2008. doi: 10.1158/0008-5472.CAN-07-6598
© 2008 American Association for Cancer Research
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 Email this article to a friend
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 Wei, S.
Right arrow Articles by Chang, A. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wei, S.
Right arrow Articles by Chang, A. E.

Immunology

Tumor-Induced Immune Suppression of In vivo Effector T-Cell Priming Is Mediated by the B7-H1/PD-1 Axis and Transforming Growth Factor β

Shuang Wei1, Andrew B. Shreiner1, Nobuhiro Takeshita2, Lieping Chen3, Weiping Zou1 and Alfred E. Chang1

1 University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan; 2 Institute of Gastroenterology of Hofu, Yamaguchi, Japan; and 3 Department of Dermatology and Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland

Requests for reprints: Alfred Chang, 3302 Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI, 48109-0932. Phone: 734-936-4392; Fax: 734-647-9647; E-mail: aechang{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
We have generated effector T cells from tumor-draining lymph nodes (TDLN) that are efficacious in adoptive immunotherapy. We now examine the effect of concomitant tumors on the generation of effector T cells. We inoculated methylcholanthrene (MCA) 205 in the flanks of normal mice and mice bearing MCA 205 lung metastases. TDLN cells from these mice were activated and expanded in vitro, and adoptively transferred to mice bearing lung metastases. Effector T cells generated from TDLN in mice with only flank tumor mediated potent antitumor activity. However, antitumor efficacy of the effector T cells generated from TDLN in mice with pre-existent lung tumor (cTDLN) was reduced. Phenotyping studies showed that dendritic cells in cTDLN expressed higher levels of B7-H1, whereas cTDLN T cells expressed higher levels of PD-1. The levels of IFN{gamma} were reduced, and the levels of CD4+Foxp3+ regulatory T cells were increased in cTDLN versus TDLN. The in vitro activation of cTDLN was increased by blocking B7-H1 or transforming growth factor (TGF)-β. Importantly, we found a synergistic up-regulation of IFN{gamma} with simultaneous blockade of B7-H1 and TGF-β that was much greater than observed with TDLN. In vitro activation of cTDLN with anti–B7-H1 and anti–TGF-β and in vivo administration of these antibodies after adoptive transfer resulted in the abrogation of the suppression associated with cTDLN. These results show a major role for the B7-H1/PD-1 axis and TGF-β as synergistic suppressive mechanisms in cTDLN. Our data have clinical relevance in the generation of effector T cells in the tumor-bearing host. [Cancer Res 2008;68(13):5432–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The clinical application of adoptive cellular immunotherapy has relied upon the generation of tumor-reactive effector cells derived from the patient bearing cancer. This requires the ability to isolate and expand tumor-reactive cells. One of the successful methods has been to isolate effector T cells from a growing tumor, namely tumor-infiltrating lymphocytes (TIL) and expand them in medium containing interleukin (IL)-2 (1–3). However, the successful generation of TIL is not always reliable nor are they always effective after adoptive transfer (4). Our laboratory has been interested in developing effector T cells from lymph nodes (LN) primed by tumor antigen administered as a vaccine (5, 6). Pre-effector LN cells are primed in vivo and subsequently activated and expanded in vitro by methods we have described previously (7, 8). In animal models and in clinical studies, these vaccine-primed LN cells can mediate the regression of established tumors (9–12). However, in clinical trials, only a subgroup of patients benefits. Clearly, there is a need to further improve these therapies, which can result in durable remissions.

The original animal models we have established, upon which we have predicated our clinical studies, have used non–tumor-bearing mice as donors for tumor-primed LN cells. However, as noted above, this does not mirror the clinical setting where patients bearing tumors need to be the donors for effector cells that can be used for adoptive immunotherapy. To accurately mimic the clinical setting, we have established tumor-bearing models where in vivo tumor priming is performed to elicit pre-effector LN cells. Not surprisingly, we have shown that the tumor-bearing host is not a favorable environment to induce pre-effector cells compared with normal non–tumor-bearing hosts. In this study, we have identified mechanisms by which this tumor-induced immune suppression occurs and some approaches to abrogate this suppression. Our findings have relevance not only to adoptive cell therapies but also to active immunotherapies involving vaccinations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Mice. Female C57BL/6 (B6, Thy1.2) and B6.PL-Thy1a/CyJ (Thy1.1) mice were purchased from The Jackson Laboratory. All the mice were maintained in specific pathogen-free conditions and were used for experiments at ages 8 wk or older. Recognized principles of laboratory animal care (NIH publication No. 85–23, revised 1985) were followed, and the University of Michigan Laboratory of Animal Medicine approved all animal protocols.

Murine tumor cells. Methylcholanthrene (MCA) 205 is a 3-methylcholanthrene–induced weakly immunogenic fibrosarcoma that is syngeneic to B6 mice. Tumors were maintained in vivo by serial s.c. transplantation in B6 mice and were used within the eighth transplantation generation. Tumor cell suspensions were prepared from solid tumors by enzymatic digestion in 50 mL of HBSS (Life Technologies) containing 40 mg of collagenase, 4 mg of DNase I, and 100 units of hyaluronidase (Sigma Co.) for 2 h at room temperature. Tumor cells were washed in HBSS three times before s.c. injection in mice to induce TDLN. MC38, a colon cancer tumor syngeneic to B6 mice was used as a specificity control.

Concomitant tumor model. To elicit tumor-draining lymph nodes (TDLN), normal B6 mice were inoculated with 1 x 106 MCA 205 tumor cells in 0.2 mL of PBS s.c. in the lower flank. In the concomitant tumor model, B6 mice were given 0.2 x 106 tumor cells by tail vein injection to establish pulmonary metastasis. Three to 6 d later, these mice were given 1 x 106 tumor cells s.c. in the lower flank.

Lymph node preparation. Nine days after flank tumor inoculation, inguinal LNs from mice bearing only flank tumor (TDLN) or mice bearing both flank tumor and lung metastasis (cTDLN) were removed aseptically. Multiple TDLN or cTDLN were pooled from groups of mice. Lymphoid cell suspensions were prepared by mechanical disruption with the blunt end of a 3-mL plastic syringe in HBSS. The resultant cell suspension was filtered through 40 µm cell strainer and washed in HBSS.

Activation of TDLN cells. TDLN cells were activated with 1.0 µg/mL anti-CD3 monoclonal antibody (mAb) plus 1.0 µg/mL anti-CD28 mAb (BD PharMingen) immobilized in 6-well plates (20 x 106 cells per 10 mL per well) at 37°C with 5% CO2 for 2 d. For antibody immobilization, each well of a 6-well cell culture plate (Costar) was coated with 4 mL of anti-CD3 plus anti-CD28 at 4°C overnight or at room temperature for 5 to 6 h. After antibody activation, the cells were harvested and counted. The cells were then expanded in complete medium (CM) containing human recombinant IL-2 (Chiron Therapeutics) starting at a concentration of 3 x 105 cells per milliliter in tissue culture flasks (Costar) for 3 d. CM consisted of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, 2 mmol/L fresh L-glutamine, 100 mg/mL streptomycin, 100 units/mL penicillin, 50 ug/mL gentamicin, 0.5 ug/mL Fungizone (all from Life Technologies, Inc.), and 0.05 mmol/L 2-mercaptoethanol (Sigma). The concentration of IL-2 was 80 IU/mL. At the end of the cell expansion, cells were harvested and counted to determine the fold of expansion and were used for adoptive transfer. The supernatant was collected. IFN{gamma} was detected with BD OptEIA mouse IFN{gamma} ELISA set (BD PharMingen).

Treatment of established pulmonary metastases. Pulmonary metastases were established via tail vein injection of viable MCA 205 cells (2 x 105) in B6 mice. Three-day tumor-bearing mice were infused i.v. with TDLN cells activated with anti-CD3/anti-CD28 and expanded in IL-2. Commencing on the day of the cell transfer (day 0), i.p. injections of IL-2 (40,000 IU) were given in 0.5 mL of PBS and continued twice daily for 8 doses. Approximately 14 d after tumor injection, all mice were sacrificed, and lungs were harvested for enumeration of pulmonary metastatic nodules. The metastases seemed as discrete white nodules on the black surface of lungs insufflated with a 15% solution of India ink.

Fluorescence-activated cell sorting analysis. All antibodies for flow cytometry were purchased from BD PharMingen except antibodies for Foxp3 and granzyme B, which were purchased from eBioscience. Foxp3 staining was performed using a commercial kit (eBioscience) according to the manufacturer's directions. Flow cytometry was performed on a LSRII (BD Bioscience), and data were analyzed using Diva software (BD Bioscience).

T-cell intracellular cytokine profile analysis. T cells were stimulated for 4 h with leukocyte activation cocktail [ready-to-use polyclonal cell activation mixture with phorbol ester Phorbol 12-Myristate 13-Acetate, a calcium ionophore (Ionomycin), and the protein transport inhibitor BD GolgiPlug (brefeldin A; BD PharMingen)]. Cells were first stained extracellularly with anti-CD4, anti-CD8, and anti-CD90, then were fixed and permeabilized with Fixation/Permeabilization solution (eBioscience), and finally were stained intracellularly with antibodies to mouse IL-2, IL-4, IL-10, perforin, granzyme B, and IFN{gamma} (BD PharMingen). Samples were acquired on a LSR II, and data were analyzed with DIVA software.

ELISPOT assay. MultiScreen filtration plates (96-wells per plate; Millipore) were coated with 100 µL per well of 4 µg/mL purified anti-mouse IFN{gamma} mAb (clone R4-6A2; BD PharMingen) overnight at 4°C and then incubated for 90 min at room temperature with 150 µL per well 1% bovine serum albumin (Sigma) in PBS. Activated lymphocytes (2 x 105 cells in 100 µL of CM) were placed into each well and incubated for 24 h at 37°C, 5% CO2 in the absence or presence of irradiated (60 Gy) specific MCA 205 tumor cells or irrelevant mouse MC38 tumor cells (5 x 104 cells in 100 µL of CM). Plates were incubated overnight at 4°C with 100 µL per well of 4 µg/mL of biotinylated rat anti-mouse IFN{gamma} mAb (clone XMG1.2; BD PharMingen) followed by a 90-min incubation with 100 µL per well anti-biotin alkaline phosphatase (Vector Laboratories, Inc.) diluted 1:1,000. Spots were visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium alkaline phosphatase substrate and counted using the ImmunoSpot analyzer (Cellular Technology, Ltd.). Data are reported as an average number of spots per 2 x 105 responders ± SE of triplicate samples.

Blockade of B7-H1 and transforming growth factor β. Anti-mouse B7-H1 mAb (clone MIH5; rat IgG2a, {lambda} isotype, eBioscience) 5 ng/mL and anti–TGF-β 1/300 (ascites raised with hybridoma B lymphocyte 1D11.16.8 purchased from American Type Culture Collection) was added to the TDLN and cTDLN cells during activation with anti-CD3/CD28 and during expansion with IL-2 to block B7-H1 and transforming growth factor (TGF)-β in vitro. Anti-mouse B7-H1 mAb (clone 10B5, hamster IgG; ref. 13) was used to block the B7-H1 for the in vivo experiments. This larger quantity of mAb was provided by Dr. L Chen (Johns Hopkins School of Medicine, Baltimore, MD). Fifty micrograms of anti–B7-H1 and 100 µL of anti–TGF-β were administered i.p. on the day of adoptive transfer and on day 3, 6, and 9 after adoptive transfer.

To determine if the in vivo blockade of B7-H1 and TGF-β was affecting the transferred cTDLN or had an effect on host immune cells, expanded cTDLN cells from Thy1.2 C57BL/6 mice were transferred to 3-d lung metastasis–bearing Thy1.1 mice. Lungs were harvested 8 d after adoptive transfer. Single-cell suspension from lungs were first stained extracellularly for CD45, CD90.1, CD90.2, CD4, and CD8 and then stained for intracellular IFN{gamma} and Foxp3 as described above. Donor T cells were gated on CD45+CD90.2+, and recipient T cells were gated on CD45+CD90.1+.

Statistical analysis. In the adoptive transfer model, the significance of differences in numbers of metastatic nodules between experimental groups was determined by the one-way ANOVA test using StatView software. P values of <0.05 were considered statistically significant. Student's t test was used to analyze flow cytometry and cytokine release data. Two-tailed P values of <0.05 were considered statistically significant between two groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Pre-established visceral or s.c. tumor will suppress the induction of TDLN effector T cells. We used the MCA 205 sarcoma tumor model to evaluate the effect of pre-existent tumor on the ability to elicit TDLN cells for adoptive immunotherapy. Groups of mice were inoculated with tumor cells i.v. to establish lung metastases 3 to 6 days before s.c. inoculation of tumor cells in the flank as described in the Materials and Methods (Fig. 1A ). Control mice received s.c. tumor but not i.v. tumor cells. Nine days after s.c. inoculation of tumor cells, TDLN were harvested from the inguinal regions. The effector T cells generated from mice receiving s.c tumor were termed TDLN. The effector T cells generated from mice receiving i.v. and s.c tumor were termed concomitant TDLN (cTDLN). The tumor-draining LN cells were activated in anti-CD3/CD28 as described in the Materials and Methods and expanded in IL-2. After the culture period, the antitumor reactivity of the cells was assessed in the adoptive immunotherapy of 3-day established lung metastases as described in Materials and Methods. As illustrated in Fig. 1B, the antitumor reactivity of the cTDLN was significantly reduced on a per cell basis compared with equivalent numbers of transferred TDLN in a dose-dependent manner. The data suggest that concomitant visceral tumor induces immune suppression in the tumor-draining LNs. In a separate experiment, we found that the reduced effector function of cTDLN was dependent on timing of the lung tumor establishment. If i.v. tumor cells were injected the same day as s.c. tumor inoculation, the effector function remained, suggesting that there was no significant immune suppression induced by the lung tumors (Fig. 1C). It indicates that the lung tumors needed to be established before s.c. tumor inoculation.


Figure 1
View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Pre-established visceral or s.c. tumor suppressed the induction of TDLN effector T cells. A, concomitant tumor model and schematic experimental design. TDLN were harvested from the inguinal regions of mice bearing 9-d flank tumor by s.c. inoculation. cTDLN were harvested from the inguinal regions of mice given tumor cells by i.v. inoculation 3 d before the s.c. inoculation of tumor cells in the flank. TDLN and cTDLN cells were activated and expanded ex vivo and then adoptively transferred to mice with pulmonary metastases. B, the antitumor activity of the cTDLN was significantly reduced compared with comparable numbers of TDLN cells as assessed by the enumeration of pulmonary metastatic nodules. C, the reduced antitumor activity of the cTDLN was dependent on the establishment of the lung tumor before (i.e., 6 d) s.c. tumor inoculation as opposed to the same day (i.e., day 0). Cells (2 x 106) were transferred. D, the establishment of s.c. flank tumors 6 and 12 d before inoculating tumor cells in the contralateral flank resulted in cTDLN with reduced antitumor reactivity compared with TDLN cells as assessed in the adoptive immunotherapy model. Cells (5 x 106) were transferred. Two independent experiments with five animals per experiment per group.

 
We proceeded to determine if pre-existent s.c. tumor, as opposed to visceral tumor, would suppress the in vivo priming of effector cells. In this experiment, MCA 205 tumor was inoculated s.c. in the flank 6 and 12 days before injecting tumor cells in the contralateral flank. Control mice were inoculated with tumor cells on one flank only. TDLN and cTDLN were harvested 9 days after the last flank injection. As before, the cells were activated and expanded and their antitumor reactivity assessed in the adoptive immunotherapy model. As shown in Fig. 1D, pre-existent s.c. flank tumors had an adverse effect in the priming of effector cells in the LNs.

Phenotypic differences between TDLN and cTDLN: dendritic cells, Tregs, and effector T cells. We proceeded to examine differences in the LN cellular subsets between TDLN and cTDLN. The latter were established using mice with 12-day lung metastases and 9-day flank tumors to prime draining LNs. TDLN were harvested from control mice that had 9-day flank tumors. TDLN and cTDLN were obtained from several mice and separately pooled. The number of cells obtained per TDLN versus cTDLN were not significantly different (data not shown). The first cell subset we examined were dendritic cells (DC) (CD11b–CD11c+) with respect to various costimulatory and coinhibitory markers. There were no differences in the expression of CD40, CD86, or MHC class II between DC from both groups. However, we did observe significant up-regulation of B7-H1 (Fig. 2A ) with a concomitant down-regulation of CD80 (Fig. 2B) in DC from cTDLN compared with TDLN. This represents an alteration in host DC induced by the presence of established tumor that has not been previously described. Because of this finding, we examined the expression of the ligand for B7-H1, namely PD-1, on TDLN and cTDLN T cells. In Fig. 3A , we found that PD-1 was significantly more expressed on activated CD4+ and CD8+ cells from cTDLN compared with TDLN. This up-regulated expression of PD-1 in cTDLN cells was maintained after expansion in IL-2 for 3 d (Fig. 3B). Furthermore, the PD-1+ T cells in cTDLN expressed low levels of L-selectin (CD62L; Fig. 3C). We examined expression of OX-40 and CTLA-4 on fresh and activated LN cells and did not find significant differences between the two groups (data not shown).


Figure 2
View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Altered expression of B7 family members in cTDLN. A, higher levels of B7-H1 on cTDLN DC compared with TDLN DC. B, lower levels of CD80 in DCs were observed in freshly harvested cTDLN compared with TDLN. The expression of B7-H1 and CD80 was determined by fluorescence-activated cell sorting (FACS) analysis, gated on lin–CD11c+CD11b– cells. Columns, mean percentage of expression; bars, SE. Results are from at least three independent experiments with four mice per group. In each experiment, LNs were pooled from each individual mouse and analyzed separately.

 

Figure 3
View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Altered expression of PD-1 expression observed in T cells from cTDLN compared with TDLN. A, freshly harvested T cells from cTDLN were increased compared with TDLN. B, after activation and expansion in culture, this increased expression of PD-1 by cTDLN cells compared with TDLN cells was still observed. The expression of PD-1 was determined by FACS analysis, gated on CD4+CD90+ or CD8+CD90+ T cells. C, majority of the PD-1+ T cells observed in expanded cTDLN expressed low levels of L-selectin. Columns, mean percentage of expression; bars, SE. Results are from at least three independent experiments with four mice per group. In each experiment, LNs were pooled from each individual mouse and analyzed separately.

 
We also looked at T-cell subpopulations from freshly harvested cTDLN versus TDLN. There were no significant differences in the percentage of CD3+, CD4+, or CD8+ lymphoid cells. However, we did observe an increased percentage of CD4+Foxp3+ cells obtained from cTDLN compared with TDLN (Fig. 4A ). In addition, there was an increased percentage of CD8+Foxp3+ found in cTDLN compared with TDLN (Fig. 4A). After antibody activation and expansion in IL-2, the CD4+Foxp3+ and CD8+Foxp3+ subpopulations persisted (Fig. 4B).


Figure 4
View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Increased Foxp3+CD4+ and Foxp3+CD8+ T cells were observed in cTDLN compared with TDLN. Increased Foxp3+CD4+ and Foxp3+CD8+ T cells were observed in freshly harvested (A) and activated/expanded (B) cTDLN compared with TDLN. The percentage of Foxp3+ T cells was determined by FACS analysis, gated on CD4+CD90+ or CD8+CD90+ T cells. Columns, mean percentage of Foxp3+ T cells; bars, SE. Results are from at least three independent experiments with four mice per group. In each experiment, LNs were pooled from each individual mouse and analyzed separately.

 
Functional differences between TDLN and cTDLN cells. We proceeded to evaluate the functional characteristics of T cells obtained from cTDLN compared with TDLN. Intracytoplasmic staining for IFN{gamma} revealed decreased levels in cTDLN compared with TDLN (Fig. 5A ; P < 0.01). This was correlated with decreased amounts of IFN{gamma} production by cTDLN effector cells compared with TDLN cells during activation and expansion of cells as described in Materials and Methods (Fig. 5B; P < 0.01). We further analyzed the response of these activated cells in response to irradiated tumor using an ELISPOT assay (Fig. 5C), and show that the number of tumor-induced IFN{gamma} spots were significantly reduced in cTDLN compared with TDLN (P < 0.01). This was a tumor-specific response as shown by the low number of spots in response to MC38 tumor cells. We also evaluated intracytoplasmic staining of IL-2, IL-4, IL-10, perforin, and granzyme B. We did not detect IL-4 or IL-10 intracytoplasmically. There were low levels of intracytoplasmic IL-2 that were similar between both groups of LN cells. Almost all the cells in both groups expressed perforin and granzyme B at similar levels (data not shown).


Figure 5
View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. cTDLN cells produced less IFN{gamma} compared with TDLN cells. A, percentage of IFN{gamma}+CD8+ cells was lower in activated/expanded cTDLN compared with TDLN, whereas the percentage of IFN{gamma}+CD4+ cells was similar in TDLN and cTDLN. The percentage of IFN{gamma}+ T cells was determined by intracellular staining and analyzed by FACS analysis, gated on CD8+CD90+ and CD4+CD90+ T cells. Columns, mean percentage of IFN{gamma}+ T cells; bars, SE (eight independent experiments). B, the secretion of IFN{gamma} was lower in expanded cTDLN compared with TDLN. Supernatants were collected from the expanded cTDLN and TDLN cells. IFN{gamma} was detected by ELISA. Columns, mean values of IFN{gamma}; bars, SE of triplicate samples from three independent experiments. C, the frequency of tumor-specific IFN{gamma}-producing effector cells was significantly less in the cTDLN population. Effector cells (2 x 105) were cultured alone or in the presence of 5 x 104 irradiated specific MCA 205 or irrelevant mouse MC38 tumor cells for 24 h in 96-well ELISPOT plates coated with anti-IFN{gamma}. The number of spots from wells containing effector cells alone was subtracted from the corresponding wells for each sample to determine the number of tumor-induced spots. Columns, mean number of spots per well; bars, SE. (five independent experiments).

 
Blockade of B7-H1 and TGF-β. Our studies thus far indicate that there is up-regulation of B7-H1 and increased numbers Treg cells in cTDLN versus TDLN. We proceeded to examine blockade of B7-H1 as well as TGF-β, an important cytokine in the function and expansion of Treg cells using neutralizing antibodies. Anti–B7-H1 and anti–TGF-β mAbs where added to cTDLN and TDLN cells during in vitro activation and expansion as described in the Materials and Methods section. At the end of the culture period, cells were harvested and analyzed for intracytoplasmic IFN{gamma}. As shown in Fig. 6A , the presence of anti–B7-H1 mAb resulted in a significant increase of intracytoplasmic IFN{gamma} for both CD4+ and CD8+ cTDLN cells compared with respective control cultures without antibody; which was not observed for TDLN cells. The presence of anti–TGF-β mAb resulted in a significant increase of intracytoplasmic IFN{gamma} for both TDLN and cTDLN cells; with a significant increase expressed by cTDLN compared with TDLN cells. The presence of both antibodies resulted in a synergistic increase of IFN{gamma} in cTDLN cells that was significantly greater than observed with TDLN cells.


Figure 6
View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Blockade of B7-H1 and TGF-β recovered the reduced antitumor activity of cTDLN. B7-H1 and TGF-β were blocked as we described in Materials and Methods. A, blockade of B7-H1 and TGF-β synergistically increased IFN{gamma}+ T cells in cTDLN and TDLN. The increase in IFN{gamma}-producing T cells was significantly larger in cTDLN than in TDLN. The percentage of IFN{gamma}+ T cells was determined by intracellular staining and analyzed by FACS. Columns, mean percentage of IFN{gamma}+ T cells; bars, SE. Gated on CD4+ or CD8+CD90+ T cells from eight independent experiments; *, P < 0.01; **, P < 0.001 compared with no blockade. B, blockade of B7-H1 and TGF-β recovered the reduced antitumor activity of cTDLN as assessed in an adoptive immunotherapy model. Results are representative of two independent experiments. Cells were transferred (8 x 106). Five animals were in each group. See Results for description of each group. C and D, blockade of B7-H1 and TGF-β increased IFN{gamma}+ T cells (C) and decreased CD4+Foxp3+ regulatory T cells (D) in cTDLN cells and recipient T cells harvested from the lungs of recipient mice. Expanded cTDLN cells from Thy1.2 C57BL/6 mice were transferred to Thy1.1 mice. Lungs were harvested 8 d after adoptive transfer. Single-cell suspension from lungs was stained for intracellular IFN{gamma} and Foxp3. Donor cells were gated on CD45+CD90.2+ and recipient cells were gated on CD45+CD90.1+.

 
Based on these results, we examined the in vivo function of cTDLN cells in conjunction with this blockade strategy in our adoptive immunotherapy model. Neutralizing mAb was used during the activation/expansion of cTDLN and also administered exogenously to the adoptive transfer hosts as described in the Materials and Methods section. Figure 6B illustrates that cTDLN cells cultured in the absence of mAbs have deficient antitumor reactivity compared with TDLN cells. However, the presence of anti–B7-H1 or anti–TGF-β during culture and in vivo administration of either mAb partially abrogated the suppressed antitumor reactivity of cTDLN cells. The combination of the two mAbs resulted in complete abrogation of the suppressed antitumor reactivity when compared with TDLN cells. These results were confirmed in a replicate experiment. In another experiment, we found that the antibody blockade needed to be applied both in vitro and in vivo to achieve maximal therapeutic efficacy of cTDLN (data not shown).

Next, as simultaneous blockade of B7-H1 and TGF-β resulted in maximal tumor reduction, we performed additional studies to determine if the in vivo blockade of B7-H1 and TGF-β was affecting the transferred cTDLN or had an effect on host immune cells. Expanded cTDLN cells from Thy1.2 (CD90.2+) mice were transferred to Thy1.1 (CD90.1+) mice bearing lung metastases. Lungs were harvested 8 days after adoptive transfer. Single-cell suspensions prepared from lungs were stained for intracellular IFN{gamma} and Foxp3. Donor cells were gated on CD90.2+ and recipient cells were gated on CD90.1+. Blockade of B7-H1 and TGF-β increased IFN{gamma}+ T cells (Fig. 6C) and decreased CD4+Foxp3+ regulatory T cells (Fig. 6D) in both cTDLN cells and recipient T cells harvested from the lungs of recipient mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In this study, we have documented that the establishment of visceral or s.c. tumors suppress the induction of pre-effector T cells in LNs primed by a subsequent s.c. tumor cell inoculation. Based on previous observations made by our laboratory, this is not simply a phenomenon of increased tumor burden. We have reported that the establishment of bilateral s.c. flank tumors at the same time does not lead to suppressed pre-effector cell induction in TDLN compared with unilateral flank-bearing mice (14). In our current model, this suppressed priming is associated with an up-regulation of the coinhibitor molecule B7-H1 expressed on CD11c+ DC and a parallel up-regulation of PD-1, the counter-receptor of B7-H1, on pre-effector T cells. We found that the up-regulation of PD-1 was maintained after in vitro T activation and expansion in IL-2. Furthermore, the majority of the PD-1+ T cells expressed low levels of CD62L. It has been reported that tumor-specific pre-effector T cells are enriched in CD62Llow T-cell population (15). The data suggest that the PD-1+CD62Llow T cells may be functionally exhausted tumor-specific effector T cells. In line with this concept, these T cells expressed significantly less intracytoplasmic IFN{gamma} compared with effector cells generated in non–tumor-bearing hosts.

B7-H1 (aka PD-L1) is a cell surface glycoprotein of the B7 family that has been described to negatively regulate T-cell functions by engagement with PD-1, a CD28 family member receptor. Besides antigen-presenting cells, B7-H1 mRNA is found in a variety of nonlymphoid parenchymal organs, including the heart, placenta, skeletal muscle, and lung (13, 16, 17). B7-H1 costimulates T-cell growth, selectively induces IL-10 during priming of T cells (16, 17), and promotes programmed cell death of effector T cells through ligation of an unknown receptor (13). In addition, B7-H1 is thought to inhibit T-cell growth and cytokine production by ligation of the PD-1 receptor (18), which is expressed on activated T and B cells (16, 19). Chen and colleagues (20) reported the presence of B7-H1 protein by immunohistochemistry in a wide range of human cancers. Tumor-associated B7-H1 induces apoptosis of effector T cells and is thought to contribute to immune evasion by cancers (13). Other reports indicating that blockade of B7-H1 enhances tumor immunity but has no direct effects on tumor cells (13, 21, 22). In our model, we found that <10% of MCA 205 tumor cells when freshly harvested expressed B7-H1, although it was inducibly expressed in >96% of tumor cells when cultured with IFN{gamma} in vitro (data not shown). In a study reported by Zou and coworkers (23), virtually all the DCs isolated from ovarian tumor tissues or tumor-draining LNs expressed high levels of B7-H1. Furthermore, blockade of B7-H1 enhanced DC-mediated T-cell activation that was accompanied by down-regulation of T-cell production of IL-10, with a concomitant up-regulation of IL-2 and IFN{gamma} production. Different soluble factors have been implicated in up-regulating B7-H1, which include IFN{gamma} (13, 21), IL-10, and vascular endothelial growth factor (23). In the context of our model, we plan to investigate the possible role of these mediators in the microenvironment of cTDLN.

Dysfunctional DCs that express up-regulated B7-H1 may lead to the induction of Treg cells (24). We have shown that there is a significantly increased number of Treg cells in cTDLN versus TDLN. TGF-β is a potent inducer of Treg cells as well (25, 26). Furthermore, Tregs can suppress effector function via TGF-β signaling (27). One of our hypotheses is that TGF-β is a critical mediator in the suppressive phenomenon observed in the development of cTDLN pre-effector cells. TGF-β may be drived from multiple cells including tumor cells and Treg cells. Indeed, we have found that freshly harvested MCA 205 tumor cells secrete TGF-β in culture when assessed 3 days later (data not shown), giving support to our hypothesis. We found that the suppressed tumor reactivity of cTDLN effector cells can be partially blocked in vitro. The presence of either anti–TGF-β or anti–B7-H1–blocking antibodies during in vitro activation and expansion of cTDLN and TDLN cells resulted in increased intracellular CD4+ and CD8+ IFN{gamma} expression. More importantly, there was a synergistic increase of intracellular IFN{gamma} in the presence of both antibodies that was significantly greater in cTDLN compared with TDLN cells. In adoptive transfer where exogenous blocking antibodies were also administered, the suppression of the antitumor reactivity of the cTDLN effector cells was completely abrogated when compared with TDLN effector cells. Although MCA 205 secretes TGF-β, we found reduced numbers of host-derived Treg cells in the tumor microenvironment when B7-H1 and TGF-β blockade was performed after adoptive immunotherapy. These studies implicate the role of B7-H1/PD-1 axis and TGF-β (or Treg cells) as important and previously unappreciated synergistic mechanisms in suppressing cellular responses to active immunization in the tumor-bearing host.

Immunotherapy often targets cancer patients with metastatic diseases. The model described in this report is very clinically relevant to the development of effective adoptive cell therapies. Effector cells have to be generated from the cancer-bearing patient. Most animal models have used normal, non–tumor-bearing hosts as donors for effector cells transferred to secondary recipients. Our current animal model shows that blockade of suppressive mechanisms induced by established tumors can optimize the generation of antitumor reactive effector cells. The use of in vitro blockade can readily be applied in clinical adoptive cellular therapies.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: NIH CA82529 and the Gillson Longenbaugh Foundation.

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 12/11/07. Revised 4/ 4/08. Accepted 5/ 1/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Rosenberg SA, Spiess P, Lafreniere R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 1986;233:1318–21.[Abstract/Free Full Text]
  2. Rosenberg SA, Packard BS, Aebersold PM, et al. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. N Engl J Med 1988;319:1676–80.[Abstract]
  3. Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol 2005;23:2346–57.[Abstract/Free Full Text]
  4. Dudley ME, Wunderlich JR, Shelton TE, Even J, Rosenberg SA. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J Immunother 2003;26:332–42.[CrossRef][Medline]
  5. Geiger JD, Wagner PD, Cameron MJ, Shu S, Chang AE. Generation of T-cells reactive to the poorly immunogenic B16-BL6 melanoma with efficacy in the treatment of spontaneous metastases. J Immunother Emphasis Tumor Immunol 1993;13:153–65.[Medline]
  6. Shu S, Krinock RA, Matsumura T, et al. Stimulation of tumor-draining lymph node cells with superantigenic staphylococcal toxins leads to the generation of tumor-specific effector T cells. J Immunol 1994;152:1277–88.[Abstract]
  7. Li Q, Yu B, Grover AC, Zeng X, Chang AE. Therapeutic effects of tumor reactive CD4+ cells generated from tumor-primed lymph nodes using anti-CD3/anti-CD28 monoclonal antibodies. J Immunother 2002;25:304–13.[CrossRef][Medline]
  8. Li Q, Carr A, Ito F, Teitz-Tennenbaum S, Chang AE. Polarization effects of 4–1BB during CD28 costimulation in generating tumor-reactive T cells for cancer immunotherapy. Cancer Res 2003;63:2546–52.[Abstract/Free Full Text]
  9. Chang AE, Yoshizawa H, Sakai K, Cameron MJ, Sondak VK, Shu S. Clinical observations on adoptive immunotherapy with vaccine-primed T-lymphocytes secondarily sensitized to tumor in vitro. Cancer Res 1993;53:1043–50.[Abstract/Free Full Text]
  10. Chang AE, Sondak VK, Bishop DK, Nickoloff BJ, Mulligan RC, Mule JJ. Adoptive immunotherapy of cancer with activated lymph node cells primed in vivo with autologous tumor cells transduced with the GM-CSF gene. Hum Gene Ther 1996;7:773–92.[Medline]
  11. Chang AE, Aruga A, Cameron MJ, et al. Adoptive immunotherapy with vaccine-primed lymph node cells secondarily activated with anti-CD3 and interleukin-2. J Clin Oncol 1997;15:796–807.[Abstract/Free Full Text]
  12. Chang AE, Li Q, Jiang G, Sayre DM, Braun TM, Redman BG. Phase II trial of autologous tumor vaccination, anti-CD3-activated vaccine-primed lymphocytes, and interleukin-2 in stage IV renal cell cancer. J Clin Oncol 2003;21:884–90.[Abstract/Free Full Text]
  13. Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 2002;8:793–800.[Medline]
  14. Sondak VK, Wagner PD, Shu S, Chang AE. Suppressive effects of visceral tumor on the generation of antitumor T cells for adoptive immunotherapy. Arch Surg 1991;126:442–6.[Abstract/Free Full Text]
  15. Kagamu H, Shu S. Purification of L-selectin(low) cells promotes the generation of highly potent CD4 antitumor effector T lymphocytes. J Immunol 1998;160:3444–52.[Abstract/Free Full Text]
  16. Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 1999;5:1365–9.[CrossRef][Medline]
  17. Tamura H, Dong H, Zhu G, et al. B7-H1 costimulation preferentially enhances CD28-independent T-helper cell function. Blood 2001;97:1809–16.[Abstract/Free Full Text]
  18. Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 2000;192:1027–34.[Abstract/Free Full Text]
  19. Agata Y, Kawasaki A, Nishimura H, et al. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol 1996;8:765–72.[Abstract/Free Full Text]
  20. Chen L. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev 2004;4:336–47.
  21. Blank C, Brown I, Peterson AC, et al. PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8+ T cells. Cancer Res 2004;64:1140–5.[Abstract/Free Full Text]
  22. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 2002;99:12293–7.[Abstract/Free Full Text]
  23. Curiel TJ, Wei S, Dong H, et al. Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity. Nat Med 2003;9:562–7.[CrossRef][Medline]
  24. Zou W. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev 2006;6:295–307.
  25. Gorelik L, Flavell RA. Abrogation of TGFβ signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity 2000;12:171–81.[CrossRef][Medline]
  26. Gorelik L, Flavell RA. Transforming growth factor-β in T-cell biology. Nat Rev 2002;2:46–53.
  27. Chen ML, Pittet MJ, Gorelik L, et al. Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-β signals in vivo. Proc Natl Acad Sci U S A 2005;102:419–24.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 JCOHome page
M. L. Disis, D. R. Wallace, T. A. Gooley, Y. Dang, M. Slota, H. Lu, A. L. Coveler, J. S. Childs, D. M. Higgins, P. A. Fintak, et al.
Concurrent Trastuzumab and HER2/neu-Specific Vaccination in Patients With Metastatic Breast Cancer
J. Clin. Oncol., October 1, 2009; 27(28): 4685 - 4692.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
V. Pulko, X. Liu, C. J. Krco, K. J. Harris, X. Frigola, E. D. Kwon, and H. Dong
TLR3-Stimulated Dendritic Cells Up-regulate B7-H1 Expression and Influence the Magnitude of CD8 T Cell Responses to Tumor Vaccination
J. Immunol., September 15, 2009; 183(6): 3634 - 3641.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
I. Kryczek, S. Wei, W. Szeliga, L. Vatan, and W. Zou
Endogenous IL-17 contributes to reduced tumor growth and metastasis
Blood, July 9, 2009; 114(2): 357 - 359.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. Ilkovitch and D. M. Lopez
The Liver Is a Site for Tumor-Induced Myeloid-Derived Suppressor Cell Accumulation and Immunosuppression
Cancer Res., July 1, 2009; 69(13): 5514 - 5521.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. H. Schreiber, V. V. Deyev, J. D. Rosenblatt, and E. R. Podack
Tumor-Induced Suppression of CTL Expansion and Subjugation by gp96-Ig Vaccination
Cancer Res., March 1, 2009; 69(5): 2026 - 2033.
[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 Email this article to a friend
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 Wei, S.
Right arrow Articles by Chang, A. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wei, S.
Right arrow Articles by Chang, A. E.

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