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The Journal of Immunology, 2008, 180, 5871 -5881
Copyright © 2008 by The American Association of Immunologists, Inc.
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Tumor-Induced Impairment of TCR Signaling Results in Compromised Functionality of Tumor-Infiltrating Regulatory T Cells1

M. E. Christine Lutsiak*, Yutaka Tagaya{dagger}, Anthony J. Adams{ddagger}, Jeffrey Schlom2,* and Helen Sabzevari*

* Laboratory of Tumor Immunology and Biology, {dagger} Metabolism Branch, and {ddagger} Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
This study demonstrates, for the first time, that murine regulatory T (Treg) cells in the tumor microenvironment display both enhanced proliferation and reduced functionality. This enhanced proliferation, combined with decreased apoptosis, leads to an intratumoral accumulation of Treg cells with a unique phenotype: CD4+CD25+FoxP3+GITRhighCD27lowCD62L–. The loss of functionality is associated with down-regulation of the TCR signaling complex, including IL-2-inducible T cell kinase. It is also demonstrated that tumor-infiltrating Treg cells have impaired TCR-mediated signaling and calcium influx. Based on these findings, this study supports the hypothesis that 1) tumor-infiltrating Treg cells lose functionality due to their diminished ability to become effectively activated and 2) intratumoral accumulation of Treg cells may compensate for the impaired functionality, thus maintaining immune tolerance to the tumor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T (Treg)3 cells, which constitutively express CD25, FoxP3, glucocorticoid-induced TNF receptor (GITR), and CTLA-4, account for 5–10% of the CD4+ T cell population in the spleen (1, 2, 3). Treg cells control key aspects of tolerance to self-Ags, rendering them crucial in the prevention of autoimmune responses (4, 5, 6). Because tumor-associated Ags are derived from self-Ags, Treg cells play an important role in the prevention of antitumor immune responses (7). The number of CD4+CD25+ T cells in peripheral blood increases in many human cancers, including colorectal carcinoma (8), epithelial cancers (9), gastric cancer (10), lymphoma (11), non-small cell lung carcinoma (12), and ovarian cancer (13). Additionally, it has been shown that Treg cells accumulate at the site of tumor in several human cancers (14, 15, 16, 17, 18, 19). In head and neck cancers, CD4+CD25+FoxP3+ cells accumulate at the tumor site (19). In breast cancer, CD4+CD25+ cells accumulate in tumor, and ~50% of these cells are FoxP3+ (16). Additionally, CD4+CD25+ T cells have been observed in non-small cell lung carcinoma (14), ovarian cancer (18), and hepatocellular carcinoma (15) tumor infiltrates. The presence of the Treg cells is also likely to hinder the development of antitumor immune responses following the delivery of an immunotherapeutic agent. For this reason, methods of abrogating the activity of Treg cells may be critical for the successful immunotherapeutic treatment of cancer.

Similar increases in peripheral Treg cell number have also been observed in the spleen in both spontaneous and transplant animal tumor models (20, 21, 22). In animal models, it has further been shown that the removal of CD4+CD25+ Treg cells enhances antitumor immune responses (23, 24). In tumor-bearing mice, CD4+CD25+ cells amass in the tumor (22). Intratumoral depletion of CD4+ or CD25+ cells leads to tumor regression, mediated by CD8+ cells, implicating the local Treg cells in the tumor environment in the lack of antitumor immune responses (23, 24).

To date, there have been few studies addressing the signaling pathways in Treg cells; however, it is known that the suppressive actions of Treg cells require activation through the TCR (6, 25). A recent report has indicated that Treg cells display altered proximal TCR signaling compared with effector CD4+ cells (26). It has been hypothesized that the induction of suppressive functions of Treg cells may require activation of upstream signaling molecules. IL-2-inducible kinase (ITK) is part of the early signaling events upon stimulation of the TCR and that it is required for TCR signaling (27). It is currently not known what role ITK plays in the activation on Treg cells.

There is a large amount of evidence illustrating the increase in the number of Treg cells in the tumor setting, but very little work has been done to address the functionality of these cells. Despite the numerous studies showing that Treg cells from tumor-bearing animals and cancer patients are functional, no studies have directly compared Treg cells from tumor-bearing and nontumor-bearing animals. It is widely understood that tumors have a localized immunosuppressive effect, but the impact of the tumor environment on Treg cells has not been described. This is the first publication directly comparing Treg cells in the spleen and the tumor infiltrate at both the molecular and functional levels. The study reported here provides evidence that tumor-infiltrating Treg cells are functionally impaired, due to a loss of ability to respond to TCR stimulation. The data further demonstrate that the accumulation of Treg cells in the tumor is caused by multiple factors, including increased proliferation, decreased apoptosis, and altered expression of chemokines, chemokine receptors, and cell-surface markers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Tumor models

All animal studies were approved by the National Institutes of Health Animal Care and Use Committee.

Female mice were injected s.c. on the abdomen with 3 x 105 tumor cells. Mice were sacrificed at varying specific intervals following tumor cell injection.

In most experiments, MC32a cells, a murine colon adenocarcinoma cell line (MC38) transduced with human carcinoembryonic Ag, as described elsewhere (28), were used in C57BL/6 mice. In the tumor strain comparison study, MC38 cells, a murine colon adenocarcinoma, and Lewis lung carcinoma cells were used in C57BL/6 mice, while 4T1 cells, a murine mammary carcinoma, were used in BALB/c mice.

Mice

Itk–/– mice, originally established in the 129/Sv mouse line (29) and backcrossed onto C57BL/6 mice for five generations, were generously supplied by the laboratory of Pamela L. Schwartzberg (National Human Genome Research Institute, National Institutes of Health, Bethesda, MD).

Flow cytometry

Cells from the spleen and tumor were stained with Abs to cell-surface markers. Abs against CD3 (145-2C11), CD4 (RM4–6), CD25 (PC61), CD27 (LG.3A10), CD62L (MEL-14), CD69 (H1.2F3), and CTLA-4 (UC10-4F10–11) were purchased from BD Biosciences. Abs against FoxP3 (FJK-16s) and GITR (DTA-1) were purchased from eBioscience. Ab against the TCR (H57–597) was purchased from Abcam. For BrdU studies, mice were given BrdU (Sigma-Aldrich) water (0.8 mg/ml) for 72 h before sacrifice. Following cell-surface marker staining, cells were stained for BrdU incorporation using the BrdU Flow Kit from BD Biosciences and anti-BrdU (B44). Annexin V staining was performed using annexin V (BD Biosciences, catalog no. 556149). FoxP3 staining was performed using the FoxP3 Staining Set from eBioscience, except in selected experiments where combined FoxP3 and BrdU staining was performed using the BrdU staining method.

Data were analyzed using CellQuest software (BD Biosciences). At 3 wk, >96% of the CD4+CD25+ cells were FoxP3+; consequently, cells were gated on CD4 and CD25, and BrdU incorporation of these cells was determined. At 4 wk, only 75% of CD4+CD25+ cells were FoxP3+, and thus the FoxP3– subpopulation was gated out before BrdU incorporation analysis. Because it is not possible to use annexin V in combination with the permeabilization required for FoxP3 staining, analysis could be performed only on CD4+CD25+ cells. At the 3 wk timepoint, virtually all CD4+CD25+ cells are FoxP3, so this is not an issue. At 4 wk, it must be considered that some FoxP3– cells are being included in the analysis. For cell-surface marker analysis, gates were placed on CD4+CD25+FoxP3+ cells and the expression of surface markers on that population was determined.

For intracellular staining of CD3 and TCR, external CD3 or TCR was first stained with high levels (5 µg) of the appropriate FITC-conjugated Ab to ensure as complete coverage of the marker as possible. Cells were permeabilized with BD Cytofix/Cytoperm (BD Biosciences), as per the manufacturer’s instructions. Cells were then stained for intracellular CD3 or TCR using the appropriate APC-conjugated Ab. External staining with APC was minimal (<1%) in samples that remained unpermeabilized, indicating that any APC seen in permeabilized samples was due only to Ab binding to internalized molecules rather than to molecules at the cell surface. Data were analyzed after gating on either the CD4+CD25+ cells or the CD8+ cells.

For intracellular staining of pITK, single-cell suspensions from either the spleen or tumor infiltrate were prepared. Cells were treated with Fc block for 15 min on ice. For activation, cells were activated with 10 µg/ml anti-CD3 (BD Biosciences) and 10 µg/ml goat anti-hamster cross linker (Jackson Immunoresearch) in a 37°C waterbath for 1, 2, or 5 min. After fixation and permeabilization, cells were stained for CD4, CD25, and pITK (pY511, BD Biosciences) for 1 h at room temperature in the dark.

Apoptosis study

Sorted CD4+CD25+ cells (>97% FoxP3+) were placed into culture overnight in either media with 0.5% serum or in normal media on plate-bound anti-CD3 (BD Biosciences). The next day cells were collected from culture and stained with annexin V (BD Biosciences).

Functional assays

For functional assays of Treg cells from spleens of tumor-bearing mice, mice were sacrificed 4 wk after tumor implant. Spleens from tumor-bearing and nontumor-bearing control animals were removed and pressed through a 70-µm filter. On pooled control spleens, a CD8 positive selection was performed using anti-CD8 beads from Miltenyi Biotec according to the manufacturer’s protocol. These CD8 cells were used in the proliferation assay. On pooled spleens from tumor-bearing mice, CD11b depletion removed excess CD11b+ cells using anti-CD11b beads from Miltenyi Biotec according to the manufacturer’s protocol. CD4+CD25+ cells from the CD8– and CD11b– fractions were isolated using the CD4+CD25+ Regulatory T Cell Isolation Kit from Miltenyi Biotec. The CD8–CD4– cells from control splenocytes were used as APCs. CD4+CD25– cells were isolated from splenocytes from a healthy control mouse.

Proliferation assays were set up in 96-well plates with each well containing 5 x 104 CD8+ or CD4+CD25– cells, 1 x 105 APC, and 5 x 104 CD4+CD25+ cells from either tumor-bearing or nontumor-bearing mice (Treg cell/effector cell ratio was 1:1). Anti-CD3 was added to each well in a final concentration of 1 µg/ml. For the CD8+ proliferation assay, 1 µCi [3H]thymidine was added to each well after 48 h, and the plate was harvested and read 24 h later. For the CD4+CD25– proliferation assay, 1 µCi [3H]thymidine was added to each well after 72 h, and the plate was harvested and read 24 h later.

The BD Cytometric Bead Array Kit was used to measure IFN-{gamma} production in supernatants removed from the CD8+ proliferation plates at 48 h.

Assays to examine the function of Treg cells from the tumor infiltrate were performed 3 wk after tumor implant, to avoid the FoxP3– subpopulation that develops at later time points. The tumor infiltrate was sorted for cells with dual expression of CD4 and CD25 because it is not feasible to sort out live Treg cells based on their FoxP3 expression. Expression of FoxP3 by the sorted CD4+CD25+ cells was checked by flow cytometry. Expression was confirmed as being >95% FoxP3+ before their use in the functional assays. This level of FoxP3 expression was identical with that of the splenic samples with respect to both percentage positive cells and mean fluorescence intensity (MFI) in all cases. Tumors were removed and digested (as described in Ref. 30). Cells were stained for CD4, CD8, and CD25. Using a FACSVantage flow cytometer (BD Biosciences), CD4+CD25+ (>95% FoxP3+) and CD8+ cells were purified. Splenic CD4+CD25+ (>95% FoxP3+) and CD8+ cells were isolated from spleens of tumor-bearing and nontumor-bearing animals as described above. Proliferation assays were set up as described above.

In all functional assays with cells from both spleen and tumor infiltrate, wells containing CD4+CD25+ cells, APC, and anti-CD3 but no effector cells were used to determine any background proliferation. The absence of background proliferation in conjunction with the expression of FoxP3 by the CD4+CD25+ cells confirmed that these cells were true Treg cells.

For the dilution study with varying ratios of Treg cells/CD8+ T cells, the cells were purified as described above. The number of CD8+ T cells (5 x 104 cells/well) and APC (1 x 105 cells/well) remained constant while the number of Treg cells per well was varied to change the ratio: 1 x 105 (2:1), 5 x 104 (1:1), 2.5 x 104 (0.5:1), and 1.25 x 104 cells/well (0.25:1). Anti-CD3 was added to each well in a final concentration of 1 µg/ml. [3H]thymidine (1 µCi) was added to each well after 48 h, and the plate was harvested and read 24 h later.

Microarray

CD4+CD25+ T cells were isolated by FACS sorting from both spleens and tumor infiltrate. Cells from four different sorting events, each representing 3–5 animals, were combined to have enough cells for the microarray procedure. RNA was isolated from sorted splenic or tumor-infiltrating CD4+CD25+ T cells (>97% FoxP3+) using the Qiagen RNeasy Plus Mini Kit. Microarray analysis of samples was performed by the Laboratory of Molecular Technology (SAIC-Frederick) using the Affymetrix GeneChip arrays with the expression analysis protocol from the company. Gene ontology analysis was performed using GoMiner software (Genomics and Bioinformatics Group of the Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health).

Quantitative PCR

The quantitative PCR conditions were as follows: 5 µl of 2x Universal TaqMan master mix from Applied Biosciences, 0.5 µl of primer probe mix from Applied Biosciences, and 2.5 µl of H2O. Eight microliters of master mix were added to 2 µl of sample to give a total of 10 µl reaction. The cycling conditions were 52°C for 2 min and 95°C for 10 min (95°C for 15 s and 60°C for 1 min) for 40 cycles.

Calcium influx study

Cells from either the spleen or the tumor infiltrate were labeled with 3.6 µM Indo-1 AM (Molecular Probes) for 30 min at 37°C. After washing, cells were incubated on ice with FITC-labeled anti-CD4, PE-labeled anti-CD25, and biotin-labeled anti-CD3 for 40 min. After washing, cells were resuspended in HBSS with calcium and 0.5% BSA and run on a flow cytometer. After 30 s of equilibration, a stimulus of 1 µg streptavidin (Zymed Laboratories) was given. Data were collected for 4 min total. Analysis was performed using FlowJo software (Tree Star). Gates were placed around CD4+CD25+ cells (samples were checked and found to be >95% FoxP3+) or CD8+ cells and the mean ratio of 405/510 nm was plotted vs time.

Statistics

Data are presented as the means ± SD. The significance of the difference between groups was evaluated with Student’s t test. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expansion and apoptosis of Treg cells in tumor-bearing animals

In this study, MC32a tumors were implanted s.c. in mice. There was an increase in the number of Treg cells in the spleen as tumor burden increased, from 8% in mice with no or very early-stage tumors, to 14% in mice with late-stage tumors. Additionally, we showed that Treg cells accumulate in the tumor and that the proportion of these cells was 2–4 times higher than that seen in the spleen. The number of tumor-infiltrating Treg cells also increased as the tumor progressed, from <20% of CD4+ cells in early-stage tumors to >40% of CD4+ cells in late-stage tumors.

Previous studies have shown that Treg cells have a higher rate of homeostatic proliferation in vivo compared with other T cells (31, 32). To determine whether this in vivo proliferation was altered in tumor-bearing mice, this study compared BrdU incorporation by splenic Treg cells from nontumor-bearing and tumor-bearing mice at various time points. Proliferation of Treg cells in the tumor infiltrate, but not those in the spleen of the same animal, was elevated (Fig. 1A). Between 23 and 43% of Treg cells in the tumor were proliferating compared with 11–16% in the spleen (Fig. 1A; p < 0.01). The elevated level of proliferation was maintained from early through late stages of tumor progression (Fig. 1A).


Figure 1
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  		FIGURE 1. Expansion and apoptosis of Treg cells in spleen and tumor infiltrate. A, The level of in vivo proliferation of CD4+CD25+ Treg cells was measured by BrdU incorporation over 72 h at various time points following tumor implant. The difference between spleen and tumor infiltrate is significant (p < 0.001). Data are representative of three experiments. B, The level of apoptosis of freshly recovered CD4+CD25+ Treg cells was measured using annexin V staining at various time points following tumor implant. The difference between spleen and tumor infiltrate is significant (p < 0.05). Data are representative of three experiments. C, Sorted CD4+CD25+ Treg cells (>96% FoxP3+) from the spleen and tumor infiltrate were cultured either in low serum media or with bound anti-CD3 to induce apoptosis. Apoptosis was measured using annexin V staining after 24 h. The experiment was repeated twice and one example is shown.

 
In splenic Treg cells, there was little or no difference in the level of apoptosis between control and tumor-bearing mice, regardless of the tumor stage (Fig. 1B). However, Treg cells in the tumor infiltrate were much less apoptotic than were those from the spleen of the same animals (Fig. 1B; p < 0.05). The drop in apoptosis (from 11 to 2%) was seen at all time points.

To examine the susceptibility of Treg cells to apoptotic stimuli, sorted Treg cells (>97% CD4+CD25+FoxP3+) from the spleen and tumor infiltrate were incubated with either low serum media or plate-bound anti-CD3 to induce apoptosis. Treg cells from the tumor infiltrate were less susceptible to both types of apoptotic stimuli than were splenic Treg cells from the same animal (Fig. 1C). These data indicate that the protective influence of the tumor environment on the Treg cells is maintained, at least in the short term, after the cells are removed from the tumor.

GITR expression on splenic Treg cells is increased in tumor-bearing mice

To further define the characteristics of tumor-infiltrating Treg cells, we examined the expression of a number of molecules involved in the activation or function of Treg cells. When we examined splenic Treg cells from both tumor-bearing and nontumor-bearing mice, the sole marker with differential expression levels was GITR (Table I). Virtually all of the Treg cells in the spleens of both tumor-bearing and nontumor-bearing mice express GITR; however, the level of expression of GITR, as indicated by MFI, was higher in tumor-bearing mice and increased with tumor progression (Table I). For all other markers studied, no differences in expression were seen between splenic Treg cells in tumor-bearing mice and their nontumor-bearing controls (Table I). There were also no differences seen in the expression of these markers as the tumors progressed (Table I).


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  		Table I. Phenotypic characteristics of splenic and tumor-infiltrating Treg cellsa

 
Tumor-infiltrating Treg cells display a unique phenotype

CD4+CD25+ cells in the spleens and tumor infiltrates of tumor-bearing mice were examined for FoxP3 expression. At 1, 2, and 3 wk posttumor implant, virtually all CD4+CD25+ cells in the tumor infiltrate were FoxP3+ and could be positively identified as Treg cells. There were no differences in the level of FoxP3 expression per cell at any time point (data not shown). At 4 wk posttumor implant, 10–15% of the CD4+CD25+ cells in the tumor infiltrate were FoxP3–. Further examination of these cells revealed that they were GITRhigh, BrdU–, CD27–, CD62L–, and ~50% CD69+. All further phenotypic analysis for the 4-wk time point was performed on the CD4+CD25+FoxP3+ population of Treg cells.

GITR was expressed at higher levels on Treg cells from the tumor infiltrate than on Treg cells from the spleens of the same animals (Table I). Additionally, the level of GITR increased on Treg cells in the tumor infiltrate as the tumor burden increased.

Approximately 80% of Treg cells in both the spleen and tumor infiltrate expressed CD27. In the tumor infiltrate, this CD27+ population separated into CD27high- and CD27low-expressing cells while Treg cells in the spleen remained ~70% CD27high as the tumor progressed (Table I). The Treg cells in the tumor infiltrate appeared to be down-regulating their expression of CD27 as the tumor progressed.

CD62L is highly expressed on naive T cells while there is little to no expression of the molecule on activated T cells or effector memory T cells (33, 34). In tumor-bearing mice, 60–70% of splenic Treg cells expressed CD62L, while in the tumor infiltrate only 15–20% of Treg cells were CD62L+ (Table I).

To determine whether the observed phenotypic changes could be generalized to other tumors and mouse strains, we performed a comparison of the phenotype of tumor-infiltrating Treg cells from several tumor lines in two different mouse strains. MC38 cells (a colon carcinoma cell line that is the parent cell line to MC32a cells) and Lewis lung carcinoma cells were injected into C57BL/6 mice, while 4T1 cells (a breast carcinoma line) were injected into BALB/c mice. FoxP3 expression on CD4+CD25+ T cells was similar in both spleen and tumor infiltrate of mice injected with MC38 and Lewis lung carcinoma tumors compared with previous results with mice injected with MC32a tumors (3 wk of growth, Table II). The number of CD4+CD25+ T cells expressing FoxP3 in both spleen and tumor infiltrate of BALB/c mice injected with 4T1 cells was lower than that seen in C57BL/6 mice injected with any of the other tumors (Table II).


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  		Table II. Comparison of Treg cell phenotypes between different mouse strains and tumor cell linesa

 
Although the level of expression of the molecules of interest showed some variability between the different tumors and mouse strains, the phenotypic changes between spleen and tumor-infiltrating Treg (CD4+CD25+FoxP3+) cells were consistent. In all cases, the GITR expression increased, and CD27 expression and CD62L expression decreased on the tumor-infiltrating CD4+CD25+FoxP3+ cells as compared with the splenic CD4+CD25+FoxP3+ cells from the same animals (Table II).

As a result of these findings, this study defines, for the first time, the phenotype of tumor-infiltrating Treg cells as predominantly CD4+CD25+FoxP3+GITRhighCD27lowCD62L–, while Treg cells in the spleens of both tumor-bearing and nontumor-bearing animals are mainly CD4+CD25+FoxP3+GITRlowCD27highCD62L+.

Interestingly, a study of cell-surface markers on CD8+ effector cells from inside the tumor revealed similar alterations to the phenotype. CD62L and CD27 were down-regulated on tumor-infiltrating CD8+ T cells compared with splenic CD8+ cells from the same animal (data not shown). GITR was up-regulated on a subpopulation of the CD8+ T cells inside the tumor, when compared with splenic CD8+ cells from the same animal (data not shown).

Treg cells from tumor-bearing mice are functionally impaired

Initially we examined the functionality of Treg cells from the spleens of tumor-bearing mice. Proliferation assays of CD8+ T cells, with or without Treg cells from spleens of control or tumor-bearing mice, demonstrated that, at a 1:1 ratio, Treg cells from tumor-bearing mice were significantly (p < 0.05) less capable of suppressing the proliferation of CD8+ T cells (from healthy animals) than were Treg cells from control animals (Fig. 2A). Analysis of the supernatant demonstrated that the same effect (p < 0.05) was seen on IFN-{gamma} production by CD8+ cells (Fig. 2B). Proliferation assays of CD4+CD25– T cells yielded similar results (data not shown). All results indicate that, on a per cell basis, Treg cells from the spleens of tumor-bearing mice are less suppressive than cells from the spleens of nontumor-bearing controls.


Figure 2
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  		FIGURE 2. Functionality of Treg cells from spleens and tumor infiltrate of tumor-bearing mice. A, Functionality of Treg cells from the spleen. CD8+ T cells from healthy animals were stimulated with anti-CD3 and APC in the absence or presence of splenic CD4+CD25+ Treg cells from tumor-bearing animals or nontumor-bearing controls. The ratio of Treg cells/CD8+ cells was 1:1. Proliferation of CD8+ cells was measured by [3H]thymidine incorporation. *, p < 0.05; **, p < 0.005. Data are representative of six experiments. B, Functionality of Treg cells from the spleen. CD8+ T cells from healthy animals were stimulated with anti-CD3 and APC in the absence or presence of splenic CD4+CD25+ Treg cells from tumor-bearing animals or nontumor-bearing controls. The ratio of Treg cells/CD8+ cells was 1:1. IFN-{gamma} production by CD8+ T cells was measured by cytokine bead array. *, p < 0.05. Data are representative of three experiments. C, Functionality of Treg cells from the tumor infiltrate. CD8+ T cells from healthy animals were stimulated with anti-CD3 and APC in the absence or presence of CD4+CD25+ Treg cells from tumor infiltrate or spleens of nontumor-bearing controls. The ratio of Treg cells/CD8+ cells was 1:1. *, p < 0.05; **, p < 0.005. Data are representative of three experiments. D, Functionality of Treg cells from the tumor infiltrate. CD8+ T cells from healthy animals were stimulated with anti-CD3 and APC in the absence or presence of CD4+CD25+ Treg cells from tumor infiltrate or spleens of nontumor-bearing controls. Multiple ratios of Treg cells/CD8+ cells were used: 2:1, 1:1, 0.5:1, 0.25:1. **, p < 0.005. The experiment was repeated twice.

 
We next examined the functionality of tumor-infiltrating Treg cells in relation to splenic Treg cells from nontumor-bearing controls. The tumor infiltrate was sorted for cells with dual expression of CD4 and CD25 because it is not feasible to sort out live Treg cells based on their FoxP3 expression. The expression of FoxP3 in the sorted CD4+CD25+ cells was checked by flow cytometry. Expression was determined to be >97% before use in the functional assays. Additionally, the expression of FoxP3 was found to be identical in tumor infiltrate and splenic samples used in the studies. In all three experiments, Treg cells, at a 1:1 ratio with the CD8+ T cells, from the tumor infiltrate proved to be significantly (p < 0.005) less suppressive than splenic Treg cells from nontumor-bearing animals (Fig. 2C).

Additionally, we performed a titration assay comparing the functionality of tumor-infiltrating Treg cells with splenic Treg cells at varying Treg/CD8+ cell ratios. At all ratios (2:1, 1:1, 0.5:1, and 0.25:1), tumor-infiltrating Treg cells were significantly (p < 0.005) less suppressive than splenic Treg cells (Fig. 2D).

Tumor-infiltrating Treg cells up-regulate gene expression of molecules implicated in the accumulation of cells at the tumor site

Microarray analyses were conducted to elucidate the factors involved in the unique characteristics of tumor-infiltrating Treg cells. We performed gene ontology analysis comparing Treg cells from the tumor infiltrate to Treg cells from the spleen. Only categories that contained more than five genes and had a p value <0.05 were considered. The analysis demonstrated altered expression of several categories of genes that are implicated in the amassing of Treg cells inside the tumor, including cell cycle, cell motility/chemotaxis, cell adhesion, apoptosis, and T cell proliferation (Table III).


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  		Table III. Gene ontology categories showing altered expression in tumor-infiltrating Treg cells compared to splenic Treg cells from nontumor-bearing micea

 
We also analyzed the microarray results on the individual gene level. Comparison of Treg cells from the tumor infiltrate to Treg cells from the spleens of nontumor-bearing animals indicated multiple factors that contribute to the accumulation of Treg cells inside the tumor (Table IV). The microarray results showed increased expression of various cell cycle-related genes that are clearly associated with the dramatic increase in the in vivo proliferation of tumor-infiltrating Treg cells. There was also up-regulation of TGF-β gene expression in these cells, which is consistent with the demonstrations that TGF-β increases proliferation of Treg cells (35, 36, 37). Note that TGF-β was also observed in the immunohistochemical analysis of the tumor microenvironment.


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  		Table IV. Altered expression of select genes by tumor-infiltrating Treg vs. splenic Treg from nontumor-bearing animalsa

 
Additionally, tumor-infiltrating Treg cells showed increased gene expression of several chemokines and chemokine receptors, as compared with splenic Treg cells from nontumor-bearing animals (Table IV). Both CCR2 and CCR5, which aid migration to sites of inflammation, were increased in tumor-infiltrating cells. Interestingly, the tumor-infiltrating Treg cells also showed increased expression of CXCL12, which has been shown to attract CXCR4-expressing cells, such as Treg cells (38).

The ability of tumor-infiltrating Treg cells to respond to stimulation through the TCR is diminished

Because tumor-infiltrating Treg cells showed altered functionality compared with splenic Treg cells from control animals, we focused our attention on unique genes displaying altered gene expression in this comparison. Gene ontology analysis revealed multiple categories of genes that are potentially responsible for the diminished activation of tumor-infiltrating Treg cells (Table III). These categories included the {alpha}β TCR complex, immune response, signal transduction, T cell activation, and TCR signaling pathway (Table III).

At the individual gene level, microarray analysis demonstrated that a number of molecules involved in both proximal and distal TCR signaling pathways, such as the TCR, CD3, CD28, ITK, NFAT, and GATA-3, were down-regulated in tumor-infiltrating Treg cells, as compared with functional Treg cells from nontumor-bearing mice (Table IV). Cell-surface staining confirmed the down-regulation of both TCR and CD3 expression on Treg cells from the tumor infiltrate (Fig. 3A). This down-regulation of TCR and CD3 on the surface of the cells was also seen in the tumor-infiltrating effector cell populations (data not shown). To determine whether the TCR and CD3 were truly down-regulated or whether the molecules had been internalized following activation of the cells, we performed intracellular staining for the molecules. We found that the intracellular expression of both CD3 and TCR was lower in terms of both percentage positive cells and MFI in tumor-infiltrating Treg cells compared with splenic Treg cells from either nontumor-bearing or tumor-bearing mice (data not shown). A similar observation was made on effector CD8+ cells (data not shown).


Figure 3
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  		FIGURE 3. Capability of CD4+CD25+ Treg cells or CD8+ T cells to respond to stimulation through the TCR. A, Cells from the tumor infiltrate or from spleen were stained for CD4, CD25, FoxP3, and either the TCR (left panel) or CD3 (right panel). During analysis, gates were placed around CD4+CD25+FoxP3+ cells and the level of expression of the TCR or CD3 was determined. The solid, filled histogram is the isotype control, the solid line histogram is the spleen, and the dotted line histogram is the tumor infiltrate. Data are representative of six experiments. B, Calcium influx in Treg cells. Indo-labeled cells from either spleen (left panels) or tumor infiltrate (right panels) were loaded with 5 (top panels) or 1 (bottom panels) µg/ml biotinylated anti-CD3. In all cases, at 30 s a stimulus of 1 µg streptavidin was given. For analysis, gates were placed around CD4+CD25+ cells. Data are presented as the mean of 405/510 nm vs time. Data are representative of three experiments. C, Calcium influx in CD8+ T cells. Indo-labeled cells from either spleen (left panels) or tumor infiltrate (right panels) were loaded with 5 (top panels) or 1 (bottom panels) µg/ml biotinylated anti-CD3. In all cases, at 30 s a stimulus of 1 µg streptavidin was given. For analysis, gates were places around CD8+ cells. Data are presented as the mean of 405/510 nm vs time. The experiment was repeated twice.

 
Additionally, quantitative PCR confirmed that gene expression of both GATA-3 (–1.6-fold) and NFAT (–1.7-fold) was down-regulated in these cells. The decrease in functionality of tumor-infiltrating Treg cells reflects a generalized inability of the cells to respond to stimuli, a situation analogous to that seen with tumor-infiltrating effector T cells.

To assess the level of ITK expression as well as the phosphorylation of ITK following activation, we performed intracellular staining for pITK. Treg cells from the tumor infiltrate had much lower levels of pITK than did Treg cells from the spleens of nontumor-bearing animals (Table V; p < 0.05). This was observed in unactivated cells and in cells that had been activated through the TCR. These results further validate the microarray analysis.


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  		Table V. Expression of pITK in splenic and tumor-infiltrating Treg cellsa

 
To establish the functional consequences of the altered expression of the TCR-proximal signaling molecules, calcium influx studies were performed. As shown in Fig. 3B, at 5 µg/ml anti-CD3, splenic Treg cells from nontumor-bearing animals (top left panel) responded to the stimulus by a calcium influx while the tumor-infiltrating Treg cells (top right panel) did not. At 1 µg/ml (bottom panel), neither set of cells responded to the stimulus.

Recently it has been reported that human Treg cells are defective in calcium flux compared with effector T cells (26). We examined the calcium flux of CD8+ T cells in our system and found that the effector T cells from the spleen did have a higher calcium flux than splenic Treg cells (Fig. 3C). Just as for the Treg cells, the calcium flux was dose-dependent and was minimal with 1 µg/ml anti-CD3. Although the Treg cells displayed less of a calcium flux than did the effector cells, the tumor-infiltrating Treg cells exhibited a further impairment of calcium flux. Additionally, the tumor-infiltrating CD8+ T cells did not respond to the activating stimulus by a calcium flux either, which implies that both the regulatory and the effector T cells in the tumor microenvironment are unable to respond to stimulation through the TCR. These data show that the end result of the down-regulation of the TCR signaling pathway is a generalized inability of tumor-infiltrating Treg cells to respond to TCR stimulation.

Regulatory T cells from Itk–/– mice display lower functionality than do cells from wild-type animals

Itk–/– mice were used to determine the functional consequences of lowered expression of ITK in Treg cells. Flow cytometry staining demonstrated that Itk–/– mice had a lower proportion of CD4+ cells in the spleen than did C57BL/6 mice (data not shown). However, CD4+ cells from both strains of mice expressed similar levels of CD25 and FoxP3 (Fig. 4A). Examination of the expression of GITR, CD3, CD27, and CD62L on the Treg cells from these animals showed few differences between the groups (Fig. 4A).


Figure 4
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  		FIGURE 4. Phenotype and functionality of Treg cells from Itk–/– mice. A, Expression of various cell-surface markers on Treg cells from Itk–/– mice was examined by flow cytometry. CD25 expression histogram is gated on CD4+ cells. All other markers were examined on CD4+CD25+ cells. The solid line histogram represents the wild type controls, and the dashed line represents the Itk–/– mice. Data are representative of three experiments. B, CD8+ T cells from C57BL/6 mice were stimulated with anti-CD3 and APC in the absence or presence of CD4+CD25+ Treg cells from C57BL/6 or Itk–/– mice. Proliferation of CD8+ cells was measured by [3H]thymidine incorporation. *, p < 0.05; **, p < 0.005. Data are representative of three experiments.

 
Functional assays using CD8+ cells from C57BL/6 mice demonstrated that Treg cells from Itk–/– mice were significantly less suppressive than Treg cells from C57BL/6 mice (p < 0.05) at both Treg cell/CD8+ cell ratios (Fig. 4B). To achieve comparable levels of inhibition as Treg cells from C57BL/6 mice (p > 0.05), twice the number of Treg cells from Itk–/– mice were required (Fig. 4B). Interestingly, the decreased functionality of Treg cells from Itk–/– mice is reminiscent of that seen in tumor-infiltrating Treg cells (Fig. 2C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
There has been considerable speculation that tumor-infiltrating Treg cells may have enhanced activity; however, this fails to explain how Treg cells could escape the acknowledged immunosuppressive effects of the tumor microenvironment on T cell function. This study is the first to describe the decline in functionality of tumor-infiltrating CD4+CD25+FoxP3+ Treg cells, which indicates that Treg cells do not escape the consequences of the growing tumor on the immune system.

The reduced functionality of Treg cells from tumor-bearing animals does not negate their putative role in preventing antitumor immune responses. It has long been known that the tumor microenvironment impairs the functionality of tumor-infiltrating effector T cells. We postulate that the preinhibited state of these CD8+ T cells renders them more susceptible to the effects of the tumor-infiltrating Treg cells; specifically, the threshold for suppression of the tumor-infiltrating CD8+ effector population has been lowered through interaction with the tumor. Additionally, the overwhelming increase in the number of tumor-infiltrating Treg cells leads to a higher Treg cell/effector cell ratio than is generally seen in the periphery, allowing the sheer volume of inhibiting cells to act, despite the overall decrease in the functionality of these cells. Thus, despite an average 2.4-fold decrease in the functionality of tumor-infiltrating Treg cells, the ~4-fold increase in the Treg cell/effector cell ratio can shift the balance in favor of immune tolerance.

The data reported here indicate a general decrease in the ability of tumor-infiltrating Treg cells to become activated as a result of decreased expression of TCR chains, costimulatory markers such as CD28 and CD27, and signaling molecules required for activation. A recent study showing that Treg cells demonstrate altered proximal TCR signaling compared with effector CD4+ cells hypothesized that the induction of suppressive functions of Treg cells may require activation of upstream signaling molecules (26). Our data demonstrate that further impairment of signaling events in tumor-infiltrating Treg cells is correlated with both an inability to respond to TCR stimulation and diminished functionality.

To explore possible mechanisms for the altered functionality of the tumor-infiltrating Treg cells, we examined changes at the gene level in these cells. Analysis of the tumor-infiltrating Treg cells reveals altered gene expression of a number of signaling molecules. It has previously been suggested that the loss of certain CD4+ effector functions is marked by decreased expression or function of transcription factors such as NFAT and GATA-3 (39, 40). Moreover, it has been demonstrated that ITK is required for CD4+ T cells to exert their function and that ITK acts, in part, through the regulation of expression and function of NFAT and GATA-3 (41, 42). The correlation of decreased functionality of tumor-infiltrating Treg cells and the down-regulation of ITK, NFAT, and GATA-3 by these cells indicates that down-regulation of these molecules plays a role in their loss of functionality. Data from this study that demonstrate a decline in functionality of Treg cells from Itk–/– mice further supports the connection between decreased ITK expression and decreased Treg cell functionality.

Two additional molecules are implicated in the loss of functionality of tumor-infiltrating Treg cells: CD27 and GITR. CD27 may act as a costimulatory factor in assisting activation of T cells, particularly Treg cells (43, 44). Additionally, there is evidence that CD4+CD25+CD27– T cells lack suppressive activity (45, 46). Thus, the decreased expression of this molecule can be indicative of the decreased functionality of the Treg population inside the tumor. Recent studies have shown that stimulation through GITR by either the ligand or a stimulating Ab can decrease Treg cell activation in vitro and in vivo (24, 47, 48). Because tumors can express the ligand for GITR, it is possible that the high levels of GITR on the surface of the tumor-infiltrating Treg cells may be a source for negative regulatory signals that adversely affect the function of the Treg cells (49). Based on these data, we suggest that CD27 down-regulation and GITR up-regulation, in conjunction with the other observed changes, suppress the activation of these cells, leading to functional impairments.

We propose the novel theory that the loss of functionality of tumor-infiltrating Treg cells is due to their diminished ability to become effectively activated. The calcium influx studies confirm that the tumor-infiltrating Treg cells are less capable of responding to activation through the TCR than are splenic Treg cells, supporting our hypothesis that the tumor microenvironment inhibits functionality of Treg cells by decreasing their ability to respond to antigenic stimuli.

It is possible that the diminished function of the tumor-infiltrating Treg cells is not due to the immunoinhibitory effects of the tumor but is rather caused by chronic activation of these cells. The continual increases in in vivo proliferation of these cells seems to indicate, however, that the cells have not reached a state of exhaustion. Additionally, intracellular staining for both CD3 and TCR showed the same down-regulation of expression of these molecules that was seen on the cell surfaces (data not shown). There was no sign of the sequestration of these molecules that would be expected in the setting of chronic activation. Furthermore, the tumor-infiltrating Treg cell data are paralleled in most cases by the data on tumor-infiltrating effector cells, which indicate that they are being affected by similar factors. These facts combine to argue against chronic activation being the primary cause of the observed diminished function in tumor-infiltrating Treg cells.

This study is the first to address the mechanism of the intratumoral accumulation of Treg cells. These results confirm that tumor-infiltrating Treg cells rapidly proliferate. They also describe a decrease in the level of apoptosis by these cells, likely due to their increase in GITR and ephrin A1 (Efna1) expression, both of which provide protection against apoptotic stimuli (47, 50, 51, 52). The increased expression of CXCL12 by these cells can attract CXCR4+ Treg cells from the bone marrow reservoir (38). Finally, our results establish that the tumor-infiltrating population of Treg cells down-regulates CD62L, which is required for homing to the lymph node (53, 54). We propose the following model to explain the intratumoral accumulation of Treg cells. Treg cells traffic to the tumor in response to chemotactic signals. Once within the tumor, Treg cells 1) down-regulate CD62L, preventing their trafficking to the lymph nodes; 2) up-regulate GITR and Efna1, protecting them from apoptosis; and 3) up-regulate expression of cell cycle mediators, enhancing their proliferation. The cumulative effect of these factors is a progressive amassing of Treg cells in the tumor. This accumulation may be a mechanism to compensate for the decreased functionality of the Treg cells, allowing the Treg population to aid in the maintenance of immune tolerance to the tumor.

Effective cancer immunotherapy requires that immune tolerance to the tumor be impeded without the induction of systemic autoimmunity. Data from our study indicate that the enhanced level of proliferation of tumor-infiltrating Treg cells can be exploited through the use of agents that preferentially affect cells with high rates of turnover such as certain chemotherapeutic agents and radiation therapy. Additionally, the high levels of GITR on the surface of tumor-infiltrating Treg cells that we describe herein may allow for a targeted depletion or inhibition of these cells without interfering with systemic maintenance of self-tolerance.

In conclusion, our data refute the theory that tumor-infiltrating Treg cells are highly suppressive. We also offer a novel explanation of both the functional deficits and the accumulation of these cells inside the tumor. These results are critical for an understanding of the impact of the growing tumor on the immune system.


   Acknowledgments
 
We thank Robert Balderas of BD Biosciences for assistance with the pITK study. We thank Susan Sharrow and Francois Van Laethem, National Institutes of Health, for valued advice on the calcium influx studies. We also thank Barbara J. Taylor for vital assistance with the cell sorting, Judith DiPietro for technical assistance, and Bonnie Casey and Debra Weingarten for assistance with manuscript preparation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Jeffrey Schlom, Building 10, Room 8B09, Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. E-mail address: js141c{at}nih.gov Back

3 Abbreviations used in this paper: Treg, regulatory T; Efna1, ephrin A1; GITR, glucocorticoid-induced TNF receptor; ITK, IL-2-inducible kinase; MFI, mean fluorescence intensity. Back

Received for publication June 14, 2007. Accepted for publication February 24, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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