Mice and Tumor Cell Lines

All animal study protocols were reviewed and approved by the institutional review committee. Six-week old female C57BL/6 mice and C57BL/6 Rag1^−/− mice were purchased from Jackson Laboratories. The dnTGF-BetaRII mice were a generous gift from Dr. RA Flavell of Yale University ³¹. The mice were housed and bred at Washington University School of Medicine Animal Facilities in specific pathogen-free conditions. Animal care and all procedures were in accordance with the guidelines of the animal care committee. The mouse pancreas adenocarcinoma cell line Pan02 is syngeneic to C57BL/6 and was obtained from DCT Tumor Repository (NCI-Frederick Cancer Research and Development Center, Bethesda, MD). The C57BL/6 syngeneic esophageal tumor cell line, Eso-2 was a gift from Dr. R. Battafarano. Both cell lines were grown in RPMI culture medium (Mediatech, Manassas, VA) supplemented with 10% (v/v) fetal bovine serum (FBS, Life Technologies, Carlsbad, CA), and 1% glutamine and antibiotics.

T cell Isolation and Preparation

The spleen and LN from C57BL/6 female mice were harvested and homogenized. The splenocytes were briefly treated with ACK buffer to remove red blood cells. Tumor tissue was digested in a buffer containing 1mg/ml collagenase, 2.5 U/ml hyaluronidase and 0.1mg/ml DNase for 30 minutes. The suspension was then separated from tissue debris by filtration through 40 μm cell strainer and centrifugation on Histopaque®-1083. All cells were then washed twice in PBS containing 0.5% FBS and passed again through 40 micron nylon mesh to obtain single cell suspensions. CD4⁺25⁻ and CD4⁺25⁺ Treg populations were separated using a mouse regulatory T cell isolation kit (Miltenyi Biotech, Auburn, CA) per manufacturer’s instructions. Briefly, CD4⁺ cells were separated from other cells by negative selection with an antibody cocktail provided by the manufacturer. The cells were then passed over a magnetic column and the CD4⁺ cells were collected. CD25⁺ cells were then selected by positive selection using anti-CD25 magnetic microbeads and then passed over a magnetic column. The initial flow through was collected as CD4⁺25⁻ T cells and the bound fraction was eluted from the column and collected as the CD4⁺25⁺ T cells. Purity (>93%) of each fraction was checked using FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) and antibodies against mouse CD4 (Cy-chrome labeled clone L3T4), CD25 (FITC labeled clone 7D4), as well as compatible isotype control antibodies (BD Biosciences, San Jose, CA).

Cell Culture, Cytokine Assays

In vitro conversion of naive T cells to Treg has been described elsewhere ¹⁸. Briefly, freshly isolated C57BL/6 naive CD4⁺25⁻ T cells were cultured in 6 well plates with anti-CD3 (0.5 μg/ml), and either irradiated APCs or soluble anti-CD28 (2ug/ml) in the presence or absence of 0.02, 0.2, 2, or 20 ng/ml of TGF-Beta1 (R&D Systems, Minneapolis, MN). The cells were kept in these culture conditions for 3 days, after which they were washed and subjected to further analysis. Cytokine assay for TGF-Beta production by our tumor cell lines was done using a commercially available ELISA kit (Biosource International, Carlsbad, CA). Pan02 and Eso2 were both plated at a density of 1×10⁶ cells per well, and kept in culture for 7 days, after which the supernatant was removed and processed according to manufacture’s instructions for TGF-Beta specific ELISA assay. Measurement of TGF-Beta in mice sera was preformed according to the manufacture’s instructions (Biosource International, Carlsbad, CA). In brief, C57BL/6 mice were injected with 0.25×10⁶ Pan02 or Eso2 cells and after 3 weeks serum was collected. Samples were stored at 70°C until ready for processing. The sera were diluted to 1/40 for ELISA.

FACS

Freshly isolated tumor infiltrating lymphocytes were labeled using antibodies against mouse CD4 (Cy-chrome labeled clone L3T4), CD25 (FITC labeled clone 7D4) and were analyzed with a FACS Calibur® Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ). At least 100,000 live events were collected per sample. The analyses were conducted using Flojo (Tree Star, Ashland, OR).

Real Time PCR

RNA was isolated from 5×10⁶ lymphocytes using the RNeasy®Protect Mini Kit (Qiagen, Valencia, CA). Synthesis of cDNA was performed on 100ng of RNA using 8 Oligo (dT)12-18 (SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA) per manufacturer’s instructions. Real-time PCR for Foxp3 normalized to hypoxanthine-guanine phosphoribosyl-transferase (HPRT) was performed on a 9600 thermal cycler and analysis of the data was performed using Sequence Detection System 5700 software (Applied Biosystems, Foster City, CA). TaqMan® Universal PCR Master Mix and TaqMan® Primer with non-fluorescent quencher probes for Foxp3 (Cat # Mm 00475156) and HPRT (Cat # Mm00446968) were purchased from Applied Biosystems.

Regulatory T cell Functional Assays

Cells were cultured in DMEM medium supplemented with 10% FBS (Aleken Biologicals, Nashville, TN) HEPES, non-essential amino acids, sodium pyruvate, 50 μM β-ME, and penicillin/streptomycin/L-glutamine. αThy1.1-PE (clone OX-7) and αCD4-PE-Cy5.5 (clone RM4-5) were purchased from BD Pharmingen (Franklin Lakes, NJ) and Biolegend (San Diego, CA) respectively. For the in vitro suppression assays, 50,000 CFSE-labeled CD4+ CD25−Thy1.1+ T cells were co-cultured in 96-well plates with 200,000 irradiated thy1.2+ splenocytes, 0.1 μg/ml and αCD3, with or without 50,000 CD4+ CD25+ Thy1.2+ cells for 72 hours. Cells were subsequently harvested, stained for Thy1.1 and CD4, and then analyzed by flow cytometry. The gates were set for Thy1.1 and CD4 and a histogram constructed based on CFSE positivity.

Adoptive Transfer Experiments

Six-week old Rag1^−/− mice received i.p. injections of 200 μl HBSS with or without 5×10⁶ freshly isolated CD4⁺25⁻ naive T cells from either normal C567BL/6 mice or dnTGFBetaRII transgenic mice. Two days after reconstitution, the mice received subcutaneous injections in the left outer thigh of 0.5×10⁶ Pan02 or Eso-2 tumor cells. Seven weeks later, the mice were sacrificed and the spleen and tumor draining lymph nodes (TDLN) were harvested and analyzed for expression of CD25 and CD4 by FACS, and for Foxp3 by real-time PCR.

Immunohistochemistry

Tumors were implanted by subcutaneous injection of 1.0×10⁶ PanO2 or ESO2 cells in the hind limb of C57BL/6 mice. At four weeks post implantation the tumors and TDLN were resected and fixed overnight in buffered formalin (Sigma, St. Louis, MO). Prior to staining antigen retrieval was performed by incubating sections in a decloaking chamber (Biocare Medical, Concord, CA) for 10 minutes at 90°C in Reveal™ buffer. Sections were stained with a monoclonal rat anti-mouse-Foxp3 IgG (FJK-16s, 50 μg/ml, eBioscience, San Diego, CA) followed by Alexa Fluor 488 conjugated goat anti-rat antibody IgG (A11029, Invitrogen, Carlsbad, CA). Sections were cover slipped with Vectashield Hard Mounting Media containing DAPI (Vector Labs, Burlingame, CA). Sections were examined using an Olympus BX61 microscope and captured digitally with an Olympus DP70 camera (Olympus, Center Valley, PA). For the CD4+ and Foxp3+ co-localized tissue sections, Pan02 tumors were section and immediately embedded in base molds (Ted Pella, Redding, CA) containing Tissue-Tek OCT medium (Sakura, McGaw Park, IL ) then frozen over liquid nitrogen. Frozen tumor sections were cut at 10μm and mounted on SuperFrost Plus slides (Fisher, Pittsburgh, PA) then stored at −80°C until use. Tumor sections were fixed in 1% paraformaldehyde for 5 minutes at room temperature then washed several times in 1X phosphate-buffered saline (PBS). The slides were blocked for 30 minutes in Serum Free Protein Block (DAKO) at room temperature and then stained with rabbit anti-mouse Foxp3 (Abcam, Cambridge, MA) and rat anti-mouse CD4 (eBioscience, San Diego, CA) primary antibodies. Sections were then stained with AlexaFluor 488 goat anti-rabbit and AlexaFluor 555 goat anti-mouse IgG secondary antibodies (Invitrogen, Carlsbad, CA). Confocal images were visualized on an Axiovert 100M microscope equipped with a LSM 510 confocal system (Axiovert, San Marcos, CA).

TGFBeta Blockade

In vivo blockade of TGF-Beta was achieved using a multi-species monoclonal antibody toward TGF-Beta1,2,3 (Clone 1D11, IgG₁, R&D Biosystems, Minneapolis, MN). On day 0 Rag1 ^−/−mice were reconstituted with 5×10⁶ naive T cells given intraperitoneally. On day 2 the mice were injected subcutaneously with 0.25×10⁶ Pan02 cells into the lateral left thigh. After two weeks the mice received intravenous injections via the tail vein of 10μg of anti-TGF-Beta without azide, low endotoxin IgG₁ isotype control (BD Biosciences, San Jose, CA). Treatments were given either weekly (Q7) or every 4 days (Q4). The treatments lasted for a total of four weeks at which time the mice were sacrificed and their TDLN analyzed for expression of Foxp3 by real time PCR. Tumor growth curves were based on weekly measurements taken with calipers, in two separate axes and were confirmed by two independent observers. The tumor volume was defined as the multiplication of the diameters taken in two separate axes. Tumor burden was defined as the percentage weight of the tumor relative to the total body weight (tumor weight ex vivo ÷ total body weight at sacrifice).

In vitro conversion of naive T cells into Treg

We first studied whether TGF-Beta induced conversion of naive T cells into Treg in vitro. Freshly isolated CD4⁺25⁻ naive T cells were purified from the spleens of normal C57BL/6 mice. The purity of these fractions were found to be >93% by FACS (data not shown). The cells were cultured with soluble anti-CD3 and either anti-CD28 or irradiated antigen presenting cells (APCs) in the presence or absence of TGF-Beta. Cells were then purified from culture after 72 hours and then analyzed for expression of Foxp3 by real time PCR. In prior studies, we validated that induction of Foxp3 expression is a marker of Treg conversion and function through the use of T cell suppression assays ⁸. For this reason functional assays of Treg suppression were not routinely performed. Figure 1 shows that Foxp3 expression, as expected, was significantly higher in freshly isolated regulatory T cells (CD4⁺25⁺) when compared with naive non-regulatory T cells (CD4⁺25⁻ ). Foxp3 expression was induced in cultures of naive T cells treated with anti-CD3/CD28 and TGF-Beta. The upregulation of Foxp3 by TCR crosslinking did not differ whether costimulation was provided by irradiated APCs or soluble anti-CD28. Neither TGF-Beta nor TCR engagement alone was enough to induce Foxp3 expression in CD4⁺25⁻ T cells. The effect of TGF-Beta was concentration dependent, where the highest expression of Foxp3 was observed at a TGF-Beta concentration of 2ng/ml.

Pancreas cancer represents a high TGF-Beta environment

Several clinical studies have demonstrated that pancreas cancer patients have significantly elevated levels of the immunosuppressive cytokines TGF-Beta and IL-10 in their systemic circulation ^3;4;6. We investigated the possibility of whether pancreas cancers induce Treg conversion in vivo through secretion of TGF-Beta. Pan02, a murine pancreas adenocarcinoma cell line, is a highly tumorigenic cell line derived from 3-methylcholanthrene (3-MCA)-induced tumors in C57BL/6 mice. ELISA studies of culture medium taken from Pan02 cells demonstrated significantly elevated levels of TGF-Beta when compared with media alone (Fig. 2a). An unrelated murine cell line of esophageal adenocarcinoma, Eso2, exhibited no difference in TGF-Beta levels when compared with media alone. Neither cell line produced significant levels of IL-10 (data not shown). Because with successive cultures cell lines can change phenotype, we also analyzed Pan02 and Eso2 production of TGF-Beta in vivo. Pan02 and Eso2 tumors were established in the left lateral thigh of C57BL/6 mice and allowed to grow for three weeks. At the time of sacrifice all mice exhibited palpable tumors of relatively similar sizes (data not shown). Serum levels of TGF-Beta in Pan02 mice were significantly elevated above levels measured in non-tumor bearing mice (Fig. 2b). There was no statistically significant difference in TGF-Beta serum levels between Eso2 and non tumor bearing mice.

Pan02 recruits regulatory T cells within the tumor microenvironment

In prior studies, we have shown that regulatory T cells are elevated in the peripheral blood and TDLN of patients and mice with pancreas cancer ^8;21. Based on these observations we hypothesized that Treg were actively being recruited to the tumor microenvironment. To test this we took tumors explanted from Pan02 and Eso2 bearing mice and stained them for Foxp3 using immunofluorescence. Hematoxylin and Eosin (H&E) staining of Pan02 and ES02 demonstrated standard morphology of murine pancreas and esophageal adenocarcinoma (Fig. 3A,B). Staining for Foxp3 in Pan02 tumors demonstrated a high number of Foxp3⁺ cells within the Pan02 tumors (Fig. 3C). These finding are consistent with our real-time PCR and FACS analysis of TDLN taken from Pan02 bearing mice. Tumors established with the non-TGF-Beta secreting Eso2 cell-line showed no tumor infiltrating Foxp3⁺ cells, suggesting that the increased prevalence of Treg in Pan02 tumors is possibly related to tumor dependent expression of TGF-Beta (Fig. 3D). To further phenotype the Foxp3+ tumor infiltrating lymphocytes we did co-localization of Foxp3 and CD4. Figure B demonstrates the Pan02 tumors have strong positivity for CD4+ tumor infiltrating lymphocytes, some of which co-localize with Foxp3. Taken together, this suggests that populations of tumor infiltrating lymphocytes in Pan02 are CD4+Foxp3+ T cells.

The induced populations of Treg in Pan02 tumors were found to have functional Treg suppressor activity similar to that of natural Treg (Figure 4). To demonstrate this, we labeled freshly naïve CD4+CD25−Thy1.1+ effector cells (Teff) with CFSE and cultured them in the presence of CD4+25+Thy1.2+ regulatory T cells (Treg) derived from the spleen and tumors of Pan02 challenged mice. FACS was used to analyze CFSE staining with gating for CD4+Thy1.1 Teff cells. These experiments demonstrate that tumor induced Treg are capable of suppressing effector T cell proliferation, a function that likely impairs the anti-tumor immune response within the tumor microenvironment.

Tumors induce conversion of naive T cells into Treg through TGF-Beta

An increased prevalence of Treg has been demonstrated within the peripheral blood, TDLN, malignant ascites, and effusions of cancer patients ^8;9;11;32;33. It is unclear whether this is mediated by recruitment or expansion of existing Treg, or whether the tumor induces de novo generation of Treg from peripherally circulating naive CD4⁺25⁻ T cells. We established an adoptive transfer model using Rag1-knockout (Rag1^−/−) who because of a defect in the recombinase gene have no mature T cells. Here we reconstituted Rag1^−/− with an intraperitoneal injection of 5×10⁶ syngeneic naive CD4⁺25⁻ T cells followed two days later by a subcutaneous injection of either 0.25 × 10⁶ Pan02 or Eso2 in the left medial thigh. The tumors became palpable after approximately two weeks and reached their maximum allowable size by six weeks. Once the mice were sacrificed the spleen and TDLN were harvested and analyzed for expression of Foxp3. There was low expression of Foxp3 in the freshly isolated CD4⁺25⁻ T cells used to reconstitute Rag1^−/− mice and in TDLN of mice that were not reconstituted (Figure 5). By contrast, CD4⁺CD25⁻ reconstituted mice challenged with Pan02 had marked upregulation of Foxp3 expression within the TDLN compared to those challenged with ES02. Additionally, Foxp3 induction within the Pan02 tumor-bearing mice was restricted to the TDLN as it was not seen in the harvested splenocytes.

To study the importance of TGF-Beta in tumor induced conversion, we utilized the same Rag1^−/− adoptive transfer model and studied the effects of systemic TGF-Beta blockade on Treg prevalence. Four weeks following tumor challenge, reconstituted Rag1^−/− mice all exhibited palpable tumors with an average size 40mm² (Fig. 6A). Starting at Week 4 mice received either no treatment, anti-TGF-Beta antibody (10 μ.g., i.v.), or IgG1 isotype-matched control. The decision to treat mice with established tumors at the fourth week was based on prior studies that indicated the maximal increase in prevalence of Treg numbers occurred at four weeks following tumor challenge ³³. The mice were treated on a schedule of either every four days (Q4) or weekly (Q7) for a total of four weeks. Tumor volume was recorded as the measured product of diameters taken in two separate axes. The growth curves in Figure 6A show that there was no difference in tumor size between treatment groups at the start of treatment. However over time mice treated with anti-TGF-Beta demonstrated a statistically significant delay in tumor growth when compared to untreated and isotype controls. FACS analysis of total cells purified from the TDLN of these mice showed that mice receiving a Q7 schedule of anti-TGF-Beta had more than a fifty percent reduction in the percentage of CD4⁺25⁺ T cells when compared with the no treatment controls (Fig. 6B). (FACS analysis of the no tumor group was not preformed due to insufficient number of cells isolated from normal LN). Analysis of Foxp3 expression by real-time PCR demonstrated that untreated tumor bearing mice had a 2.5 fold induction in Foxp3 expression compared with the non-tumor bearing control (Fig. 6C). Treatment with anti-TGF-Beta given on a Q7 schedule resulted in a significant reduction in Foxp3 expression whereas treatment with isotype or antibody given more frequently than Q7 did not reduce tumor mediated induction of Foxp3. We speculate that the Q4 frequency was less effective at blockade given that the tail veins by often the 4^th treatment and mice were unlikely receiving an appropriate dose of the antibody.

Intact TGFBetaRII signaling is necessary for Pan02 mediated conversion of CD4⁺25⁻ naive T cells into Treg

TGF-Beta plays an important role in the induction and maintence of immune tolerance. Mice expressing a dominant negative TGF-Beta receptor II (dnTGF-BetaRII) transgene have a mutation that prevents downstream signaling via the receptor and renders T cells insensate to TGF-Beta ³¹. To study the effect of TGF-Beta receptor signaling on Pan02 mediated conversion of naive T cells, we isolated naive CD4⁺25⁻ T cells from dnTGFBetaRII transgenic mice, adoptively transferred them into Rag1^−/− mice, and subsequently challenged the mice with Pan02. Mice that were reconstituted with wildtype CD4⁺CD25⁻ naive T cells and then challenged with tumor expectedly showed a marked induction of Foxp3 expression, demonstrating that these naive T cells had undergone conversion (Fig. 7). In contrast, when tumor-challenged Rag1^−/− mice were instead reconstituted with dnTGFBetaRII CD4⁺25⁻ naive T cell there was no observed induction of Foxp3 expression. This suggests that intact TGF-Beta receptor signaling is necessary for tumor induced conversion of naive T cells into Treg. Interestingly, there was no statistically significant difference in the extent of tumor burden in mice reconstituted with wildtype versus those transgenic CD4⁺25⁻ naive T cells (Fig. 8A). By contrast, if the transgenic line of mice themselves were challenged with tumor versus the wildtype controls, the transgenic mice had statistically less tumor burden than their wildtype controls (Fig. 8B). Previously published studies with dnTGFBetaRII transgenic mouse had shown that these mice were resistant to tumor challenge with thymoma and melanoma cell lines ³⁴.