2.4.1 General culture conditions
All cell lines were cultured at 37°C and 5% CO2 in tissue culture flasks. Cell culture procedures were executed with sterile reagents under a laminar flow hood. The concentration and viability of cells was determined by trypan blue staining. Trypan blue is a reagent that is not absorbed by viable cell. In dead cells, by contrast, the dye could pass the membrane and thereby stains cells blue. Thus, dead cells are visible in a distinctive blue color under the micro- scope and can be distinguished from viable cells. In order to stain death cells and count viable cells, 0,25% trypan blue in PBS was added in an appropriate dilution to cell suspensions. Cells were counted in a Neubauer hemocytometer under the microscope. The total number of cells per ml was calculated by multi- plying the total cell number in one hemocytometer grid by the dilution factor and 104.
2.4.2 Murine tumor cell line
4T1 breast cancer (kindly provided by Dr. M. Wartenberg, Friedrich-Schiller- University, Jena, Germany) Colon-26 (CT26) (Cell Lines Service, Heidelberg, Germany), Colon-26 with inducible CCL22 expression (rtTA-CCL22-CT26), B16 melanoma F1 (LGC Promochem, Teddington, UK), mGC8 gastric cancer (kindly provided by Dr. med. J. Nöckel, LIFE-Center, University Clinic, Grosshadern, Germany) MethA sarcoma (kindly provided by Prof. W. Zimmermann,
LIFE-Center, University Clinic, Grosshadern, Germany) as well as Panc02 (kindly provided by Prof. C. Bruns, Department of Surgery, University of Munich, Germany) were maintained in complete DMEM or RPMI medium as described above. Tumor cells were split in a 1:10 dilution two times a week. Cells were detached with Trypsin-EDTA (0.25%), centrifuged (400 g, 7 minutes, 4°C), resuspended in fresh medium and transferred to culture flasks. All tumor cell lines were expanded for two to three passages upon receipt, stored in liquid nitrogen and freshly thawed prior each experiment.
2.4.3 Isolation of DC and T cells by magnetic cell separation
MACS (magnetic cell separation or magnetic-activated cell sorting) Technology from Miltenyi was used for the separation of magnetic labeled viable cells from lymphoid and non-lymphoid tissues.
For MACS separation cells are labeled with MACS MicroBeads which are superparamagnetic particles of approximately 50 nanometers in diameter and coated with antibodies against particular surface antigens. After cell labeling with these MicroBeads cells are applied on MACS columns. By a strong permanent magnet a high-gradient magnetic field is induced on the column matrix. Therefore, all labeled cells are retained in the column and all unlabeled cells pass through the column and can be collected. After removal of the column from the magnet, labeled cells can be released from the column and can also be collected. With MACS separation cells can be sorted by negative selection (= unbound fraction) and positive selection (= bound fraction).
Murine DC were separated from total spleen cells, lymphocytes or ex vivo tumor cell suspension with CD11c beads (= positive selection). Regulatory T cells were sorted from total spleen cells via a two-step procedure. First, non-CD4+ T cells were depleted by indirectly labeling with a cocktail of biotin-conjugated antibodies against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCR γ/δ and CD235a (glycophrin A) and Anti-Biotin MicroBeads (= negative selection). In a second step, the CD4+ flow-through fraction was labeled with CD25 MicroBeads for subsequent positive selection of CD4+CD25+ regulatory T cells.
In this study reagents from Miltenyi Biotec were used according to the manufacturer’s instructions, and all cell separations were done according to manufacturer’s protocol. In brief: Cells were labeled with the appropriate microbeads in MACS buffer for 15 minutes at 4°C and washed with MACS buffer. MS columns were placed in a magnet and equilibrated with 0.5 ml MACS buffer prior to loading the labeled cells in a volume of 0.5 ml. After washing the column with three times 0.5 ml MACS buffer, the column was removed from the magnet, and the bound fraction was eluted by flushing the cells in 1 ml MACS buffer through the column with the supplied plunger. If high purity of sorted cells was needed a second column purification step was performed. The purity of CD11c-sorted cells was 90% on average after one column purification. After a second column purification this purity was increased to more than 96%.
In the case of the two-step Treg separation procedure cells were incubated with a biotin antibody cocktail (including CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCR γ/δ and CD235a). After 10 minutes at 4°C anti-biotin microbeads and CD25-PE antibody were added and incubated for another 15 minutes at 4°C. Afterwards, cells were washed with MACS buffer. A LD column was placed in a magnet and equilibrated with 2 ml MACS buffer prior to loading the labeled cells in a volume of 0.5 ml. The column was washed with three times 1 ml MACS buffer, and the flow through was collected. In the second separation step these CD4+ cells were incubated with anti-PE microbeads for 15 min at 4°C, washed with MACS buffer and applied in a volume of 0.5 ml onto a MS column (equilibrated with 0.5 ml MACS buffer). After washing the column, the column was removed from the magnet, and the bound fraction was eluted by flushing the cells in 1 ml MACS buffer through the column with the supplied plunger.
2.4.4 Toll-like receptor ligands treatment in vitro
For in vitro cultures murine tumor-infiltrating cells of CT26, B16 and Panc02 tumors were treated with 5 μg/ml CpG 1826 (Coley Pharmaceutical Group), 1000 U/ml murine interferon-alpha (IFN-α) (R&D Systems), 50 ng/ml murine interferon-gamma (IFN-γ) (Peprotech), 10 ng/ml murine IL-1β, IL-2, IL-6, IL-10 or IL-12 (all Peprotech), respectively.
2.4.5 Cell transfection
For cell transfection in vitro we cultured 1 to 4 x 104 cells per well in 96 well plates. For one well following transfection mixture was prepared: 2.0 to 5.0 μg plasmid was added to 10 μl Opti-MEM. The transfection reagent Lipofectamine 2000 was mixed with 10 μl Opti-MEM in a ratio of 5:1 to the amount of used plasmid DNA. Both mixtures were incubated for five minutes at room temperature, then combined and incubated for another 20 minutes. After 20 minutes the DNA and transfection reagent complex was added directly into the supernatant of cultured cells. Transfection efficiency was verified 24 hours post transfection.
2.4.6 Conditioned medium
Conditioned medium (CM) was produced by stimulating 5 x 105 splenocytes of C57BL/6 mice with 5 μg/ml CpG 1826 (Coley Pharmaceutical Group) for 2 hours, followed by extensive washing and culture in fresh medium for an additional 20 hours before removal and transfer of the supernatant into stim- ulation assays of murine tumor-infiltrating cells.