The wildtype C57BL/6 mice were obtained from Charles River WIGA (Sulzfeld, Germany) Gmbh. TLR2 knockout on C57BL/6 background mice were kindly obtained from S. Akira (Osaka University, Osaka, Japan). All animal experiments were approved by the Animal Experimental Committee of Radboud University Nijmegen Medical Centre and were performed in accordance with institutional and (inter)national guidelines.
Antibodies and flow cytometry
Directly labeled monoclonal antibodies used for staining by anti-CD4-APC or -FITC (clone L3T4), anti-CD25-FITC or -PE (clone 7D4), anti-CD11c-FITC (HL3), anti-Streptavidin-PerCp, and their isotype controls were obtained from BD Biosciences - Pharmingen. Anti-mTLR2-biotine (clone T2.5), anti-mFoxP3-biotine (clone FJK-16S) and the isotype control were obtained from eBioscience. Analysis of cell surface markers on lymphocytes was performed using FACScalibur (BD) and CELLQuest software (version 3.3; BD Biosciences - Pharmingen).
T cell purification and analysis
Spleens from wildtype or TLR2 knockout mice were mashed and filtered, and CD4+ T cells were purified using anti-mouse-CD4 Microbeads (MACS, Miltenyi Biotec), resulting in a enriched CD3+CD4+ T cell population. Naive CD4+CD25low Teff cells and naive CD4+CD25high Treg cell subsets were obtained by flow cytometry purification of the pre-sorted CD4+ T cells; CD4 cells were stained with FITC-conjugated CD4 mAb (BD bioscience, clone L3T4) and PE-conjugated CD25 (BD biosciences, clone 7D4). Cell sorting was performed on a Coulter Altra HyPerSort cell sorter. Both naive CD4+CD25low T cells and naive CD4+CD25high T cells were 98% pure, based on CD25 expression pattern. Determining the purity by using biotinilated-FoxP3 Ab (eBiosciences, clone FJK-16S) and streptavidine-PerCp Ab (BD bioscience) showed that from the CD25high FACSsorted cells, 67% of the cells were FoxP3+, therefore Tregs. Sorted cells were used directly in several assays or kept in culture as described below. After several weeks of culture the purity of the Treg cell line as well as the Teff cell line (referred in this paper to the cultured Tregs or cultured Teff cells) was 96% or higher (see Additional file 2).
Treg and Teff cell culture and suppression assay
FACS-sorted purified CD4+CD25+ T cells and CD4+CD25-T cells are kept in culture for several weeks. Each cell-line is cultured in 10^4 cells per well of a 96-well plate, and were stimulated weekly with 5*10^4 irradiated CD4-MACS bead depleted splenocytes per well, in 2 μg/ml Pam3Cys (EMC microcollections, Germany), 1 μg/ml anti-CD3 (145-2C11; BD Biosciences - Pharmingen), and 120 IU (international units) IL-2/ml complete medium. The cells were washed 3 days after each stimulation and maintained in culture medium supplemented with 120 IU IL-2/ml. When necessary, dead cells were removed by ficoll density gradient. Cultured Tregs or Teff cells were used in assays at least 6 days after stimulation, in this case the Tregs and Teff cells are in a resting state when used in all assays.
Suppression assays were performed as follows; freshly sorted (wildtype or TLR2 knockout) CD4+CD25-naive T cells (20*10^3 per well) and either cultured or freshly isolated (wildtype or TLR2 knockout) Tregs (20*10^3 per well) were mixed and cocultured for 3 days with 20*10^3 irradiated (wildtype or TLR2 knockout) APCs per well. If indicated, the T cells were stimulated with TLR ligand Pam3Cys (5 μg/ml, EMC microcollections GmbH) or HKLP (10^6-10^9 CFU/ml of heat-killed Legionella pneumophila; InvivoGen) with or without soluble anti-CD3 (1 μg/ml, 145-2C11; BD Biosciences - Pharmingen) in complete medium. After 3 days of coculture, supernatant was collected for cytokine analysis. In addition, the suppression of proliferation was monitored by analyzing the CFSE-labeled (1 μM) freshly sorted (wildtype or TLR2 knockout) CD4+CD25-naive T cells. CFSE fluorescence intensity was measured by flow cytometry.
Calculation of suppression
The amount of suppression is calculated by the amount of proliferation or cytokine production. In each assay we include 5 conditions (see Additional file 3) by which we calculate the suppression, using the mean fluorescent intensity of the CFSE-labeled Teff cells. The first condition (background) is a coculture of CFSE-labeled Teff cells, APCs and anti-CD3 stimulation (1 μg/ml). This condition indicates the level of Teff cell proliferation induced by anti-CD3 in the presence of APC. The second condition (0% suppression) is a coculture of CFSE-labeled Teff cells, APCs, anti-CD3 stimulation (1 μg/ml) and Pam3Cys (concentration used between 10 to 0.01 μg/ml). This condition represents maximum of Teff cell proliferation. The next condition consists of a coculture of CFSE-labeled Teff cells, APCs, Tregs and anti-CD3 stimulation (1 μg/ml), which is used to achieve maximal suppression (set to 100% as a reference). The last condition consists of a coculture of CFSE-labeled Teff cells, APCs, Tregs, anti-CD3 stimulation (1 μg/ml) and Pam3Cys (concentration used between 10 to 0.01 μg/ml), indicated as 'x% suppression'. This x% suppression is calculated by the following formula; ((MFI x% - MFI 0%)*100)/(MFI max - MFI 0%). For all data presented the difference between the 0% suppression and max suppression was at least 150 in MFI. In the case of cytokine production, we used the same formula with amount of cytokines produced as principle parameter.
For the detection of cytokines in culture supernatant we used the Mouse Th1/Th2 Bio-Plex Cytokine Assay (#171-F11081, Bio-Rad, Hercules, CA). All procedures were performed according to the manufacturer's instructions.
Freshly FACS-sorted or cultured CD4+CD25+Treg and CD4+Tconv cells were cultured for 4 days with a range of Pam3Cys concentrations (between 0 μg/ml-5 μg/ml) or HKLP (10^6-10^9CFU/ml of heat-killed Legionella pneumophila; InvivoGen) with or without soluble CD3 stimulation (1 μg/ml). After 4 days the proliferation was measured by overnight (20 hours) thymidine incorporation.
The data are analysed using a one-way ANOVA test, to test the differences between groups (PRISM software version 4.0; GraphPad, San Diego, CA, USA). Statistical significance is inferred at P < 0.05. Significant differences are indicated with an asterisks (*).