Cd11c microbeads

Single splenic cell suspensions were prepared by grinding the mouse spleens through nylon mesh. After the lysis of red blood cells, splenocytes were incubated with CD11c MicroBeads (130–052-001, Miltenyi Biotec) for 15 min. After washes, CD11c + dendritic cells were isolated by positive selection using MACS separation columns (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions.

Dendritic cells were isolated from splenic cell suspension using CD11c Microbeads (Miltenyi Biotec Inc., San Diego, CA) following the manufacturer’s suggested protocol. Total RNA was isolated directly from cells using Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA) as described in the manufacturer’s protocol. Residual DNA was removed after on-column DNase treatment. RNA integrity was confirmed on 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). 250–1000 ng of RNA was reverse-transcribed using GoScript Reverse Transcription System (Promega Corp., Madison, WI) as described in the manufacturer’s protocol. Aliquots (1 µl) from the reverse transcribed samples were PCR amplified on Stratagene MX3005P using RT² SYBR Green ROX qPCR Mastermix (QIAGEN, Valencia, CA) and primers for beta-actin (QIAGEN), Indoleamine 2,3-dioxygenase (forward primer: 5′-TGGCGTATGTGTGGAACCG-3′ and reverse primer: 5′-CTCGCAGTAGGGAACAGCAA-3′) or CD11c (forward primer: 5′-CTGGATAGCCTTTCTTCTGCTG-3′ and reverse primer: 5′-GCACACTGTGTCCGAAC TCA-3′) genes.

Splenocytes were isolated as described before. CD4⁺CD25^– Tconv and CD4⁺CD25⁺ Treg were isolated as described above using the CD4⁺CD25⁺ Regulatory T cell Isolation Kit (MACS, Miltenyi Biotec). Tconv were stained with CFSE to track cell proliferation and co-cultured with Treg (4:1 ratio, stimulation with 0.5 µg/mL plate-bound anti-CD3, 1 µg/mL soluble anti-CD28). Cells were cultured for 3 days (37 °C, 5% CO₂) prior to FACS analysis. For antigen-specific suppression assays, Treg from the respective mouse line and Tconv from 2D2 mice were isolated and labeled with CFSE as described before. For isolation of APCs from WT mice, the spleen was homogenized by enzymatic digestion. Therefore, 0.1 mg/mL collagenase D was injected into the spleen and incubated for 15 min at 37 °C. Spleen was homogenized by a 70 µm cell strainer. To isolate CD11c⁺ APCs, CD11c MicroBeads (MACS, Miltenyi Biotec) were used in a MACS separation according to the manufacturer’s protocol. APCs were incubated with 20 µg/mL MOG_35–55 for 10 min at 37 °C and washed with PBS before setting up the culture. A co-culture of 5 × 10⁴ APCs and in total 2 × 10⁵ Tconv and Treg (ratios of 1:1, 2:1 and 4:1) was set up for 3 days. Flow cytometry analysis of CFSE-labeled Tconv was used to determine the suppressive capacity of Treg.