Recombinant interleukin 2

100-mm dishes were coated with anti-CD3 antibody (Ab) (cat. no. 553057, clone 145-2C11, BD Pharmingen, San Diego, CA, USA) for 24 h and washed before use. A single cell suspension was prepared from the draining lymph node (DLN) of GFP transgenic mice (Figure S1). These DLN cells (DLNC), which stably express GFP, were cultured in RPMI-1640 (PAN Biotech) supplemented with 10% FBS (PAN Biotech) in the presence of 1000 U/mL recombinant interleukin-2 (rIL-2) (R&D system, Minneapolis, MN), 2 μg/μL rCD28 (BD Pharmingen), and rTGF-β (R&D system) to promote differentiation into Tregs. At 7 days of culture ex vivo in supplemented medium, CD4⁺ T cells were isolated using MagCellect Mouse CD4⁺ T cell isolation kit (R&D system) according to the manufacturer’s protocol. After magnetic cell separation, these cells were further sorted by cell sorting using a FACSAria^TM III sorter (BD Biosciences, San Jose, CA, USA).

HaCaT cells, a spontaneously immortalized human keratinocyte cell line, were cultured in 5% CO₂ at 37 °C in a humidified culture incubator in a high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics (10,000 units/mL penicillin and 10 mg/mL streptomycin mixture, Welgene, Daejeon, Korea). THP-1 cells, a human promonocytic cell line, were purchased from Korean Cell Line Bank (KCLB 40202) and incubated in RPMI-1640 with 10% heat-inactivated FBS and 1% penicillin and streptomycin mixture. For differentiation, phorbol-12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) was added at a final concentration of 10 ng/mL in RPMI 1640 + 1% FBS for 24 h to prime the THP-1 monocytes into macrophage-like cells. The cells were then washed to remove the PMA, given a 24 h rest, and then allowed to differentiate for 2–3 days. NK 3.3 cells, a human natural killer cell line, were kindly provided by Dr. Ranjit and incubated in RPMI-1640 with 15% heat inactivated FBS, 1% penicillin and streptomycin mixture, and 200 IU/mL of recombinant interleukin-2 (R&D Systems, Minneapolis, MN, USA) at 37 °C in a 5% CO₂ incubator. The cells were kept at 70–80% confluence and subcultured every 2–3 days.

Resting CD4+ T cells were purified from two healthy blood donors by Ficoll gradient separation followed by negative selection and magnetic separation using the human CD4+ T Cell enrichment kit supplemented with anti-HLA-DR, anti-CD25 and anti-CD69 (Stem Cell Technologies). Cells (10⁶) were incubated for 24 hours, either with medium only, or with 100 or 1000 IU/ml interferon α (Roferon-A, Roche) or interferon γ (R&D Systems), or with 2 μg anti-CD3/1 μg anti-CD28 and 100 U/ml recombinant interleukin-2 (R&D Systems) to mimic TCR stimulation. After 24 hour incubation, total RNA extraction was performed using Illustra RNAspin mini isolation kit (GE Healthcare) and further processed for mRNA-Seq library preparation (TruSeq RNA sample prep kit, Illumina). 100 bp paired-end sequencing was performed using Illumina HiSeq2000. About 140 mio read pairs were obtained for the samples. Sequencing reads were quality filtered before alignment using Cutadapt [56 ] and PrInseq [57 (link)]. Cleaned sequencing reads were aligned to a build genome using SOAPsplice. Gene expression was assessed by determining the number of mapped reads per gene using HTSeq-count tool, followed by DESeq R package normalization. An additional normalization step taking into account the gene coding length was performed so that the resulting counts could be compared across samples for a given gene, and also between genes.

To assess the proliferation of T cells, freshly purified CD4⁺CD25⁻ T cells or CD8⁺ T cells were stained with 5 mmol/L carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) according to the manufacturer’s instructions. The stained cells were then cultured in a 24-well plate alone or with Tregs at a ratio of 4:1 at 37 °C with 5% CO₂ in X-VIVO™15 medium (Lonza) supplemented with 5% FBS and recombinant interleukin-2 (100 units/ml; R&D systems) and stimulated with anti-CD3/CD28 beads (Miltenyi Biotec) at a ratio of 1:1. After 5 days, proliferating CD4⁺CD25⁻ T cells or CD8⁺ T cells were identified as CFSE-diluted subsets. The control group consisted of CD4⁺CD25⁻ T cells that were not cocultured with Tregs but were stained with CFSE.

To assess the apoptosis of T cells, freshly purified CD4⁺CD25⁻ T cells or CD8⁺ T cells were cultured in a 24-well plate alone or with Tregs at a ratio of 4:1 at 37 °C with 5% CO₂ in X-VIVO™15 medium (Lonza) supplemented with 5% FBS and recombinant Interleukin-2 (100 units/ml; R&D Systems) and were stimulated with anti-CD3/CD28 beads (Miltenyi Biotec). After 3 days, apoptotic CD4⁺CD25⁻ T cells or CD8⁺ T cells were assayed by an Annexin v/PI apoptosis kit (BD Biosciences) according to the manufacturer’s instructions.