Regulatory t cell isolation kit 2

Human PBMC were purified from healthy blood donors by density gradient centrifugation at 2000 rpm for 20 min with Biocoll Separating Solution (Biochrom, Darmstadt, Germany). Erythrolysis was performed after which the cell suspension was passed through a 0.45 mm filter (BD, Franklin Lakes, NJ, USA). Human Treg (CD4 + CD25 + CD127dim/- cells) were obtained from freshly isolated PBMC by magnetic-activated cell sorting (MACS) using a CD4 + CD25 + CD127dim/- Regulatory T-cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer`s protocol in a two-step isolation. Firstly, non-CD4 + and CD127high cells were removed by negative magnetic selection and, secondly, CD25 + cells were collected using CD25 positive selection magnetic beads. Purity of the isolation was confirmed by flow cytometry and found to be around at 75% SD (Supplement 2). Next, the cells were labelled with CD4-PE, CD127-APC, CD25-FITC, CD19-PacBlue and CD3-APC (all BioLegend, San Diego, CA, USA). Intracellular staining for FoxP3 was performed using a PacBlue anti-human FoxP3 Antibody and FoxP3 Fix/Perm Buffer Set (BioLegend, San Diego, CA, USA) following the manufacturer’s protocol. To check the quality of the isolation, a Zombie NIR Fixable Viability Kit (BioLegend, San Diego, CA, USA) was added once after which a viability over 90% was found in each isolation fraction.

To examine the suppressive activity of CD45RA⁺ naive Tregs and CD45RA⁻ memory Tregs, blood samples (50 ml) from 40 healthy controls and 37 SLE patients were collected in EDTA-containing tubes. Ficoll-Hypaque (inno-train Diagnostik GmbH) density gradient centrifugation was used to separate the PBMCs. The “Regulatory-T-cell Isolation Kit II” (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to purify CD4⁺CD127^low+/–CD25⁺ Tregs according to the manufacturer’s instructions. First, CD4⁺CD127^low+/− T cells were isolated by magnetic depletion of non-CD4⁺CD127^high+ T cells. In the second step, the CD4⁺CD127^low+/–CD25⁺ Tregs were isolated by positive selection over two consecutive columns. The CD4⁺CD127^low+/–CD25⁻ T cells were obtained in the flow-through fraction and used as Tresps. The CD4⁺CD127^low+/–CD25⁺ Tregs were subsequently retrieved from the columns.

CD4⁺CD25⁺CD127^dim/− regulatory T cells will be separated from responder T cells by magnetic-activated cell sorting using a CD4⁺CD25⁺CD127^dim/− Regulatory T Cell Isolation Kit II (Miltenyi Biotec). Responder T cells will be labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen) according to the manufacturer’s instructions. Afterwards regulatory T cells and CFSE⁺ responder T cells will be cocultured at a ratio of 1:2 and 1:1, respectively. Cultures will be stimulated with anti-human CD3 (2 μg/ml; OKT3) and soluble mouse anti-human CD28 (1 μg/ml; eBioscience) for 4 days. After the stimulation period proliferation will be assessed by flow cytometry.