Macs magnetic beads

T cells from spleen or lymph nodes were enriched by MACS magnetic beads (Miltenyi Biotec) and were sorted by FACS Aria (BD Biosciences) before in vitro stimulation experiments. Purified T cells were cultured in custom ordered Dulbecco’s Modified Eagle Medium (D-MEM, KOHJIN BIO) supplemented with 10% fetal bovine serum (FBS) (Hyclone). For in vitro culture, 2.0 × 10⁵ cells were stimulated in 96-well plates by precoated 2 µg/ml anti-CD3e antibody (553058, BD Biosciences) with 2 µg/ml soluble anti-CD28 antibody (553295, BD Biosciences) for 2 days. Activated T cells were maintained with 40 U/ml rmIL-2 (402-ML, R&D Systems). 2B4 cell line was maintained in RPMI medium (GIBCO) supplemented with 10% FBS and B16-F10 cell line in D-MEM medium (GIBCO) supplemented with 10% FBS. All cell culture medium was supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin.

ChIP assays using anti-CBFβ^{26 (link)} and SATB1 (ab70004, Abcam) antibodies were performed as follows^{58 (link)}. In brief, CD4⁺ or CD8⁺ T cells were enriched by MACS magnetic beads (Miltenyi Biotec) and crosslinked with 1.0% formaldehyde for 10 min at room temperature. The reaction was stopped by adding final 0.15 M glycine and the cells were washed extensively with PBS. Fixed cells were lysed with lysis buffer containing 0.5% NP-40 and 0.25% Triton X-100, and nuclei pellets were resuspended in sonication buffer containing 0.1% Sodium deoxycholate and 0.5% N-laurylsarcosine sodium salt. The nuclei were sonicated using XL2000 ultrasonic cell disruptor (Microson) at output level 6 for 15 s between 8 and 10 times to yield 200–300 bp fragments. Sonicated chromatin was immunoprecipitated by either 1 μg of anti-CBFβ or anti-SATB1 antibody. Captured DNA fragments were washed with RIPA buffer and purified by the ChIP DNA Clean and Concentrator Kit (Zymo Research). The primers used for the ChIP-qPCR is provided in Supplementary Table 1.

Human CD14⁺ peripheral blood mononuclear cell (PBMC) monocyte-derived macrophages were isolated from buffy coats (German Red Cross Blood Donation Service, Ulm, Germany) as described previously [31 (link)]. Isolation was performed by density centrifugation on Biocoll separating solution (1.077 g/mL; Biochrom, Cambridge, UK). PBMCs were resuspended in ammonium–chloride–potassium buffer to lyse residual red blood cells. CD14⁺ PBMCs were isolated using MACS magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). After washing the isolated CD14⁺ PBMCs with PBS, cells were seeded into Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, St. Louis, Missouri, USA) supplemented with 10% fetal bovine serum (Biochrom, Cambridge, UK), 1× nonessential amino acid-mix (Sigma-Aldrich, St. Louis, MO, USA), 1 mM pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 1× l-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and 1× penicillin-streptomycin (Biochrom, Cambridge, UK). After 48 h, granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the medium to a final concentration of 0.01 µg/mL to induce differentiation of PBMCs to macrophages.