Cell isolation and cell culture

For experiments intended for gene editing, pre-enrichment of CD4+ cells from PBMCs was performed using CD4 microbeads (Miltenyi Biotech) according to the manufacturer´s protocol. Due to their higher stability61, CCR7+ Tregs (CD4+CD25+CD127lowCCR7+) were sorted using a Tyto sorter (Miltenyi Biotech) after staining with aCD4 VioBlue (Miltenyi, REAL103), aCD25 APC (Miltenyi, REAL128), aCD127 PE-vio770 (Miltenyi, REAL102), aCD45RA FITC (Miltenyi, REAL164) and CCR7 PE (BioLegend, G043H7). Sorted Tregs were cultured in a 96 U well plate with 100,000 cells in 200 µl Treg medium per well. Treg medium consists of X-Vivo 15 (Lonza) medium supplemented with 10% heatinactivated FCS, 500IU/mL of recombinant human interleukin-2 (IL-2) (Miltenyi, Bergisch Gladbach, Germany) and 100 nMRapamycin (Pfizer).

Gene editing to modulate HLA surface expression on primary human Treg
Gene editing in Treg was performed as previously described40,41. After 7 days of culture MACS® GMP ExpAct™ Treg Kit beads were depleted using a MACSiMAG™ Separator (Miltenyi). Cells were removed from the magnet, counted, and then washed twice in sterile PBS by centrifugation at 100 × g for 10min at room temperature (RT). In parallel, Cas9 RNP was prepared. For the electroporation of 106 primary Tregs, 0.5 µL of poly(L-Glutamic acid) (PGA) (molecular weight 15,000- 50,000, Sigma-Aldrich, 100 µg/µL), 0.48 µL of synthetic modified sgRNA (Suppl. Data 3; 100 µM in TE buffer; IDT), and 0.4 µL recombinant SpCas9 protein (Alt-R S.p. Cas9 Nuclease V3; IDT; 61 µM) were mixed by thorough pipetting. Themixture was incubated for 15min at RT and placed on ice. For HLA-E KI conditions, 0.5 µL of HDRT (stock concentration: 1 µg/µL) was added prior to electroporation. 10^6 harvested Tregs were resuspended in 20 µL ice-cold P3 electroporation buffer (Lonza) just before electroporation to keep the exposure time to the electroporation buffers to a minimum. 20 µL of resuspended cells were transferred to the RNP/HDRT suspension, mixed thoroughly, and transferred into a 16-well electroporation strip (Lonza) without any air bubbles. The cellswere electroporated using the EH-115 program on the 4D-Nucleofector (Lonza). Immediately after electroporation, 90 µL of pre-warmed Tregmediumwas added per well. After 10 min, the cells were carefully resuspended and transferred to two 96- well round-bottom plates (50 µL/well) containing 150 µL pre-warmed Treg medium per well. The gene editing of HLA-E KI into B2M plus KO of CIITA Tregs (H/C Tregs) was performed in two subsequent editing steps. Seven days afterHLA-E KI into B2Ma second electroporationwas performed using adenine base editor ABE8.20-m30 mRNA produced in
house by in vitro transcription as described previously55. Tregs were harvested, beads were magnetically removed, and cells were washed twice in PBS. 5 × 10^6 cells were resuspended in 100 µl P3 electroporation buffer (Lonza) and mixed with 2 µg of ABE8.20-m mRNA and 0.48 µL of synthetic modified sgRNA (100 µM in TE buffer; IDT). The suspension was electroporated in 100 µl Nucleocuvette Vessels (Lonza) using a Lonza 4D nucleofector device (program EH-115). 900 µL of pre-warmed Treg medium was added per cuvette. After 10 min, the cells were carefully resuspended and transferred to a 24 well cell culture plate.