Cd25 pe cy7 m a251

Following 4-day co-cultures of CAR T cells and SKBR3 cells at a ratio of 4:1, cells were extracted by centrifugation and prepared for flow cytometry analysis of immunological markers. First, cells were stained for viability (Zombie Aqua, Biolegend) in PBS, washed and stained in FACS buffer (2% FBS, 0.1% NaN3 in PBS) for the following markers: HLA-DR-Alexa Fluor 647 (L243), CD69-Pacific Blue (FN50), CD25-PE/Cy7 (M-A251), CD137/4-1BB-PE/Dazzle 594 (4B4-1), CD45RA- PE/Dazzle 594 (HI100), CCR7-APC/Cy7 (3D12), CD27-BV570 (O323), CD39-FITC (A1), CD127-PE (A019D5), CTLA-4-BV785 (L3D10), LAG-3-BV711 (11C3C65), TIGIT-BV421 (A15153G) from Biolegend; and CD3-BUV395 (UCHT1), CD4-BUV496 (SK3), CD8-BUV805 (SK1), CD62L-BV650 (SK11), PD-1-BB700 (EH12.1), TIM3-BV480 (7D3) from BD Biosciences (Supplementary Table 3). After washing, cells were analyzed using the Cytek Aurora full-spectrum flow cytometry technology. Data were further processed with FlowJo 10 software (BD Biosciences; Supplementary Fig. 12).

The African green monkey kidney cell line Vero 10–87 was obtained from WHO (world health organization, Geneva, Switzerland). The Jurkat cell line (JE6.1) and the mouse thymoma cell line BW5147 were derived from in house stocks and cultured in RPMI1640 supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) (from Sigma-Aldrich, St. Louis, MO). For transfection, HEK293T cells (kindly provided by A. Carmo, Porto, Portugal) were used. Mycoplasma contaminations were detected by using a recently described reporter system based on a human monocytic THP-1 cell line [31 (link)]. The following antibodies were purchased from Biolegend (San Diego, CA): 4-1BB (CD137)-PE (4B4-1), m4-1BB (CD137)-PE (17B5), 4-1BBL (CD137L)-PE (5F4), m4-1BBL (CD137L)-PE (TKS-1), CD14-APC (M5E2), CD4-BV421 (OKT4), CD8-PerCP (HIT8α), CD56-PE (5.1H11), CD56-BV510 (5.1H11), CD25-PeCy7 (M-A251). A DyLight-649-conjugated goat-anti-mouse IgG(H + L) antibody from Jackson ImmunoResearch (West Grove,PA) was used for anti-CD3 single chain fragment detection. The Strep-tag was detected by using the monoclonal mouse NWSHPQFEK-Tag (biotin) antibody (GeneScript, Leiden, The Netherlands) in conjunction with a Streptavidin-PE (Biolegend, San Diego, CA). The agonistic anti-human 4-1BB antibody urelumab (human, IgG4) was obtained from Creative Biolabs (Shirley, NY).

Tregs were isolated from peripheral blood mononuclear cells (PBMCs) by negative selection of CD4 (Miltenyi Biotec) followed by positive CD25 selection (Miltenyi Biotec) using magnetic beads according to manufacturer instructions. Next, Tregs were sorted using a FACSAriaII or FACSAria Fusion (BD Biosciences) using the following antibodies: CD4 APC-eFluor780 (OKT4) and CD127 PE (eBioRDR5; both from Thermo Fisher Scientific) as well as CD25 PE/Cy7 (M-A251), CD25 Kiravia Blue 520 (M-A251), CD127 BV421 (A019D5), CD127 PE/Cy5 (A019D5), CD4 AF700 (RPA-T4), and CD4 BV421 (RPA-T4; all from BioLegend). The sorting strategy is depicted in Supplemental Figure 13, and purity of all sorts was confirmed to be > 95%. Tregs (CD4⁺CD25^hiCD127^lo) and Teff (CD4⁺CD25^–) were used in the Boyden chamber migration assay and the suppression assay.