35 m strainer

Buffy coat samples were washed with PBS 2% FCS and stained for 60 min at 4 °C with Rituximab CD20-FITC, CD3-APC (eBioscience), CD8a -APC-eFluor780 (eBioscience), CD45RO- PE-Cy7 (eBioscience), CCR7-BV421 (BD), and markers CD4-PE (eBioscience), CD56-PE (eBioscience) and CD19-PE (BD). Cells were washed and filtered using a 35 µm strainer (Falcon). Propidium Iodide (1 µg/ml) was used for exclusion of dead cells. From the CD3 + CD8 + CD45RO + CCR7- population, 1000 live CD20 + and 1000 live CD20-cells were sorted using a Beckman Coulter MoFlo Astrios cytometer. UltraComp eBeads (Thermo Fisher Scientific) were used as compensation controls.

Endometrial cancer tumor digests from three endometrial cancer patients were thawed in RPMI + 10% fetal calf serum. Each digest was split four ways; one part remained untreated, one part was incubated with phorbol myristate acetate (PMA)/Ionomycin (1× Cell Stimulation Cocktail, consist of 81 nM PMA and 1.34 µM Ionomycin, eBioscience, 00-4970-93, Waltham, MA, USA) for four hours, one part was treated with actinomycin D for 4.5 h (5 µg/mL, A1410-2mg, Sigma-Aldrich), and one part was pre-treated with 30 min of actinomycin D followed by four hours of PMA/Ionomycin. All conditions were incubated at 37 °C. Next, samples were washed with PBS 2% FCS and stained with CD103-FITC, CD39-APC, CD3-PE, and CD8a-BV421 for 30 min at 4 °C (Supplementary Table S6). Cells were washed and filtered using a 35 µm strainer (Falcon). Propidium iodide (1 µg/mL) was used to exclude dead cells. Per treatment condition, 100 live CD3+CD8+ or 100 live CD3+CD8+CD39+CD103+ cells were sorted from all digests. Cells were sorted on a Beckman Coulter MoFlo Astrios sorter.