Khyg 1

All cell culture reagents were purchased from Biowest and Serana. Tumor cell lines ZR-75-1, MCF-7, HSC-4, CaoV-3 and T-47D as well as the human NK cell line KHYG-1 and murine T cell line CTLL-2 were obtained from DSMZ or ATCC and cultured in the respective recommended medium under standard conditions. KHYG-1 and CTLL-2 were cultured in presence of 10 ng/mL IL-2 (PeproTech, Rocky Hill, NJ, USA).
For in vivo studies, the murine tumor cell lines B16.F10 and CT26.wt (ATCC) were stably transfected by electroporation (Amaxa) with a plasmid coding for human MUC1 (40 tandem repeats) and confirmed for TA-MUC1 expression in vitro and in vivo in mice by flow cytometry and immunohistochemistry using GT-00A. Cells were cultured in standard medium +100 nM methotrexate (Hexal, Holzkirchen, Germany) as selection pressure. For the metastasis model, hMUC1-B16.F10 cells were further transduced to stably express firefly luciferase (110 RLU/cell) enabling detection of metastasis. Origin, TA-MUC1 and IL-15R expression status of cell lines are indicated in Table S1.
Peripheral blood mononuclear cells (PBMCs) were prepared from commercially available buffy coats (DRK Berlin) or leukapheresates (Charité Berlin) of healthy donors by Biocoll separation (Biochrom, Cambridge, UK) and density gradient centrifugation. PBMCs from leukapheresate products were stored frozen in liquid nitrogen.

The cell lines 5637 (DSMZ, #ACC-35), DU-145 (DSMZ, #ACC-261), KHYG-1 (DSMZ, #ACC-725), Ramos (DSMZ, #ACC-603), and ZR-75-1 (ATCC, #CRL-1500) were obtained from the DSMZ or the ATCC. All cell cultures were free of Mycoplasma and maintained in RPMI1640 medium (Biochrom, #F1215) supplemented with 10% FCS (Biochrom, #S0115) and 1% l-glutamine (Biochrom, #K0283). KHYG-1 medium additionally contained 10 ng/ml IL-2 (PeproTech, #200-02). KHYG-1 cells were stably transfected with hFcγRIIIa 158V (KHYG-1-CD16aV), cloned, and maintained in medium containing 25 nmol/l methotrexate (Sigma-Aldrich, #M8407).
Human PBMCs were isolated from leukapheresis products (Charité University Hospital Berlin) or commercially available buffy coats (DRK Berlin) of healthy donors via density gradient centrifugation using Biocoll Separating Solution (Biochrom, #L6113). In case of leukapheresis products, isolated PBMCs were stored in AIM-V medium (Life Technologies, #31035025) supplemented with 75% FCS (Biochrom, #S0115) and 10% DMSO (Sigma-Aldrich, #D2650) in liquid nitrogen for future usage. All leukapheresis donors were informed of the purpose of the donation and gave their written consent prior to the study.
PBMC subpopulations were isolated using magnetic cell separation: monocytes (Invitrogen, #11350D or Miltenyi Biotec, #130-096-537), B cells (Invitrogen, #11351D), and T cells (Invitrogen, #11344D).