Acd28

RPMI 1640 was from Lifetechnologies. X-Vivo15 media was supplied by Lonza. aCD3, aCD4, aCD8 (recognizing the α chain), aCD28, aCD45RO, aCD56, aCCR7, a-IL17, aTCRα24-Jα18 (clone: 6B11), cytokine secretion assays for IFNγ and IL-4, a-fibroblast microbeads and Cytostim were purchased from Miltenyi. Collagenase, Hyaluronidase and DNAse were all from Sigma-Aldrich. aCD45 and aCD38 were from Immunotools. CFDA-SE was purchased from Molecular Probes/Invitrogen. Intra staining Kit, aCD16, aCD8β and aCD3 were from Beckton Dickinson. aCXCR5 was supplied by R&D Systems and aIL21 was from ebioscience. The Beta Mark TCRVβ Repertoire Kit was supplied by Beckman Coulter. The antibodies were used in different conjugates of FITC, PE, PerCp, APC, APC-Vio770 and PE-Cy7.

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from whole blood by density centrifugation via Ficoll-Paque PLUS (GE-Healthcare; GE17-1440-03) according to manufacturer’s protocol. Isolated PBMCs were activated using 50ng/ml of aCD3 (Miltenyi Biotec; 130-093-387)/aCD28 (Miltenyi Biotec; 130-093-375) followed by 50U/ml of IL2 (Miltenyi Biotec; 130-097-743) after 24 hours. Forty eight hours post activation, 1x10⁶ PBMCs were plated on retronectin (Takara Clonetech; T100B) on 6 well plates (Corning; 351146) with retrovirus vector and spun at 1000xg for 40 minutes at RT. Tumor cell lines were transduced with retroviral supernatants on retronectin-coated 6 well plates at a concentration of 0.25x10⁶ cells/ml. Expression of the transgene was assessed by flow cytometry 72 hours post transduction. In order to generate cells expressing PDL1 and/or GD2, cell lines were transduced with retroviral constructs encoding PDL1 and/or the biosynthetic enzymes GD3 synthase and GD2 synthase linked by a viral 2A sequence. Expression of these two synthases results in the synthesis and expression on the cell surface of GD2 (12 (link)). Transduced cells expressing PDL1 and/or GD2 were isolated by FACS.