Human il 2

Peptide-specific T cells were expanded using an aAPC as described previously23 (link)24 (link). PBMCs were isolated from healthy volunteers and stimulated with 50 ng/ml anti-CD3 mAb (clone OKT3) in the presence of 100 IU/ml human IL-2 (Novartis) 3 days before transduction. Activated T cells were retrovirally transduced with TCR genes by centrifuging 1 hour at 1,000 g at 32 °C. Following transduction, CD4⁺ or CD8⁺ T cells were purified using anti-CD4 or anti-CD8 Microbeads (Miltenyi Biotec) and plated at 2 × 10⁶ cells/well in RPMI 1640 supplemented with 10% human AB serum. The stimulator aAPC was pulsed with 10 μg/ml A2-restricted wild-type MART1_27–35 for 6 hours at room temperature. The aAPC was then irradiated at 200 Gy, washed, and added to the responder T cells at a responder to stimulator ratio of 20:1. Starting the next day, 10 IU/ml IL-2 (Novartis) and 10 ng/ml IL-15 (Peprotech) were added to the cultures every 3 days. T cells were harvested, counted, and restimulated every week. T cell analysis was performed one day prior to or on the day of restimulation.

Heparinized whole blood was collected from healthy donors under informed consent by venipuncture and transported at room temperature from Ottawa Hospital Research Institute. Blood was diluted 1:1 with Hank’s balanced salt solution (HBSS) and PBMCs were isolated by Ficoll-Paque™ density gradient centrifugation. Briefly, samples layered on Ficoll-Paque™ gradient were centrifuged for 20 min at 700 × g without applying a brake. The PBMC interface was carefully removed by pipetting and was washed twice with HBSS by stepwise centrifugation for 15 min at 300 × g. PBMCs were resuspended and counted by mixed 1:1 with Cellometer ViaStain™ acridine orange/propidium iodide (AOPI) staining solution and counted using a Nexcelom Cellometer Auto 2000 (Nexcelom BioScience, Lawrence, MA, USA). T cells from were then activated with Miltenyi MACS GMP T cell TransAct™ CD3/CD28 beads and seeded 1x10⁶ T cells/mL in serum-free StemCell Immunocult™-XF media (StemCell Technologies, Vancouver, Canada) with clinical grade 20 U/mL human IL-2 (Novartis, Basel, Switzerland).

The EGFRvIII third generation MSGV1 retroviral CAR construct contains the CD28, 4-1BB, and CD3z moieties, in tandem with the scFv derived from the human monoclonal antibody 139, and the marker Thy1.1^{21 (link)}. Alternatively, the EGFRvIII specific 139 scFv was replaced with the FMC63 CD19 specific scFv. CAR T cells were prepared according to previously reported protocols⁵³ . Briefly, splenocytes were isolated from donor C57BL/6 CD45.2 or CD45.1 or IFNAR1 KO mice, made into a single cell suspension, and cultured in RPMI (HyClone) supplemented with 10% FBS, 50 μM 2-Mercaptoethanol (Sigma), 1% PenStrep (Corning), 1% NEAA (Corning), 1% Sodium Pyruvate (Corning), 50 U/mL human IL2 (Novartis) and 2.5 μg/mL Concanavalin A (Sigma). Retroviral supernatant was produced from 293T cells co-transfected with the MSGV1 retroviral plasmid and the helper plasmid pCL Eco (Imgenex) and T cells were transduced on RetroNectin-coated plates (Takara) 2 days after stimulation. Cells were split one day after transduction and used for in vitro analysis or in vivo administration on day 4 or 5. Transduced cells were identified by the expression of Thy1.1 and a representative gating scheme identifying murine CAR T cells is shown in Supplementary Fig. 10.

The cell lines used in the study were previously described [69 (link),70 (link),71 (link),72 (link)]. Myla 2059 and Myla 2000 were cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA #R2405) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Cromwell, CT, USA #04-007-1A) and 1% Penicillin/Streptomycin (Sigma, #P7539). HH cells were cultured in RPMI-1640 medium supplemented with 20% FBS and 1% Penicillin/Streptomycin. SeAx, was cultured in RPMI-1640 medium supplemented with 10% human serum (HS) (Copenhagen University Hospital Blood Bank), 1% Penicillin/Streptomycin and 1000 U/mL human IL-2 (Novartis, Basel, Switzerland #004184). Cells were maintained in an incubator at 37 °C with 5% CO₂ and were replenished with fresh medium every two days. Isolation of PBMC from SS patients was done by Ficoll-based density-gradient centrifugation. CD4⁺ T cells were isolated from PBMC using EasySep™ Human CD4⁺ T Cell Isolation Kit (StemCell, Vancouver, BC, Canada #17952) by following the manufacturer’s protocol. Necessary approvals were obtained from the Committee on Health Research ethics (H-16025331) prior to using SS patients’ samples and the work was performed in accordance with the Declaration of Helsinki.

The antibodies for flow cytometry included anti-human ErbB2-allophycocyanin (APC) (Biolegend; 324408), anti-human CD3 (Biolegend; APC-300439 or pacific blue (PB-344824), anti-human CD8-PB (Biolegend; 301023), anti-human CD4-brilliant green (BG) (Biogems; 06111-40-100), anti-human CD45RA-APC (Biolegend; 304112), anti-human CCR7-Percp 5.5 (Biolegend; 353220), and anti-human cleaved Caspas3. For the preparation of activation plates, we used anti-human CD3 (Biolegend; 317326) and anti-human CD28 (Biolegend; 302934) (Invitrogen; PA5-114687). RetroNectin (Takara; T100B) was used for the preparation of pre-coated transduction plates. Transduced lymphocyte medium was supplemented with human IL-2 (Novartis-Pharma; 4764111 U57).

The transduction using the retroviral vector pMP71 and culture of primary murine T cells has been previously described [47 (link)]. In brief, 1.2 × 10⁶ virus producing 293Vec-Eco cells were seeded into a 6-well plate 24 h prior to splenocyte isolation. After 48 and 72 h, the virus-containing supernatant was used to transduce murine T cells. Murine T cells were expanded from murine splenocytes by activation with anti-CD3 and anti-CD28 antibodies (clones 145-2C11 and 37.51, Thermo Fisher Scientific, Waltham, MA, USA) and human IL-2 (10 IU/ml, Novartis, Basel, Switzerland) for 24 h. Subsequently murine T cells were stimulated Dynabeads™ Mouse T-Activator CD3/CD28 (Thermo Fisher Scientific) and human IL-15 (50 ng/ml, Peprotech). Prior to some experiments Dynabeads™ were removed.