Hil 2

To generate T-bodies, PBMC from healthy donors were activated 48 hours with OKT-3 (50 ng/ml; Ortho Biotech Inc, Raritan, NJ, USA) and human IL-2 (hIL-2, 300 U/ml; Proleukin; Novartis Pharmaceuticals, Horsham, UK). T cells were then infected with the viral supernatant for 18 hours at 37°C and 5% CO₂, in the presence of protamine sulfate (40 µg/ml; Sigma-Aldrich) and hIL-2 (500 U/ml). The supernatant was then changed with fresh complete medium containing hIL-2 (100 U/ml). Seventy-two hours later, PBMC were analyzed for CAR and eGFP expression, and for luciferase activity. PBMC were referred to as T-body-hPSMA/eGFP and T-body-hPSMA/fluc when transduced with hPSMA/eGFP LV CAR or hPSMA/Luciferase LV CAR, respectively. T-bodies were re-stimulated once a week with irradiated (60 Gy) PC3-PIP at a 10∶1 ratio. Complete medium with fresh IL-2 was replenished twice a week.

GFP⁺ Treg cells were sorted from pooled LNs and spleens isolated from Foxp3^GFP mice and cultured in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (GE Healthcare Life Sciences), 2 mM l-glutamine, 1% penicillin-streptomycin (both from Gibco), and 50 μM β-mercaptoethanol (Sigma-Aldrich) at 37 °C with 5% CO₂. For in vitro expansion, Treg cells were cultured in 96-well plates coated with 1 μg/ml anti-CD3 antibody (clone 17A2, prepared in-house) and 1 μg/ml anti-CD28 antibody (clone 37.51, eBioscience) in RPMI medium supplemented with 300 U/ml hIL-2 (Sigma) for 14 days. The cells were refed with fresh medium every second day. Total CD4⁺ T cells were isolated using the MACS CD4 Negative Isolation Kit II (Miltenyi Biotec) and labeled with the cell proliferation dye eFluor^TM 670 (eBioscience) according to the manufacturer’s instructions. To assess the suppressive capacity of Tregs in vitro, total CD4⁺ cells were stimulated with Dynabeads^TM Mouse T-Activator CD3/CD28 (Thermo Fisher) in the presence of WT or Cd25^Y129H Treg cells for 72 h.

PBMC preparation and storage were performed according to previously established methods [18 (link)]. In addition, 10⁷ PBMC cells were thawed from P7 and P9 patients, cultured in enriched MEM as previously described [32 (link)], and supplied with 10% heat-inactivated premium FBS (VWR, PA, USA, product 97068-085, lot 214B17). Furthermore, 100 μM of NiSO₄ (Sigma, Germany, product 31483-250G, Lot SZBB0900V) was added to the culture for 7 days. Then, PBMCs were washed twice with BSS, and cultured in media with 20U/mL hIL2 (Sigma, Germany, product 11011456001) for 7 days. Next, PBMCs were washed twice with BSS and returned to media with 100 μM NiSO₄ and cultured for another 7 days. At this point, PBMCs were stained with CellTrace violet proliferation dye (Invitrogen, City, CA, USA, product C34571) and irradiated PBMCs (40 Gy) from the same patient were added. After 7 days of the second round of Ni²⁺ stimulation, the proliferative cells were sorted based on CellTrace fluorescence.

MSCs and PBMCs were used from frozen stocks for all experiments. Assays were performed in 24-well plates at the specified volume and cell concentrations per experimental condition. MSCs were seeded in Transwell inserts (GBO, Kremsmunster, Austria) and allowed to adhere overnight. To detect cell proliferation, PBMCs were stained with carboxyflourescein succinimidyl ester (CFSE, Sigma, St. Louis, MO) at 2.5uM. T cell activation was achieved through incubation of PBMCs with 10ug/mL of concanavalin A (ConA, Sigma, St. Louis, MO) and 100 ng/mL of hIL-2 (Sigma, St. Louis, MO) for a period of 4 days. Transwell inserts with seeded MSCs were added to the PBMC wells during the experimental period. Flow cytometry was used to assess CFSE dilution associated with proliferation (LSRII, BD Biosciences, Franklin Lakes, NJ).