Click s medium

PRAME-specific T-cell products were generated from total PBMCs by previously established protocol [28 (link)]. Briefly, mature dendritic cells (DC), generated by adherence, were used as antigen-presenting cells. Non-adherent cells were stimulated with PRAME peptide-pulsed (200 ng/200 uL/5 x 10⁶ cells), irradiated (25 Gy) DC at an effector-to-target ratio of 10:1 and cultured in complete media consisting of 50% Click’s medium (Irvine Scientific, Santa Ana, CA), 40% RPMI (GE Healthcare, Logan, UT), 10% human AB serum (Gemini BioProducts, West Sacramento, CA), and supplemented with 2 mM GlutaMax (Gibco, Grand Island, NY). Second and third restimulations of T-cells were carried out weekly with PRAME peptide-pulsed, irradiated DC at an effector-to-target ratio of 20:1. On day 28, cells were harvested and evaluated for antigen specificity and functionality.

Lymphocytes were isolated from spleen and peripheral lymph nodes (including inguinal, auxiliary and cervical lymph nodes) or thymus. Naïve CD4⁺ T cells (CD4⁺CD25⁻CD44⁻CD62L⁺), Treg cells (CD4⁺YFP⁺ or CD4⁺CD25⁺ as indicated in the figures and text) and thymic mature CD4⁺ T cells (CD4⁺CD8⁻CD69^loCD62L^hi) were sorted on a MoFlow (Beckman-Coulter) or Reflection (i-Cyt). Sorted cells were cultured in plates coated with anti-CD3 (2C11, 10 μg/ml) and anti-CD28 (37.51, 10 μg/ml) for 4.5 days in Click's medium (Irvine Scientific) supplemented with β-mercaptoethanol, 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin and IL-2 (200 U/ml). In certain experiments, Treg cells were stimulated with IL-2 (0-500 U/ml) as indicated in the figures and legends. CD62LhiCD69lo mature thymocytes from WT and Cd4^CreStk4^f/fStk3^f/f mice were differentiated under induced Treg skewing condition (2 μg/ml CD3, 2 μg/ml CD28, 1 ng/ml TGF-β, and 100 U/ml IL-2) with irradiated antigen-presenting cells for 5 days, and Foxp3 expression was examined by flow cytometry. For activated CD4⁺ T cell blast generation, peripheral CD4⁺CD25⁻CD44⁻CD62L⁺ naïve T cells were sorted and stimulated with plate-bound 10 μg/ml CD3⁺CD28 for 3 days and rested in 100 U/ml IL-2 for another 3 days. Live T cells were purified using Ficoll, and rested without IL-2 for 8 h and then stimulated with IL-2 for the indicated time points.

aATCs for antigen presentation were generated by stimulation of PBMCs (5×10⁵ cells per well) on non-tissue culture treated 24-well plates coated with a CD3 antibody produced by the OKT3 hybridoma (ATCC #CRL 8001, Manassas, VA) and CD28 antibody (Becton Dickinson BD, Franklin Lakes, NJ) (CD3/28 MAbs, each at 1 μg/ml). ATCs were maintained in T-cell medium (TCM) (RPMI 1640, (Hyclone, Waltham, MA) supplemented with 45% Click's medium (Irvine Scientific, Santa Ana, CA), 2 mmol/l GlutaMAX TM-I (Invitrogen, Carlsbad, CA), and 5% Human AB Serum (Valley Biomedical, Winchester, VA)] and supplemented with IL2 (50U/ml, NIH, Bethesda, VA), which was replenished every 3 days. Two days before antigen-specific T-cell restimulation, aATCs were re-activated on CD3/28 MAb-coated plates to upregulate costimulatory molecules and then irradiated and peptide-pulsed immediately before being used as APCs.

CD4⁺ T cells were isolated from the spleen and lymph nodes using the EasySep Mouse CD4 Positive Selection Kit II (Stemcell Technologies). Naive (CD25⁻CD44^hiCD62L^lo) CD4⁺ T cells were sorted by flow cytometry from the bead purified CD4⁺ T cells. The naive CD4⁺ T cells were resuspended in Click’s medium (Irvine Scientific) at 1 million cells per ml, and then plated on day 0 in 24 well plates coated with goat anti-hamster IgG antibody (200 ng ml⁻¹; MP Biomedicals) with the addition of soluble anti-CD3 (1 μg ml⁻¹; 145-2C11) and anti-CD28 (1 μg ml⁻¹; 37.51) from Bio X Cell. Polarizing conditions for different T helper subsets are as following: T_H1: human IL-2 (100 U ml⁻¹; PeproTech), mouse IL-12 (20 ng ml⁻¹; PeproTech) and anti-IL-4 (5 μg ml⁻¹; Bio X Cell); T_H2: human IL-2 (100 U ml⁻¹; PeproTech), mouse IL-4 (20 ng ml⁻¹; Biolegend), anti-IFN-γ and anti-IL-12 (5 μg ml⁻¹; Bio X Cell); T_H17: mouse IL-6 (20 ng ml⁻¹; Biolegend), human TGF-β (2 ng ml⁻¹; PeproTech), anti-IFN-γ and anti-IL-12 (5 μg ml⁻¹; Bio X Cell).

Buffy coats from healthy donors were purchased from the Gulf Coast Regional Blood Center, Houston, TX. Peripheral blood mono-nuclear cells (PBMCs) isolated with Lymphoprep density separation (Fresenius Kabi Norge) were activated on plates coated with 1 μg/mL CD3 (Miltenyi Biotec) and 1 μg/mL CD28 (BD Biosciences) agonistic mAbs. On day 2, T lymphocytes were transduced with retroviral supernatants using retronectin-coated plates (Takara Bio Inc., Shiga, Japan). Briefly, non-tissue culture treated 24-well plates are coated overnight with 7 mg/mL retronectin in the cold room, washed once with 1 mL medium, coated with 1 mL of the retroviral supernatant per well and centrifuged at 2000 g for 90 min. After removal of the supernatant, 5 × 10⁵ activated T cells are plated, and centrifuged at 1000 g for 10 min. Three days later, T cells are collected and expanded in complete medium (45% RPMI-1640 and 45% Click’s medium (Irvine Scientific), 10% FBS (Hyclone), 2 mM GlutaMAX, 100 unit/mL of Penicillin and 100 mg/mL of streptomycin) with IL-7 (10 ng/mL; PeproTech) and IL-15 (5 ng/mL; PeproTech), changing medium every 2–3 days (Xu et al., 2014 (link)). On day 12–14, cells are collected for in vitro and in vivo experiments. T cells were cultured in IL-7/IL-15 depleted medium for two days prior to being used in in vitro functional assays.

The human melanoma WM115 cells were tagged with the fusion protein eGFP–firefly-luciferase. Human T cells were engineered with CSPG4.CAR as previously described^{41 (link)}. Here we used a CSPG4-specific CAR that encodes the CD28 endodomains with enhanced functionality. WM115 cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco; Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U ml⁻¹ penicillin (Invitrogen) and 100 U ml⁻¹ streptomycin (Invitrogen). CAR-T cells were cultured in complete medium containing 45% RPMI 1640 and 45% Click’s medium (Irvine Scientific) with 10% FBS (HyClone), 2 mmoll⁻¹ GlutaMAX, human recombinant IL-7 (5 ng ml⁻¹, Pepro Tech) and human recombinant IL-15 (10 ng ml⁻¹, Pepro Tech). Cells were cultured in an incubator (Thermo Fisher Scientific) at 37 °C under an atmosphere of 5% CO₂ and 90% relative humidity. The cells were subcultivated approximately every 2–3 d at 80% confluence at a split ratio of 1:3.

All chemicals were obtained from Sigma-Aldrich and used without any purification. Human melanoma WM115 cells and WM115-luc cells were obtained from Dr. Gianpietro Dotti at UNC and cultured in RPMI 1640 (HyClon) medium containing 10% heat inactivated fetal calf serum (F Invitrogen, Carlsbad, CA), 2 mmol/L GlutaMAX (Invitrogen), 200 IU/mL penicillin, and 200 mg/mL streptomycin (Invitrogen) in an incubator at 37 °C in 5% CO₂. The CD19-specific CAR T lymphocytes and CSPG4-specific CAR T lymphocytes also were generated in Dr. Gianpietro Dotti’s lab at UNC^{[12b (link)]}. CAR T cells were cultured and expanded in complete medium containing 45% RPMI 1640 and 45% Click’s medium (Irvine Scientific) with 10% FCS (HyClone), 2 mmol/L GlutaMAX, 100 IU/mL penicillin, and 100 mg/mL streptomycin. Cells were fed twice a week with recombinant interleukin-7 (IL-7, 5 ng/mL; Pepro Tech, Inc.) and interleukin-15 (IL-15) (10 ng/mL; Pepro Tech, Inc.). Female NSG mice (6–10 weeks) were purchased from Jackson Lab. All mouse studies were carried out following the protocols approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill and North Carolina State University and complied with all relevant ethical regulations.