Rhil 7

H1-CAR ILC2s were isolated from week 5–9 ATOs as described above. For proliferation assays, up to 1×10⁵ cells were plated in 200 μl AIM V (ThermoFisher Scientific, Cat. 12055091), 5% human AB serum (Gemini Bio, Cat. 100–512) with 20 ng/mL rhIL-2 (Peprotech) and 20 ng/mL rhIL-7 (Peprotech) plus indicated cytokines at 20 ng/mL (Peprotech) in the absence or presence of irradiated NALM6 cells at a 3:1 effector to target (E:T) ratio. Fresh cytokines were replenished at day 3 via half-media change, and cells were replated into larger wells when confluent, approximately every 2–3 days. Irradiated NALM6 cells were added again on day 7 of expansion. Cells were counted twice a week on a hemocytometer.
H1-CAR T cells were expanded at 5×10⁵ cells/mL in AIM V (ThermoFisher Scientific, Cat. 12055091) supplemented with 5% human AB serum (Gemini Bio, Cat. 100–512), 5 ng/ml rhIL-7 (R&D), and 100 IU/ml rhIL-2 (Miltenyi Biotec, Cat. 130–097-748) with irradiated NALM6 cells added at a 3:1 E:T ratio 5 days prior to functional assays.

TRAC-1928z T cells were generated as previously described^{28 (link), 57 (link)}. In brief, αβTCR-T cells were purified from PBMCs with the Pan T cell Isolation kit (Miltenyi Biotec) on the AutoMACS Pro according to manufacturer instructions. Purified cells were activated with CD3/CD28 Dynabeads (1:1 beads:cell) in X-Vivo 15 media (Lonza) supplemented with 5% Human Serum (HS) (Gemini Bioproducts) with 5ng/mL rhIL-7 (R&D Systems) and 5ng/mL rhIL-15 (R&D Systems). 48 h after αβTCR-T cell activation, CD3/CD28 beads were magnetically removed, and T cells were transfected by electrotransfer of TRAC ribonucleoprotein using the Nucleofector II device (Lonza). Then 2×10⁶ T cells were resuspended in P3 buffer (Lonza) and mixed with 60pmol TRAC ribonucleoprotein in a total volume of 20μL. Following electroporation and considering 66.7% viability, cells were diluted into culture medium and 1×10⁶/mL and incubated at 37°C, 5% CO₂. Recombinant AAV6 donor vector pAAV-TRAC-1928z^{28 (link)} was added to the culture 30min after electroporation at a multiplicity of infection of 3×10⁵ genome copies. Twenty-four h after targeting, T cells were reprogrammed as described above (WT-TiPS) and TRAC-1928z-TiPS colonies were established and cloned on MEF feeder cells. PCRs were performed to determine biallelic, specific target transgene integration into the TRAC locus.

PBMC were expanded over four rounds of peptide-specific stimulation, with cells being analysed by flow cytometry at the end of rounds one and four. Briefly, 3×10⁶ PBMC were initially resuspended in NGM at 1.5×10⁶ cells/mL in a 24-well plate, in the presence of rhIL-2 (20 U/mL, Roche, Basel, Switzerland), rhIL-7 (2 ng/ml, R&D Systems, Abingdon, Oxfordshire, UK), rhIL-15 (5 ng/ml, R&D Systems) and rmIL-21 (0.5 ng/ml, R&D Systems). Peptide-specific stimulation was performed by adding pCEA691 or CMV pp65 directly into specific wells, at a final concentration of 10μM. After a first round of 7–9 days duration, cells were moved to re-stimulation. Three rounds of re-stimulation were performed as described below, each of which were 7–9 days duration.
Re-stimulation was conducted by re-suspending 5×10⁵ cells in 2 ml of cytokine-supplemented NGM in a new 24-well plate, and stimulating them with irradiated (70 Gy) T2 cells (2×10⁵) pulsed with the appropriate peptide, at a final concentration of 10μM. Irradiated (35 Gy) autologous PBMCs or PBMC from healthy HLA-A2⁺ donors obtained from the National Blood Service (Colindale, UK) were used as feeder cells (2×10⁶).