Flow Cytometry
Flow cytometry analysis of exploratory samples was conducted in a non-GCLP research laboratory at TCB, for research purposes only. Fluorophore-labelled antibodies for HLA-A2, HLA-B7, CD3, TCR Vγ9, CD45, CD33, CD14 and CD16 were purchased from Biolegend (San Diego, CA). Fluorophore-labelled antibodies for HLA-A3 and TCR α/β were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Antibody staining was performed according to the relevant manufacturers’ instructions. Measurements were done on a NovoCyte flow cytometer (Agilent, Santa Clara, CA). Data were analyzed with NovoExpress software (Agilent, Santa Clara, CA). For analysis of patient trial samples, PBMCs were cryopreserved at the clinical site and were subsequently shipped to TCB for assessment of detection of product-specific HLA-type, indicating presence of donor cells. The proportion of CD3+ / Vγ9+ cells in PBMCs samples from patient PRA1-5006 were analyzed for the expression of HLA-B7, HLA-A2, and HLA-A3 over time, up to 100 days post infusion. Donor-specific cells were identified based on differential expression of HLA markers between donor and recipient over time, up to 100 days post infusion.