Supporting online information
Materials and Methods
Retroviral vector construction and production of viral supernatant
HA-2-TCRα and TCRβ chain sequences were cloned intro retroviral constructs, linked by an IRES sequence to unmodified CD20. Cysteine modification of PRAME TCR was performed by introducing a cysteine residue in the constant domains of TCRα and β chains at positions 48 and 57, respectively1-3. Codon optimization (CO) of PRAME-TCR and CD20 was performed by GENEART®. PRAME-TCRα and TCRβ chain sequences4 were cloned into retroviral constructs, linked by a T2A sequence and were coupled to CD20 linked via a P2A sequence5, resulting in concordant stochiometric expression of both TCRαβ-chains and CD20.
HLA Class I tetrameric complexes, flow cytometric analyses and cell sorting
Tetramers used for isolation and staining of CMV-specific T-cells consisted of HLA-A1 molecules in complex with pp50 peptide (VTEHDTLLY; pp50), or HLA-B7 molecules in complex with pp65 peptide (RPHERNGFTVL; pp65). Tetramers used for isolation and staining of EBV-specific T-cells consisted of HLA-A2 molecules in complex with BMLF-1 peptide (GLCTLVAML; BMLF-1). In addition, tetramers consisting of HLA-A2 molecules in complex with PRAME peptide (SLLQHLIGL; PRAME) were used. Monoclonal antibodies used are: fluorescent isothiocyanate (FITC) conjugated CD20 (BD Biosciences), allophyocyanin (APC) conjugated NGF-R (Cedarlane Laboratories, Hornby, Ontario, Canada), and phycoerythrin-cyanin 7 (PE-Cy7) conjugated TCRαβ (Beckman Coulter, Mijdrecht, The Netherlands).
Reference List
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