Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections

Banu, Nasirah; Chia, Adeline; Ho, Zi Zong; Garcia, Alfonso Tan; Paravasivam, Komathi; Grotenbreg, Gijsbert M.; Bertoletti, Antonio; Gehring, Adam J.

Flow cytometry analysis of TCR expression. Monitoring expression of the introduced TCR was dependent on available reagents. In most cases both anti-Vbeta antibodies (Beckman Coulter) and PE labeled HLA pentamers (ProImmune)/ tetramers (prepared in-house) were available. However, there was no TCR V beta antibody available for the HBV Env 370-79 TCR and no multimer available for the EBV EBNA-4NP 399-408 . Pentamer staining was performed for 15 minutes at room temperature in PBS, 1% BSA and 0.1% sodium azide (staining buffer). Cells were washed and stained with CD8-PeCy7 (BD Bioscience, RPA-T8) for 30 min on ice. In parallel, transduced T cells were stained with anti-V beta antibodies 1 CD8-PECy7 for 30 min on ice. Cells were immediately fixed with PBS 1 1% formaldehyde and analyzed on FACs Canto flow cytometer. To determine optimal TCR alpha/beta orientation we selected 2-3 retroviral clones carrying the TCRs in either the alpha-2A-beta or beta-2A-alpha orientation and used these retroviral clones to transduce T cells as described above. Each experiment was performed at least twice and the best clone from each orientation was used to test their functionality.