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.

This demonstrates that there is a positive (high frequency) and a negative (low frequency) orientation for each TCR alpha and beta gene pair. When we classified expression based on this positive/ negative orientation we observed a statistically significant increase in the frequency of TCR1 T cells, with a mean of 35.3 vs 22.45 for beta chain expression (Fig. 2D ) and 19.49 vs 8.74 for HLA multimer staining respectively (Fig. 2G ). The fold increase in expression differed for each TCR but overall we observed a mean fold increase of 1.9 in beta chain expression and 4.2 for multimer staining (Table 1) in the optimal orientation. TCR cassette orientation and T cell function. Next, we determined if the increased TCR expression correlated with an increase in the frequency of functional cells. Primary human T cells transduced with both orientations of the TCR cassette were stimulated with specific peptides presented on either HLA-A21 T2 cells or HLA-matched EBV transformed B cell lines. IFN-c production, analyzed by flow cytometry, was used to monitor the frequency of virus-specific CD8 T cells (Fig. 3A) . T cell cytokine production mirrored TCR expression and there was clearly a positive and negative orientation for each TCR construct. Nine out of 10 TCRs resulted in higher frequencies of IFN-c1 T cells in the beta-P2A-alpha orientation whereas only one TCR was superior in the alpha-P2Abeta orientation (Fig. 3B ). When TCRs were grouped according to their positive and negative orientation, we observed a significant difference in their overall functionality, 26.86% vs 12.89% IFN-c1 CD8 T cells (Fig. 3C) . This translated to a 2.7 fold increase overall in the frequency of IFN-c producing T cells with the optimal orientation of the TCR cassette (Table 1) .