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.

The difference in expression of the TCR beta chain between the different orientations was less pronounced than that observed for HLA multimer staining. There was only a 1.9 fold increase in TCR beta expression dependent on the orientation, suggesting that translation of the proteins was similar despite orientation of the TCR cassette. In contrast, we observed a 4.2 fold increase in HLA multimer staining that was dependent on the orientation. The discrepancy between these two observations remains unclear. Unlike internal ribosome entry sites (IRES), the P2A linker results in nearly equimolar production of both the alpha and beta chains 13 . Therefore, there should be equal production despite the orientation. The difference may be related to the stability of individual TCR alpha and beta proteins. The TCR alpha chain is rapidly targeted for ER associated degradation. This is the rate-limiting step in TCR complex expression in developing thymocytes 33 and this instability carries over to TCR expression in mature T cells 34, 35 . Therefore, translating the beta chain first may facilitate TCR alpha/beta heterodimer formation and stability; as the downstream alpha chain is translated it can rapidly pair with beta.