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.

Enhancing the functionality of engineered T cells using synthetic TLR agonists. Once the optimal orientation of the TCR was identified, we focused on augmenting the function of engineered T cells. Adjuvants based on Toll-like receptor ligands can have direct effects on T cells 20 and be selected to mimic infecting pathogens 21 . We used TLR ligands that frequently signal viral infection: polyI:C (TLR-3), Imiquimod (TLR-7), ssRNA40 (TLR-8) and CpG (TLR-9). The TLR ligands were added to the PBMC during the first 48 h of activation prior to transduction. At the time of transduction, the media was changed, the primary T cells were transduced and allowed to expand for 10 days. Following their expansion, we tested TCR expression using HLA-pentamers to determine if the TLR ligands affected TCR expression compared to the standard protocol with IL-2 alone. The TCR presented in Figure 4 (HBV core 18-27) has high affinity for its cognate peptide-MHC, allowing us to monitor TCR expression in both the CD8 and CD4 T cell population using the HLA-pentamer (Fig. 1A) . We found that the addition of TLR ligands to the culture during the first 48 h of activation had little impact on HLA pentamer staining in either the CD8 or CD4 T cell population ( Fig. 4 A,B ,E). However, we observed clear differences in the frequency of functional cells. The addition of polyI:C or ssRNA40 increased the frequency of IFN-c producing CD8 and CD4 T cells compared to IL-2 alone, IL-2 plus CpG or Imiquimod (Fig. 4 C,F ). This increase in the frequency of IFN-c producing cells translated into a 60% and 80% increase in functional CD8 T cells following addition of polyI:C and ssRNA40 respectively to the culture medium (Fig. 4D ). Similar results were observed with CD4 T cells, with ssRNA40 stimulating the greatest increase in IFN-c1 T cell frequency (Fig. 4G) .