iPSC-derived epstein-barr virus-specific T cells with dual alpha T cell receptors

Abstract T cells play a critical role in immune defense by eliminating virus-infected and malignant cells. Their antigen specificity is determined by T cell receptors (TCRs), heterodimers composed of α and β chains, whose diversity is generated through V(D)J recombination and junctional nucleotide insertions. It is well known that, unlike the β chain which follows the principle of allelic exclusion and is expressed from only one allele, the α chain can undergo rearrangement at both alleles and may be expressed from both loci. Identifying the TCRαβ sequences of cytotoxic T lymphocyte (CTL) clones with robust cytotoxicity is essential for advancing TCR-engineered T cell therapies. Although the TCR repertoire is highly diverse, dominant clones occasionally emerge that possess strong reactivity against specific antigens. Analyzing these dominant clones offers opportunities to identify antigen-specific TCRs with therapeutic potential and to understand the principles of immunological memory. We have established a platform to generate rejuvenated antigen-specific CTLs (rejTs) by reprogramming T cells into induced pluripotent stem cells (T-iPSCs) and redifferentiating them into CTLs. These rejTs proliferate vigorously and exhibit strong antigen-specific cytotoxicity, allowing efficient functional evaluation and potential application in adoptive T cell therapies. In our attempt to generate Epstein-Barr virus (EBV) LMP2 (419–427, TYGPVFMSL)-specific rejTs, we obtained CTL clones with identical TCRαβ sequences in two independent experiments. RejTs derived from these clones demonstrated potent cytotoxicity, suggesting that they represent dominant clones. Interestingly, these clones expressed two functionally rearranged TCRα chains (α₁ and α₂), both paired with the same TCRβ chain. To determine which TCRα chain mediated LMP2 recognition, we knocked out endogenous TCRs in HLA-matched donor T cells using CRISPR/Cas9 and transduced them with either TCRα₁β or TCRα₂β. Only TCRα₁β-transduced T cells bound the LMP2/HLA-A24 tetramer and exhibited specific cytotoxicity against LMP2-expressing, HLA-matched lymphoblastoid cell lines (80% vs 0.5% lysis at an E:T ratio of 40:1), indicating that TCRα₁β mediates the observed anti-EBV activity. To explore whether TCRα₂β also recognizes a different epitope of , we applied three deep learning–based TCR specificity prediction tools (MixTCRpred, TCRex, and ERGO-II), which identified candidate EBV epitopes. MHC binding predictions (NetMHC v4.0) confirmed strong binding to the donor's HLA alleles. We are currently validating the predicted epitopes to assess the contribution of TCRα₂β to the cytotoxicity observed in this dominant clone.