RNA-based co-transfer of human CD8αβ with WT1-specific TCRαβ redirects tumor recognition by CD4 and γδ T-cells towards MHC class I-restricted WT1 epitopes and boosts CD8 T-cell responses with or without CD3 mRNA.

We evaluated the redirection of CD4, γδ and CD8 T-cells towards the Wilms' tumor protein (WT1) tumor-associated antigen, using a major histocompatibility complex (MHC) class I-restricted WT1-specific T-cell receptor (TCR) introduced via RNA-based engineering. We also studied whether co-transfection of TCR mRNA in combination with CD8αβ mRNA in CD4 and γδ T-cells or with CD8αβ and CD3γδεζ mRNAs in CD8 T-cells improves antigen-specific T-cell functional activity. We transfected primary human CD4 and CD8 T-cells following our in-house-developed protocol, in which electroporation with Dicer-substrate silencing RNA (DsiRNA) suppresses de novo expression of native TCR, followed by DsiRNA-resistant transgenic TCR mRNA transfection. This method allows minimal mispairing between native and introduced TCR chains. High frequencies of transgenic MHC class I-restricted WT1-specific TCR-positive cells were obtained in expanded CD4 and γδ T-cells. Only co-electroporation of CD8 mRNA led TCR mRNA-electroporated CD4 and γδ T-cells to MHC class I-restricted antigen-specific recognition of tumor cells. Co-electroporation of CD8 T-cells with WT1-specific TCR, CD8 and CD3 mRNAs also enhanced CD8 T-cell activation and antigen-specific recognition as compared to either TCR-engineered or TCR- and CD8-engineered cells. In summary, RNA electroporation is a fast and efficient method to engineer primary human CD8, CD4 and γδ T-cells for redirecting T-cell specificity. Transgenic CD8 expression in CD4 and γδ T-cells and co-electroporation of CD8 and CD3 mRNA in CD8 T-cells enable antigen recognition when T-cells are redirected with TCRs of low/intermediate avidity, showing the potential of TCR co-receptors to improve T-cell functional activity against tumor-associated antigens in adoptive TCR-T-cell therapies.