WT1-TCR gene transfer into haematopoietic stem cells: a tumour immunotherapy model

T cell receptor (TCR) gene transfer into primary T cells can redirect antigen specificity towards a known tumour antigen. We have demonstrated both in vitro and in vivo that peripheral T cells transduced with genes encoding a high avidity, HLA-A2 restricted TCR specific for Wilms Tumour 1 (WT1) antigen, can kill HLA-A2+ human leukaemia cells overexpressing WT1. However, mispairing of the introduced and endogenous TCR alpha and beta chains can result in potentially autoreactive neo-specificities. The in vitro culture and expansion protocols used to generate TCR-transduced T cells can result in accelerated loss of a naive phenotype, which may compromise subsequent in vivo function and persistence. We hypothesize that the introduction of TCR genes into haematopoietic stem cells (HSCs) may prevent endogenous TCR expression by allelic exclusion in T cells derived from TCR-transduced HSCs and generate naive T cells of the desired antigen-specificity. We have introduced the WT1-TCR genes into lineage negative murine haematopoietic stem cells (HSCs) using a lentiviral vector. WT1-TCR transduced HSCs were transferred into lethally irradiated HLA-A2 transgenic mice. Murine WT1 contains the identical pWT126 peptide epitope as human WT1, and the tissue distribution is similar in mice and humans. WT1 is markedly over-expressed in leukaemia cells and is expressed at low levels in some normal tissues. This system can therefore provide important information about central and peripheral tolerance mechanisms against high avidity, WT1-specific T cells. We have shown that mice transplanted with WT1-TCR transduced syngeneic HSCs, engraft normally and give rise to two distinct populations of Vbeta2.1+, WT1-specific CD8+ T cells in the peripheral blood, lymph nodes and bone marrow (Fig.1), either CD8hi or CD8lo. Further phenotypical analysis confirmed that the CD8hi, WT1-specific T cells were CD44hi and CD62L+. In contrast, the CD8lo, WT1-specific T cells were CD44lo and CD62L+, suggesting in vivo activation following antigen exposure. Importantly, in vivo killing assays performed in recipients of TCR-transduced HSCs have demonstrated antigen-specific killing of WT126ploaded A2Kb splenocytes (Fig.2), indicating that the WT1-specific T cells generated in vivo from modified HSCs are functional. Further experiments are in progress to compare the in vivo function of such WT1-specific T cells with adoptively transferred WT1 TCR-transduced central memory and effector T cells.