The use of adoptive T-cell therapies is often limited by barriers imposed by MHC disparity, resulting in rejection, alloreactivity and impaired antigen presentation. This study shows for the first time that lymphoid precursor cells from any donor can be transferred to any individual irrespective of MHC disparities. Our data demonstrate that allogeneic T-cell precursors, when adoptively transferred (with or without syngeneic HS cells) to irradiated recipients, develop into functional mature T cells in the absence of a complete donor hematopoietic system. MHC restriction of these cells is determined by host MHC molecules, which are encountered during T-cell development43–45
. Tolerance of the resulting T cells is dual (donor and host) as a consequence of antigen-presenting cell chimerism (owing to the dendritic cell lineage potential of T-cell precursors up to the DN2 stage46,47
). The major advantage of T cell lineage committed precursor cells with limited dendritic cell capacity is their potential to reconstitute an immunosuppressed host with host MHC restricted allogeneic T cells, while preserving a predominantly host antigen-presenting cell chimerism. Controlling antigen-presenting cell chimerism translates into host tolerant and functional TCR selection, averting GVHD and promoting immunity. Therefore, cell therapy with T-cell precursors allows for universal T-cell immunotherapy, which is currently impossible with ex vivo
generated or manipulated mature T cells. We provide evidence for the efficacy of this strategy as tumor immunotherapy in combination with prior cytostatic conditioning, as well as a countermeasure for lymphopenia due to radiation-induced injury. True off-the-shelf therapy requires the use of cryopreserved cells, which is routinely done in the clinical setting with human hematopoietic progenitor cells. Using our mouse model, we found that adoptive transfer of cryopreserved allogeneic T-cell precursors to irradiated recipients resulted in stable thymic engraftment (Supplementary Fig. 11
Genetic engineering is a powerful strategy to design tumor-associated antigen-specific cells. Potential targets for transgenic receptors include immunogenic tumor proteins21,48,49
or viral antigens in virus-associated cancers such as Epstein-Barr virus (EBV)-related lymphoma or lymphoproliferative disease16,19,50
. HS cells have been transduced with TCR genes to obtain tumor-specific T cells42,51
. However, HS cells are usually quiescent, which makes them more difficult to transduce than rapidly proliferating cells such as early T-cell precursors (in particular DN1). An important issue in engineering mature T cells to express antigen-specific receptors is the possibility of generating potentially harmful novel receptor specificities as a consequence of combining transgenic receptor and endogenous TCR chains52
. This scenario is of no concern when engineering early T-cell precursors, because these cells have not yet selected their TCR. The main challenge of this approach is to generate antigen-specific T-cell precursors that can be positively selected and are not subject to negative selection. The former can be accomplished by transferring genes encoding chimeric antigen receptors instead of TCRs, allowing endogenous TCR expression and resulting in intact positive selection of transgenic cells. The risk of clonal deletion can be reduced by targeting antigens that are distinct from self-antigens so that auto-reactivity (if any) displayed by the antigen receptor is below the threshold that induces negative selection. Because the mechanism of negative selection is very sensitive, additional strategies to overcome this barrier should be explored, such as using promoters that allow temporal control of transgene expression (conditional expression, or even better, maturation-dependent expression). Furthermore, suicide switches as a safety feature and costimulatory signals to prevent T-cell anergy and apoptosis could be incorporated to ensure optimal functionality, while avoiding unwanted adverse effects of engineered cells39,52
. However, our strategy to transduce T-cell precursors to express a chimeric antigen receptor targeting hCD19 resulted in excellent transduction efficiency and the in vivo
generation of high numbers of appropriately selected T cells expressing the chimeric antigen receptor, which were capable of significant anti-tumor activity without any undesirable auto/alloreactivity. Combining tumor-specific T-cell precursors with tumor vaccination strategies could further enhance the efficacy of this approach.
We conclude that adoptively transferred ex vivo generated allogeneic T-cell precursors can develop into host-MHC restricted T cells characterized by dual tolerance and selection of a functional TCR repertoire, even in a fully mismatched thymic epithelial MHC environment. The advantages of using T-cell precursors for immunotherapy are obvious: T-cell precursors do not have to be MHC matched because GVHD is not an issue, and the use of allogeneic precursor cells instead of autologous cells eliminates the risk of contamination with residual malignant patient cells and enables the generation and storage of virtually unlimited quantities of precursor cells for universal use. This procedure has not only substantial logistic and technical advantages, but it facilitates the use of ex vivo manipulation protocols, in particular genetic engineering, to generate antigen-specific or otherwise enhanced designer cells. Adoptive transfer of MHC-mismatched and genetically enhanced T-cell precursors therefore represents a labor-saving and cost-effective strategy for targeted immunotherapy for patients with malignant diseases and/or T-cell deficiencies.