| Summary: | Graft versus Host Disease (GvHD) remains one of the main complications after haematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, CD4+CD25highFoxp3high regulatory T (Treg) cells have been proposed as a cellular therapy to prevent GvHD. However it has been shown that Treg cells can inhibit natural killer (NK) cell functions. NK cells are key effectors of the Graft versus Leukaemia (GvL) effect post-transplant; therefore, it is plausible that a Treg cell therapy may impact on NK cell function and differentiation from haematopoietic stem cells (HSC). This study sought to elucidate the effects of Treg cells on NK cell function and differentiation using umbilical cord blood (CB) as a cell source. Herein, it is confirmed that CB CD4+CD25highFoxp3high Treg cells are fully functional and upon TCR-stimulation express CTLA-4 and LAP, and secrete TGF-β and IL-10. Also, they express receptors associated with trafficking to lymphoid tissues and the bone marrow, which are potential NK cell/Treg cell interaction sites. Furthermore, it is shown that CB Treg cells can suppress CB NK cell functions after TCR-stimulation in steady state but not in the presence of exogenous cytokines. Lastly, in an in vitro model of NK cell differentiation, a 90% reduction in total NK cells was observed when TCR-stimulated Treg cells were added at the time when HSC commitment to the NK cell lineage occurs. Interestingly, the few NK cells that developed in these cultures showed normal phenotype, IFN-γ secretion and cytotoxicity. Notably, the addition of human recombinant TGF-β to HSC cultures caused a similar reduction in NK cell differentiation as shown when TCRstimulated Treg cells were added to HSC cultures. Moreover, the Treg cellmediated effect was contact-dependent and cytokine competition-independent. Collectively, these results demonstrate for the first time that TCR-stimulated CB Treg cells inhibit NK cell differentiation through TGF-β, providing information for optimisation of the time of delivery for an adoptive Treg cell therapy post-HSCT to prevent GvHD.
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