The use of longterm immunosuppression leads to downside side effects for transplanted patients, including drug toxicity, increased susceptibility to infection and development of neoplasies. Recent reports have supported the use of Tr1 cells, a subset of Foxp3- regulatory Tregs, as one of the best candidates for use in new therapeutic protocols, based on their high production of IL-10. Tr1 cells can be identified by the co-expression of CD49b and LAG-3, which facilitates their purification. A recent protocol based on the co-culture of naïve T cells with allogeneic DC-10, which coexpress the co-inhibitory molecules ILT4 and HLA-G and allows efficient Tr1 differentiation in vitro, has been approved for clinical trials. However, recent data indicate that this subpopulation is heterogeneous and that only a proportion of CD49b+ LAG3+ cells actually express IL-10. Here, we show an improved method to increase the purity of functional allospecific Tr1 cells which maintain their phenotype and function. Monocyte derived dendritic cells were differentiated in the presence of GM-CSF, IL-4 and IL-10 for 8 days, obtaining more than 80% of ILT4+/HLA-G+ DC (DC10). Co-cultures of DC10 with sorted CD4+ CD45RA+ CD25- allogeneic T naïve cells (1:10) resulted in 30% of CD4+CD49b+LAG-3+ cells after 9 days in culture, which were sorted by FACS and polyclonally expanded, leading to an enrichment of 90% of CD49b+LAG-3+ cells. These cells were able to suppress 40% of the proliferation of allogeneic CD4+T cells, but not of polyclonal T cells. We are currently investigating the stability of these Tr1 under different inflammatory conditions.