Although growing evidence demonstrates the feasibility of using allospecific T reg-based immunotherapy for transplantation tolerance, there is still little knowledge about Treg long term stability and functionality in vivo. Our group has established several methods for generating large numbers of allospecific tTregs, iTregs and Tr1, which preserve their suppressive phenotype and function in the presence of pro-inflammatory cytokines.  Allo- tTregs, iTregs or Tr1 were obtained after in vitro stimulation with allogeneic monocyte-derived DCs or DC10 (for Tr1), they were FACS-sorted to a purity higher ≈90% and then, polyclonally expanded for 4-6 weeks. For thymic Tregs, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expression of CTLA-4, LAG-3, and CD39. Expanded thymic alloTregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of IFN-γ, IL-4, IL-6, or TNF-α. In the case of alloantigen-induced Foxp3+ Tregs, they were also polyclonally expanded for 6 weeks in the presence of TGF-β1, IL-2, and rapamycin, giving rise to 4,600 million Tregs (230,000 x). Finally, Tr1 cells CD49b+ LAG-3+ (≈80%), where generated, which produced high levels of IL-10 (≈90%) and expressed co-inhibitory receptors (PD-1, CTLA-4, TIM-3, TIGIT, CD39), as well as chemokine receptors CCR2, CCR4 and CXCR3. Most importantly tTregs, iTregs and Tr1 specifically suppressed allospecific, but not third party, T cell proliferation, in the presence of pro-inflammatory cytokines. Our data indicate that expanded-allo-Tregs are stable and express relevant chemokine receptors, suggestive of their potential homing to the kidney allograft.