Thymus-deriving natural regulatory T cell generation in vitro: Role of the source of activation signals

In this research we have examined different sources of activation signals in order to optimize culture conditions for in vitro generation of thymus-deriving natural regulatory T cells (nTregs). We have established a novel model using JAWS II dendritic cell line of immature phenotype and compared it to commonly used methods for the generation of Tregs from peripheral lymphoid organs or blood T cells. In our model the first activation signal is provided by anti-CD3 monoclonal antibodies while the second is delivered by costimulatory molecules expressed on JAWS II cells. The presence of JAWS II cells co-cultured in vitro with unsorted thymocytes directly isolated from the thymus gland creates environment favoring SP CD4+ differentiation, provides the apoptotic cells clearance, maintains the survival of thymocytes and facilitate nTreg generation. Moreover the usage of immature dendritic cells stimuli enables to conduct research on agents affecting nTreg survival, proliferation and development in conditions of cell-to-cell contact of undifferentiated thymocytes with dendritic cells.