Đedović, Neda (57208524504)Neda (57208524504)ĐedovićPaunović, Verica (24342012700)Verica (24342012700)PaunovićStojanović, Ivana (56186289000)Ivana (56186289000)Stojanović2025-06-122025-06-122019https://doi.org/10.1016/j.jim.2019.04.011https://www.scopus.com/inward/record.uri?eid=2-s2.0-85065012351&doi=10.1016%2fj.jim.2019.04.011&partnerID=40&md5=562bae193b9b0d98be019762e7192a24https://remedy.med.bg.ac.rs/handle/123456789/5526Polyclonal T regulatory cells (Treg - CD4+CD25+CD127lowFoxp3+)are used in several protocols for the treatment of type 1 diabetes (T1D), multiple sclerosis and graft-versus host disease in clinical trials. However, general opinion is that autoantigen-specific Treg could be more efficient in autoimmunity suppression due to their direct effect on pathogenic autoantigen-specific effector T cells. This study describes isolation and expansion of insulin-specific Treg in vitro. Insulin-specific Treg are uniformly distributed in lymphoid tissues however their number is extremely low. To enrich the proportion of insulin-specific Treg, pure CD4+ cells were co-cultured with insulin B chain peptide-loaded dendritic cells, isolated from mice that develop T1D spontaneously – NOD mice. Insulin-specific CD4+ cell expansion peaked after 48 h of incubation and was in favour of Treg. These cells were then sorted using insulin peptide-loaded MHC class II tetramers and cultured in vitro for 48 h in the presence of TCR stimulators, TGF-β and IL-2. The proportion of gained insulin-specific cells with T regulatory phenotype (CD4+CD25highCD127lowGITR+FoxP3+)was in average between 93% and 97%. These cells have shown potent in vitro suppressive effect on T effector cells, produced IL-10 and TGF-β and expressed PD-1 and CD39. Further proliferation of these insulin-specific Treg required the presence of dendritic cells, anti-CD3 antibody and IL-2. This study provides new, reproducible experimental design for the enrichment and expansion of insulin-specific Treg that can be used for the cell-based therapy of autoimmunity. © 2019 Elsevier B.V.ex vivo protocolExpansionImmunoregulationInsulin-specific T regulatory cells (Treg)Type 1 diabetes (T1D)