Isolation and enrichment of mouse insulin-specific CD4+ T regulatory cells.
Article (Published version)
© 2019 Elsevier B.V.
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Polyclonal 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.
Keywords:Expansion; Immunoregulation; Insulin-specific T regulatory cells (Treg); Type 1 diabetes (T1D); ex vivo protocol
Source:Journal of Immunological Methods, 2019, 470, 46-54
- Molecular mechanisms of physiological and pharmacological control of inflammation and cancer (RS-173013)
- The Iacocca Family Foundation, Boston, MA, USA