Phenotypic and functional characterisation of expanded antigen-specific regulatory T cells (Ag-sp Treg), towards clinical translation

Abstract

Abstract Treg infusion for graft-versus-host-disease treatment has been increasingly investigated. Yet, Treg of unknown specificity may suppress graft-versus-leukemia. To ascertain the factors affecting Ag-sp Treg potency and specificity of suppression, we assessed Ag-sp Treg phenotype and function within different expansion conditions and suppression assay (SA) milieus. Ex vivo Treg were co-cultured with monocyte-derived dendritic cells (DC) presenting allogeneic Ag directly (allo-DC) or indirectly (self-DC loaded with allo-lysate). Suppression of effector proliferation and cytokine secretion was assessed in SA with the same DC as in expansion (original) or 3rd party DC (3P) from various donors. Ag-sp Treg significantly suppressed Tcon and CD8+ T cell responses to original DC. Directly expanded cells suppressed responses to Ag presented direct and indirectly; however, indirectly expanded cells were less suppressive of responses to allo-DC. More potent Treg were less specific in their suppression of Tcon. Still, responses to 3P donors with MHC matches to original DC donor were significantly more suppressed than responses to unmatched 3P donors. Potency of suppression was also negatively correlated to expression of CD86 and CD83 in DC. Finally, FlowSOM analysis showed minimal overlap between Ag-sp Treg and activated Tcon phenotypes, suggesting Ag-sp Treg were pure. K-means clustering of Ag-sp Treg expression of CD137, CD154, CTLA-4, PD-1, CTLA-4 and HLA-DR identified clusters that may be correlated to Treg function. We propose the specificity of Ag-sp Treg function in the inflammatory milieu is a result of Treg phenotype, type of responders, DC co-stimulation and degree of MHC match between expansion DC and DC present in the milieu.