Generation of BTNL8+ Inducible Tregs By Allogeneic Human Regulatory Macrophages Is IDO- and B7-Dependent.

Abstract

Purpose: This study identifies the phenotypic and functional changes that human T cells undergo when exposed to regulatory macrophages. Methods: Human regulatory macrophages (M regs) represent a unique and stable state of macrophage activation, characterized by their ability to suppress activated T cells. M regs arise from CD14+ monocytes over a 6-day culture period in the presence of M-CSF, human serum, and a final 24-hour pulse of IFN-γ. In preclinical models, pre-transplant administration of donor-derived M regs prolonged allograft survival. This graft-protective effect initially depended upon metabolically-competent cells, but persisted beyond the lifespan of the M regs, implying regulation of recipient T cells by M regs. Here, mechanisms by which human M regs influence allogeneic T cell responses in vitro are examined. Results: Phenotypic and functional changes imposed by human M regs on allogeneic T cells were investigated by flow cytometry, whole-genome gene expression profiling, and secondary suppression assays. After exposure to M regs, CD4+ T cells were induced to express FoxP3, CD25, CTLA4, OX40, GITR, ICOS, EBI3, and BTNL8. Such FoxP3+ T cells generated in M reg cocultures suppressed polyclonal T cell proliferation and inhibited TNFα-induced dendritic cell maturation. The Treg-specific demethylated region (TSDR) of the FOXP3 gene in M reg-cocultured CD4+ T cells was 5% demethylated. The M reg-induced regulatory T cell (Treg) phenotype was not alloantigen-dependent, but was contact-dependent, and could be abolished by B7 blockade with CTLA4-Ig or by indoleamine 2,3-dioxygenase (IDO) inhibition with 1 mM 1-L-methyltryptophan. Tacrolimus and rapamycin did not affect FoxP3+ T cell development. Conclusions: M regs drive an antigen non-specific, B7- and contact-dependent induction of activated induced Tregs through a mechanism partially dependent on IDO. This finding supports the concept that pre-transplant treatment of recipients with donor-derived M regs engenders a pro-tolerogenic state and hints at a feedforward mechanism by which it could be perpetuated. Clinical applications of M reg immunosuppressive therapy are currently being pursued within The ONE Study framework.