Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15

Abstract

Expansion of Ag-specific naturally occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloantigen-specific nTregs. A highly enriched nTreg fraction (CD4+CD25brightCD127− T cells) was alloantigen-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDCs), or PBMCs. The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the Treg-specific demethylation region of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4, and cytokines. In addition, the Ag-specific suppressive capacity of these expanded nTregs was tested. Allogeneic mature moDCs and skin-derived DCs were superior in inducing nTreg expansion compared with immature moDCs or PBMCs in an HLA-DR– and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2 could facilitate optimal mature moDC-induced nTreg expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloantigen-induced proliferation without significant suppression of completely HLA-mismatched, Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the Treg-specific demethylation region within the FOXP3 gene and highly expressed of FOXP3, HELIOS, and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-γ, and TNF-α, but few IL-17–producing nTregs were found. Next-generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Human allogeneic mature moDCs are highly efficient stimulator cells, in the presence of exogenous IL-15, for expansion of stable alloantigen-specific nTregs with superior suppressive function.

Deep sequencing of the TCR-β repertoire of human forkhead box protein 3 (FoxP3)+ and FoxP3– T cells suggests that they are completely distinct and non-overlapping

“…Further research to identify the unique thymic compartments of tT reg versus T conv development will improve our understanding of tolerance. Furthermore, as has been observed in limited studies thus far , distinct monocyte‐derived dendritic cells may have the potential to stimulate preferentially and expand a pool of tT regs (Helios + ; TSDR demethylated), resulting in tolerance to a specific autoantigen without relying upon the conversion of antigen‐specific T conv cells into pT regs .…”

Transcriptomic profiling of bovine blood dendritic cells and monocytes following TLR stimulation

“…Similar to IL‐12 and IL‐23, IL‐15 is also one of the cytokines that promote the development and maintenance of effector T cells [81‐83], and Tregs [84], and it was reported to enhance immune responses of natural killer (NK) cells and macrophages [85]. Moreover, IL‐15 expression in murine macrophages [83] and human DCs [86] was shown to increase upon treatment with IFN‐α/β in vitro.…”

Ex vivo IL-15 replenishment augments bone marrow precursor cell-mediated adaptive immunity via PI3K-Akt pathway

“…In that condition, IL‐15 DCs differentiated by GM‐CSF and IL‐15, exhibited low antigen uptake and high capacity of T cell priming 40 . As DCs with weak antigen uptake promote regulator T cell priming, 41 it was no surprise to find that monocyte‐derived DCs and skin‐derived DCs were superior in inducing natural regulatory T cell expansion 42 . In this study, we replenished IL‐15 in the middle period of BMPC differentiation and explored the phenotype and function of IL‐15 conditioned BMPC.…”