Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15.
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(+)CD25(bright)CD127(-) 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.
Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands; n.litjens@erasmusmc.nl.
Nicolle H R
NH
Litjens
Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands;
Karin
K
Boer
Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands;
Joke M
JM
Zuijderwijk
Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands;
Mariska
M
Klepper
Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands;
Annemiek M A
AM
Peeters
Department of Dermatology, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands; Department of Rheumatology, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands; and.
Errol P
EP
Prens
Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands;
Wenda
W
Verschoor
Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands;
Rens
R
Kraaijeveld
Erasmus Medical Center, Erasmus Center for Biomics, 3000 CA Rotterdam, the Netherlands.
Zeliha
Z
Ozgur
Erasmus Medical Center, Erasmus Center for Biomics, 3000 CA Rotterdam, the Netherlands.
Mirjam C
MC
van den Hout-van Vroonhoven
Erasmus Medical Center, Erasmus Center for Biomics, 3000 CA Rotterdam, the Netherlands.
Wilfred F J
WF
van IJcken
Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands;
Carla C
CC
Baan
Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands;
Michiel G H
MG
Betjes
D019264:Adoptive Transfer; D018122:B7-1 Antigen / Q000378:metabolism; D051940:B7-2 Antigen / Q000378:metabolism; D001483:Base Sequence; D015704:CD4 Antigens / Q000378:metabolism; D060908:CTLA-4 Antigen / Q000096:biosynthesis / Q000235:genetics; D002454:Cell Differentiation / Q000276:immunology; D049109:Cell Proliferation; D002478:Cells, Cultured; D019175:DNA Methylation; D003713:Dendritic Cells / Q000187:drug effects* / Q000276:immunology; D051858:Forkhead Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D006684:HLA-DR Antigens / Q000276:immunology; D006801:Humans; D051740:Ikaros Transcription Factor / Q000096:biosynthesis / Q000235:genetics; D007371:Interferon-gamma / Q000096:biosynthesis; D016753:Interleukin-10 / Q000096:biosynthesis; D019409:Interleukin-15 / Q000276:immunology / Q000494:pharmacology*; D020381:Interleukin-17 / Q000096:biosynthesis; D007376:Interleukin-2 / Q000096:biosynthesis / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D053699:Interleukin-7 Receptor alpha Subunit / Q000378:metabolism; D017423:Sequence Analysis, RNA; D012867:Skin / Q000166:cytology / Q000276:immunology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D018122:B7-1 Antigen; D051940:B7-2 Antigen; D015704:CD4 Antigens; C497964:CD86 protein, human; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D006684:HLA-DR Antigens; C111431:IKZF2 protein, human; C508609:IL10 protein, human; C569274:IL15 protein, human; C508594:IL2 protein, human; C508017:IL2RA protein, human; D019409:Interleukin-15; D020381:Interleukin-17; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D051740:Ikaros Transcription Factor; D007371:Interferon-gamma