Generation and Accumulation of Immunosuppressive Adenosine by Human CD4+CD25highFOXP3+ Regulatory T Cells

Magis Mandapathil,Benedict Hilldorfer,Miroslaw J Szczepanski,Malgorzata Czystowska,Marta Szajnik,Jin Ren,Stephan Lang,Edwin K Jackson,Elieser Gorelik,Theresa L Whiteside

doi:10.1074/jbc.m109.047423

Magis Mandapathil, Benedict Hilldorfer + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m109.047423

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Abstract

Naturally occurring regulatory T cells (nTreg) are crucial for maintaining tolerance to self and thus preventing autoimmune diseases and allograft rejections. In cancer, Treg down-regulate antitumor responses by several distinct mechanisms. This study analyzes the role the adenosinergic pathway plays in suppressive activities of human nTreg. Human CD4(+)CD25(high)FOXP3(+) Treg overexpress CD39 and CD73, ectonucleotidases sequentially converting ATP into AMP and adenosine, which then binds to A(2a) receptors on effector T cells, suppressing their functions. CD4(+)CD39(+) and CD4(+)CD25(high) T cells express low levels of adenosine deaminase (ADA), the enzyme responsible for adenosine breakdown, and of CD26, a surface-bound glycoprotein associated with ADA. In contrast, T effector cells are enriched in CD26/ADA but express low levels of CD39 and CD73. Inhibitors of ectonucleotidase activity (e.g. ARL67156) and antagonists of the A(2a) receptor (e.g. ZM241385) blocked Treg-mediated immunosuppression. The inhibition of ADA activity on effector T cells enhanced Treg-mediated immunosuppression. Thus, human nTreg characterized by the presence of CD39 and the low expression of CD26/ADA are responsible for the generation of adenosine, which plays a major role in Treg-mediated immunosuppression. The data suggest that the adenosinergic pathway represents a potential therapeutic target for regulation of immunosuppression in a broad variety of human diseases.

Highlights

CD4ϩ T Effector Cells—Lymphocytes in PBMCs were identified based on their forward scatter (FS) and sideward scatter (SS) characteristics (Fig. 1A)
We conclusively showed that in humans, adenosine is a byproduct of Treg activity, which is used by used these cells isolated by AutoMACs to confirm that adeno- these cells to suppress immune responses
Treg-derived adenosine produced during ATP cleavage by the associated ecto- sine is a hydrolysis product of ATP cleaved in tandem by two nucleotidases is responsible for suppression of responder cells (RCs)

Summary

EXPERIMENTAL PROCEDURES

Healthy Volunteers—Peripheral venous blood samples for phenotypic and functional analyses of lymphocyte subsets were obtained from 15 normal controls. Cells were treated with a serum-free protein block (Dako) for 1 h at room temperature, followed by washing with PBS and an overnight incubation at 4 °C in the dark with the primary Ab. The following Abs were used: unconjugated anti-human CD73 antibody (1:100 dilution, Santa Cruz Biotechnology); rabbit anti-human CD26 Ab; mouse antihuman ADA or appropriate isotype controls. Suppression Assay—Single cell-sorted CD4ϩCD39ϩ, CD4ϩCD26neg or CD4ϩCD25high, and CD4ϩCD39neg or CD4ϩCD26ϩ cells were tested for suppressor activity in a co-culture with CD4ϩCD25neg responder cells (RCs) To evaluate their proliferation, RCs separated by single cell sorting were stained with 1.5 ␮M CFSE (Molecular Probes/Invitrogen) and incubated at 37 °C for 15 min. ATP Hydrolysis Assay—MACS-sorted CD4ϩCD25neg or CD4ϩCD25ϩ cells (25 ϫ 103/well) were incubated in wells of flat-bottom 96-well plates for 30 min with various concentrations of exogenous ATP (10 ␮M, 25 ␮M, and 50 ␮M, SigmaAldrich).

RESULTS

Effects of an Adenosine Receptor

DISCUSSION