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Logo of bmcimmBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Immunology
 
BMC Immunol. 2012; 13: 47.
Published online Aug 21, 2012. doi:  10.1186/1471-2172-13-47
PMCID: PMC3482559
In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function
Volker Daniel,corresponding author1 Mahmoud Sadeghi,1 Haihao Wang,1,2 and Gerhard Opelz1
1Department of Transplantation-Immunology, Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, Heidelberg, 69120, Germany
2Institute of Organ Transplantation, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, 430030, China
corresponding authorCorresponding author.
Volker Daniel: Volker.Daniel/at/med.uni-heidelberg.de; Mahmoud Sadeghi: Mahmoud.Sadeghi/at/med.uni-heidelberg.de; Haihao Wang: Haihao.Wang/at/med.uni-heidelberg.de; Gerhard Opelz: Gerhard.Opelz/at/med.uni-heidelberg.de
Received February 9, 2012; Accepted August 16, 2012.
Abstract
Background
IFNγ-producing CD4+CD25+Foxp3+ PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins.
Methods
PBL of healthy control individuals were stimulated polyclonally in-vitro in the presence of monoclonal antibodies or recombinant proteins against/of CD178, CD152, CD279, CD28, CD95, and HLA-DR. Induction of IFNγ+ iTreg and proliferation of effector cells was determined using four-color fluorescence flow cytometry. Blockade of iTreg function was analyzed using polyclonally stimulated co-cultures with separated CD4+CD25+CD127-IFNγ+ PBL.
Results
High monoclonal antibody concentrations inhibited the induction of CD4+CD25+Foxp3+IFNγ+ PBL (anti-CD152, anti-CD279, anti-CD95: p < 0.05) and CD4+CD25+CD127-IFNγ+ PBL (anti-CD178, anti-CD152, anti-CD279, anti-CD95: p < 0.05). Effector cell proliferation increased with increasing antibody concentrations in culture medium (anti-CD178 and anti-CD279: p < 0.05). Conversely, high concentrations of recombinant proteins induced formation of CD4+CD25+Foxp3+IFNγ+ PBL (rCD152 and rCD95: p < 0.05) and decreased cell proliferation dose-dependently (rCD178 and rCD95: p < 0.05). Our data suggest an inverse association of iTreg induction with effector cell proliferation in cell culture which is dependent on the concentration of monoclonal antibodies against iTreg surface determinants. 3-day co-cultures of polyclonally stimulated PBL with separated CD4+CD25+CD127-IFNγ+ PBL showed lower cell proliferation than co-cultures with CD4+CD25+CD127-IFNγ- PBL (p < 0.05). Cell proliferation increased strongly in CD4+CD25+CD127-IFNγ- PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p < 0.05) but remained low in co-cultures with CD4+CD25+CD127-IFNγ+ PBL (with the exception anti-CD28 monoclonal antibody: p < 0.05). Monoclonal antibodies prevent iTreg induction in co-cultures with CD4+CD25+CD127-IFNγ- PBL but do not efficiently block suppressive iTreg function in co-cultures with CD4+CD25+CD127-IFNγ+ PBL.
Conclusions
CD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ+ iTreg.
Keywords: IFNγ+ iTreg, IFNγ+Foxp3+, IFNγ+CD127-, CD178, CD152, CD279, CD28, CD95, HLA-DR, Inhibition, Cell proliferation
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