In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function.
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.
Department of Transplantation-Immunology, Institute of Immunology, University of Heidelberg, Germany. Volker.Daniel@med.uni-heidelberg.de
Volker
V
Daniel
Mahmoud
M
Sadeghi
Haihao
H
Wang
Gerhard
G
Opelz
D019138:Antibodies, Blocking / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000954:Antigens, Surface / Q000276:immunology; D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006684:HLA-DR Antigens / Q000276:immunology; D006801:Humans; D007165:Immunosuppression Therapy; D007371:Interferon-gamma / Q000276:immunology*; D008213:Lymphocyte Activation; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*
D019138:Antibodies, Blocking; D015703:Antigens, CD; D000954:Antigens, Surface; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D006684:HLA-DR Antigens; D007371:Interferon-gamma