In-vitro inhibition of IFNγ⁺ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

• Main Authors: Daniel Volker, Sadeghi Mahmoud, Wang Haihao, Opelz Gerhard
• Format: Article
• Language: English
• Published: BMC 2012-08-01
• Series: BMC Immunology
• Subjects: IFNγ⁺ iTreg; IFNγ⁺Foxp3⁺; IFNγ⁺CD127^-; CD178; CD152; CD279; CD28; CD95; HLA-DR; Inhibition; Cell proliferation
• Online Access: http://www.biomedcentral.com/1471-2172/13/47
• ISSN: 1471-2172

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>CD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ⁺ iTreg.</p>