Figure 1.

Induction of CD4⁺CD25⁺Foxp3⁺IFNγ⁺and CD4⁺CD25⁺CD127^-IFNγ⁺PBL and cell proliferation in the presence of PMA/Ionomycin and monoclonal antibodies against cell surface molecules. (a, b) PBL of 5 healthy control individuals (HC1-HC5) were incubated in medium or stimulated with PMA/Ionomycin for 16 h in the presence of monoclonal antibodies against CD178, CD152, CD279, CD28, CD95, and HLA-DR in final concentrations of 0.1 μg/ml and 1 μg/ml. The assay was measured using four-color fluorescence flow cytometry. Compared to PMA-Ionomycin-stimulated cell cultures without monoclonal antibody, addition of anti-CD178, anti-CD152, anti-CD279, anti-CD95, and anti-HLA-DR monoclonal antibody blocked induction of CD4⁺CD25⁺Foxp3⁺IFNγ⁺ PBL (p < 0.05) but not induction of CD4⁺CD25⁺CD127^-IFNγ⁺ PBL (p = n.s.). When cell cultures with a 1-log difference in antibody concentration were compared, higher concentrations of anti-CD152, anti-CD279, and anti-95 monoclonal antibody blocked induction of CD4⁺CD25⁺Foxp3⁺IFNγ⁺ PBL (p < 0.05) whereas higher anti-CD178, anti-CD152, anti-CD279, and anti-CD95 concentrations blocked induction of CD4⁺CD25⁺CD127^-IFNγ⁺ PBL (p < 0.05) compared to lower concentrations of these antibodies in the cell cultures. (c) PBL of 5 healthy control individuals (HC1-HC5) were incubated in medium or stimulated with PHA for 3 days in the presence of monoclonal antibodies against CD178, CD152, CD279, CD28, CD95, and HLA-DR in final concentrations of 0.1 μg/ml and 1 μg/ml. The proportion of CFSE^low PBL was determined using flow cytometry. With the exception of anti-CD28, all monoclonal antibodies reduced cell proliferation (all p < 0.05) and cell proliferation tend to normalize in the presence of high antibody concentrations, especially of anti-CD178 and anti-CD279 (p < 0.05). In the figure, statistical significant differences of assays with vs without monoclonal antibodies are indicated by lines, whereas statistical significant differences of assays with low vs high monoclonal antibody concentrations are indicated by arrows. Data are given as mean±SD. *p < 0.05.