NY-ESO-1-Specific Circulating CD4+ T Cells in Ovarian Cancer Patients Are Prevalently TH1 Type Cells Undetectable in the CD25+FOXP3+Treg Compartment
Figure 5
Assessment of the suppressive activity of ESO-specific CD4+ T-cell polyclonal populations.
ESO-specific cells were isolated from peptide-stimulated CD25−CD127+ and CD25−CD127− CD4+ T-cell cultures (Figure 4) by tetramer-guided (NA017) or IFN-γ-guided (NA114) flow cytometry cell sorting and expanded in vitro. The resulting polyclonal cultures contained >80% ESO-specific cells. A. ESO-specific polyclonal cultures as well as polyclonal cultures from the ESO− fraction and control cultures of in vitro-expanded conventional memory CD4+ T cells (M) and memory Treg (MTreg), isolated ex vivo from healthy individuals, were stained with FOXP3-specific mAb and analyzed by flow cytometry. Numbers in dot plots correspond to the mean fluorescence intensity (MFI) of FOXP3 staining. B. The suppressive activity of ESO-specific and control polyclonal populations was assessed by co-culture with CFSE-labeled conventional CD4+ T cells, at a responder:suppressor ratio of 1∶1, in the presence of irradiated monocytes and PHA. Dot plots show the CFSE-dilution profile in the absence of test population (left) and in the presence of the indicated test populations. Numbers in histograms correspond the percentage of undivided cells. Results corresponding to the calculated% suppression are shown for all tested populations.