Additional file 2 of Peripheral blood CD4posCD25posFoxP3pos cells and inflammatory cytokines as biomarkers of response in rheumatoid arthritis patients treated with CTLA4-Ig

Additional file 2: Supplementary Fig. 1. Flow cytometry gating scheme to identify Th17/Treg subpopulations in human CD4pos cells from peripheral blood mononuclear cells of RA patients. Single-cell suspension from RA patients were stained with a combination of 6 antibodies as described in Materials and methods. After gating the singlets, lymphocytes were identified based on their side-scatter properties and expression of CD45. CD4 was used to identify CD4pos T cells among the previously selected lymphocytes. Subsequently, Tregs were analyzed on CD4 according to the commonly used Treg definitions: CD4posCD25posCD127low (Treg1) and CD4posCD25posFoxP3high (Treg2). Moreover, IL17-A expression was measured on CD4pos cells (Th17). All stained cells were acquired on a Navios flow-cytometer (Beckman Coulter, Marseille France) and data were analysed using Kaluza Software (Beckman Coulter, Marseille France). Percentage of Treg1, Treg2 and Th17 cells is given as percentage within the CD4pos population.