Figure 1. Scheme of flow analysis (representative 7-day experiment shown).

5x10⁵ CFSE labeled responding PBMC from healthy volunteer A were cultured with 5x10⁵ PKH26 labeled irradiated stimulator cells from laboratory volunteer B in the absence or presence of indicated concentrations of BEL. After 7 days, flow cytometric analyses were performed using monoclonal antibodies CD127-PE, CD4-ECD, CD25-PC7 and FOXP3-PC5. Viable lymphocytes were gated (column A) followed by CFSE bright and dim cells which were negative for either CD127-PE or PKH26 (column B), thus gating out CD127⁺ responders and any residual stimulators. This was followed by gating for CD4⁺ cells that were either non-proliferating (CFSE high) or proliferating (CFSE low) (column C). The cells in the non-proliferating (Column D) and proliferating (Column E) populations were analyzed by dot plots for CD25⁺ and FOXP3⁺ cells (among other subsets; not shown). Please note that when compared to the medium control (top row), fewer CD4⁺ cells proliferated in presence of BEL (column C). Additionally, there was a dose dependent reduction in the proportion of both total CD25⁺FOXP3⁺ Tregs and CD25^highFOXP3⁺Tregs in the proliferating CD4⁺CD127⁻ responder cells (column E). Since only the CFSE diluted proliferating fraction (as opposed to non-proliferating fraction; column D) demonstrated differences under various culture conditions, the results from only these are shown in subsequent experiments.