FOXP3+T-REGS CELLS SENSITIVITY TO LOW DOSES OF IL-2 CAN BE RAPIDLY AND EASILY MONITORED BY P-STAT-5 DETECTION IN EX VIVO WHOLE BLOOD SAMPLES

Abstract Introduction: Phospho-epitopes are difficult to detect by flow cytometry, currently requiring dedicated techniques and buffers, a total work time of 2 to 4 hours, 37°C and ice incubations, and 4 to 8 wash steps. The reference methods also uses methanol which is toxic for the user and harmful for the other cell markers, what compromises some multi-parametric studies. The FoxP3 marker is incompatible with “harsh” P-epitopes detection methods, rendering even more challenging the studies of Tregs functions. Methods: Here, we used a new commercially available kit; named PerFix EXPOSE (Phospho Epitopes Exposure Kit; for Research Use Only) based on multiple innovations in the permeabilization, staining, and wash steps. It is devoid of methanol and supports staining of all common P-epitopes with one single procedure of about 1 hour. Since many extra-cellular markers can be combined together with the anti-phospho markers, we evaluated also various FoxP3 clones and conjugates, and tried some procedure optimisations. The best conditions were then used to analyse the ex-vivo functionality of FoxP3+ cells: The IL-2 signalling pathway especially. Results: PerFix EXPOSE allows for the simultaneous detection of some antigens that are otherwise incompatible, such as FOXP3 and p-STATs. An IL-2 dose-response curve could be easily generated directly from fresh whole blood that confirmed on normal donors a 10 to 100-fold difference in sensitivity to IL-2 between Tregs and other T cells.