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.
Abstract In this report we show that it is possible to induce the expression of HLA‐DR antigens on K562 cells, previously reported to be unresponsive to interferon‐gamma (IFN‐γ). However, only low cell concentrations and a high dose of IFN‐γ allowed the induction of HLA‐DR antigens. Furthermore, the recombinant glycosylated IFN‐γ is 100‐fold more efficient than the unglycosylated form. This induction of HLA‐DR antigens on K562 was not related to a stage of differentiation or to the presence of cells subsets specifically sensitive to IFN‐γ, since repeated sorting of K562 HLA‐DR‐positive and negative cells did not lead to the selection of a cell subset with a different potential of induction for HLA‐DR. The difficulty in obtaining induction is due to the production of a soluble endogeneous inhibitor of proteic nature, whose action is not restricted to the K562 cell line since it operates also on both epithelial and fibroblastic cells. Treatment of normal human epithelial and fibroblastic cells with conditioned medium from K562 cultures caused a marked decrease in the expression of HLA class II antigens (DR and DP) induced by IFN‐γ (10000 U/ml), but had no effect on cell growth; however, it also affected expression of HLA class I antigens. This inhibition is not mediated by prostaglandin or an IFN‐α or IFN‐β‐dependent mechanism. Production of this inhibitor by pluripotent human leukemic cells could cause an unbalance in the complex control exerted by the immunological system during hematopoietic differentiation or leukemic progression.
Drug discovery often requires the screening of compound libraries on tissue cultured cells. Some major targets in drug discovery belong to signal transduction pathways, and PerFix EXPOSE* allows easy flow cytometry phospho assays. We thus investigated the possibility to further simplify and automate this assay, to allow the direct screening of drugs targeting signaling pathways. We show here the sensitivity of this fully automated assay on human growth hormone (hGH)-driven JAK/STAT5-activated IM-9 cells, and we discuss the throughput of this system, which is compatible with medium-throughput drug screening. Because the kit works directly on whole blood samples, ex-vivo assays are also possible with this approach, which could allow for the screening of drugs under more physiological conditions.
Growth hormone (GH) and insulin‐like growth factor‐1 (IGF1) play an important role in mammalian development, cell proliferation and lifespan. Especially in cases of tumor growth there is an urgent need to control the GH/IGF1 axis. In this study we screened a 38,480-compound library, and in two consecutive rounds of analogues selection, we identified active lead compounds based on the following criteria: inhibition the GH receptor (GHR) activity and its downstream effectors Jak2 and STAT5, and inhibition of growth of breast and colon cancer cells. The most active small molecule (BM001) inhibited both the GH/IGF1 axis and cell proliferation with an IC50 of 10‐30 nM of human cancer cells. BM001 depleted GHR in human lymphoblasts. In preclinical xenografted experiments, BM001 showed a strong decrease in tumor volume in mice transplanted with MDA‐MB‐231 breast cancer cells. Mechanistically, the drug acts on the synthesis of the GHR. Our findings open the possibility to inhibit the GH/IGF1 axis with a small molecule.
