Experimental Hematology

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Date of download: 5/31/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Toll-like Receptors in Regulatory T Cells of Patients.

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Experimental Hematology Expression of CD25 on leukemic stem cells in BCR-ABL1+ CML: Potential diagnostic value and functional implications Irina Sadovnik, Harald Herrmann, Gregor Eisenwort, Katharina Blatt, Gregor Hoermann, Niklas Mueller, Wolfgang R. Sperr, Peter Valent Experimental Hematology Volume 51, Pages 17-24 (July 2017) DOI: 10.1016/j.exphem.2017.04.003 Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 1 Expression of CD25 on CD34+/CD38− LSCs in patients with CML and on KU812 cells. (A) Primary CML cells were stained with fluorochrome-labeled monoclonal antibodies against CD34, CD38, CD45, CD25, CD122, and CD132. LSCs were defined as CD45+/CD34+/CD38− cells. As assessed by multicolor flow cytometry, LSCs were found to co-express CD25 (left panel), but did not express CD122 (middle panel) or CD132 (right panel) (red histograms). The isotype-matched control antibody is also shown (black open histograms). (B) The Ph+ cell line KU812 was stained with antibodies against CD25, CD122, and CD132 (blue histograms). It can be seen that KU812 cells also express CD25 and do not express substantial amounts of CD122 or CD132. Experimental Hematology 2017 51, 17-24DOI: (10.1016/j.exphem.2017.04.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 2 Effects of BCR-ABL1-targeting TKIs on CD25 expression on CD34+/CD38− CML LSCs. CML MNCs were incubated in control medium or TKIs (left histogram: imatinib, middle histogram: nilotinib, right histogram: ponatinib; each 1 μmol/L) at 37°C for 24 hours. Thereafter, CD25 expression on CD34+/CD38− LSCs was analyzed by multicolor flow cytometry. Black open histograms represent the matched isotype control; blue histograms represent CD25 expression on CML LSCs kept in control medium; and red histograms represent expression of CD25 on CML LSCs after treatment with TKI. As illustrated, the expression of CD25 on CML LSCs is not altered by imatinib, but decreases on exposure to nilotinib or ponatinib. MNCs = Mononuclear cells. Experimental Hematology 2017 51, 17-24DOI: (10.1016/j.exphem.2017.04.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 3 CML basophils express higher levels of CD25 than do basophils obtained from healthy donors. Expression of CD25 on CD123++/CD45+ basophils was analyzed by multicolor flow cytometry using fluorochrome-labeled antibodies against CD45, CD123, and CD25. Cells were obtained from five patients with CP CML (left red bar) and five healthy controls (right blue bar). Expression of CD25 was determined as the MFI and is expressed as the SI according to the following formula: SI = MFI test antibody: MFI isotype-control antibody. *p < 0.05 compared with CD25 expression on normal basophils. MFI = Median fluorescence intensity; SI = staining index. Experimental Hematology 2017 51, 17-24DOI: (10.1016/j.exphem.2017.04.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 4 Evaluation of CD25 as a functional target on CML cells. CD25 shRNA-transduced cells were mixed 1:1 with KU812 cells transduced with a RDM shRNA and cultured at 37°C for 14 days. Expression of CD25+ cells was analyzed three times a week by flow cytometry using a phycoerythrin-conjugated antibody against CD25. Results are expressed as the percentage of CD25+ cells and represent the mean ± standard deviation from three independent experiments. *p < 0.05 compared with the percentage of CD25+ cells measured on day 0. RDM = Random. Experimental Hematology 2017 51, 17-24DOI: (10.1016/j.exphem.2017.04.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 5 The PI3K/mTOR blocker BEZ235 synergizes with BCR-ABL1-targeting TKIs in inhibiting the proliferation of KU812 cells. KU812 cells were incubated in control medium, medium supplemented with BEZ235 (90 nmol/L), or medium containing BCR-ABL1-targeting drugs (imatinib, 108 nmol/L; nilotinib, 6.3 nmol/L; ponatinib, 0.27 nmol/L) alone or in combination at a fixed ratio of drug concentrations at 37°C for 48 hours (BEZ235 + imatinib, left panel; BEZ235 + nilotinib, middle panel; BEZ235 + ponatinib, right panel). After incubation, 3H-thymidine uptake was measured. Results are expressed as a percentage of control (100%), and represent the mean ± standard deviation from triplicates. Experimental Hematology 2017 51, 17-24DOI: (10.1016/j.exphem.2017.04.003) Copyright © 2017 ISEH - International Society for Experimental Hematology Terms and Conditions