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Bone marrow niche dysfunction in ET
the MSC cultures. We thereby observed that ET MSC secreted less IL-6, leptin, and GM-CSF than HD MSC (Figure 3C).
BM-MSC from patients with JAK2V617F-positive ET show an impaired immunomodulatory capacity
CD4-positive T cells from patients with JAK2V617F- positive ET showed enhanced proliferation and activation compared to normal controls (Figure 4A-B). No changes were observed in the levels of GATA3, T-bet, ROR-γt, or FOXP3 (Figure 4C). Notably, Th2 cell counts were signifi- cantly decreased in patients with JAK2V617F-positive ET (Figure 4D). The level of the anti-inflammatory factor IL-4 was lower in the patient samples, while pro-inflammatory factors, such as IL-1β, and sCD40L, were upregulated. No difference was observed in IL-6 levels between the patient and healthy samples (Figure 4E).
We next evaluated whether MSC play a role in the immune disorder mentioned above. We observed that CD4-positive T-cell proliferation and activation were remarkably enhanced, and Th2 cell counts were signifi- cantly lower when cocultured with ET MSC relative to those observed with HD MSC (Figure 4 F-H). Additionally, IL-4 was downregulated and sCD40L was upregulated with the ET MSC coculture (Figure 4I).
Downregulation of WDR4 is correlated with enhanced proliferation, decreased senescence, and impaired differentiation in BM-MSC of patients with JAK2V617F-positive ET
WDR4 was one of the differentially expressed genes according to our RNA sequencing results and was recently reported to regulate the immunosuppressive microenvi- ronment of solid tumors.25 Downregulation of WDR4
A
B
C
Figure 3. Bone marrow derived mesenchymal stromal cells (BM-MSC) from patients with JAK2V617F-positive essential thrombocythemia (ET) show insufficient ability to support normal hematopoiesis. A. Representative micrographs of CFU formed by purified normal CD34-positive cells in the presence of BM-MSC from HD (n=12) and patients with JAK2V617F-positive ET (n=16). B. Numbers of BFU-E, CFU-E, CFU-GM, CFU-Mix, CFU-Total, and CFU-MK formed by purified normal CD34- positive cells after coculture with BM-MSC from HD (n=12) or from patients with JAK2V617F-positive ET (n=16) for 7 or 14 days. After 7 or 14 days of coculture of purified normal CD34-positive cells and MSC from patients with JAK2V617F-positive ET, the numbers of CFU-GM and CFU-Total were significantly lower, with no sig- nificant changes in the numbers of BFU-E, CFU-E, CFU-Mix, and CFU-MK. C. Cytokines secreted by BM-MSC into the culture medium analyzed by ELISA (control, n=16; JAK2V617F-positive ET, n=16). MSC used in each assay were at passage four. *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001. Data are presented as the mean ± SEM. ET: essential thrombocythemia; HD: healthy donors; ELISA: enzyme-linked immunosorbent assay; n: number of unique donors in each group; ns: not signif- icant; BFU-E: burst-forming unit-erythroid; CFU-E: colony-forming unit-erythroid; CFU-GM: colony-forming unit-granulocyte and macrophage; CFU-Mix: colony-forming units mixed; CFU-Total: total colony-forming units; CFU-MK: colony-forming unit-megakaryocyte; SEM: standard error of mean.
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