A TIM-3/Gal-9 autocrine stimulatory loop drives self-renewal of human myeloid leukemia stem cells and leukemic progression. to the advanced stage in 70 MDS/acute leukemia transformed from MDS patients and was a prognostic factor in 40 MDS patients. Our data demonstrated that the Tim-3-galectin-9 pathway is associated with the pathogenesis and disease progression of MDS. These findings provide new insight into potential immunotherapy targeting the galectin-9CTim-3 pathway in MDS. mRNA was expressed in all cell lines (Figure ?(Figure1C),1C), and the percentage of Tim-3+ cells was the highest in F-36P cells (Figure ?(Figure1D),1D), although Tim-3 protein expression in whole-cell lysis was detected in all cell lines by Western blotting (Figure ?(Figure1E1E). Open in a separate window Figure VU661013 1 Tim-3 expression in MDS patients and MDS cell lines(A) Cell surface expression of Tim-3 in BM cells from an MDS patient was analyzed by FCM. Granulocytes (a), monocytes (b), lymphocytes (c), blasts (d), and CD34+ blasts (e) were gated using the CD45/side-scatter and CD34/CD45 gating methods. Solid line, staining with antibody to cell surface antigen; filled area, staining with isotype-matched control Ig. The numerical values in the lower right of each histogram are represented by relative MFI. (B) Comparison of cell surface Tim-3 expression on blasts among those from VU661013 normal controls, patients with low-grade MDS (BM VU661013 blasts 5%), high-grade MDS (BM blasts 5C19%), and AL-MDS. Tim-3 mRNA (C) and protein (D) expression in MDS cell lines was determined by qPCR and FCM, respectively. The data are mean SD. (E) Western blot analysis of Tim-3 and -actin in MDS cell lines. Numbers under the Tim-3 band indicate the relative intensity of Tim-3 normalized to the signal intensity of -actin. Tim-3, 45 kDa; -actin, 42 kDa. Tim-3 induction in the BM microenvironment To investigate whether Tim-3 expression on blasts could be induced by soluble factors in the BM microenvironment of MDS, we evaluated its expression on MDS cells cultured in complete medium containing culture supernatant of the human BM stromal cell line HS-5 (HS-5 sup.) or MDS-associated cytokines. Tim-3 expression was increased by HS-5 sup. in the F-36P and MDS-L cell lines (Figure ?(Figure2A).2A). To identify the intracellular signaling pathway of Tim-3 induction by HS-5 sup., we evaluated mRNA expression in F-36P cells by HS-5 sup. in addition to various Mouse monoclonal to CD16.COC16 reacts with human CD16, a 50-65 kDa Fcg receptor IIIa (FcgRIII), expressed on NK cells, monocytes/macrophages and granulocytes. It is a human NK cell associated antigen. CD16 is a low affinity receptor for IgG which functions in phagocytosis and ADCC, as well as in signal transduction and NK cell activation. The CD16 blocks the binding of soluble immune complexes to granulocytes signal transduction inhibitors. The increases in mRNA and cell surface expression induced by HS-5 sup. in F-36P cells were inhibited by the MEK 1/2 inhibitor U0126 (Figure ?(Figure2B2B and ?and2C2C). Open in a separate window Figure 2 Upregulated Tim-3 expression in MDS cell lines(A) MDS cell lines were cultured with or without HS-5 sup. for 48 h. The cells were pretreated with signal transduction inhibitors of STAT3, MEK1/2, JAK2, Akt/PI3K, and NF-B for 2 h and then cultured in complete medium containing VU661013 HS-5 sup., after which Tim-3 mRNA expression (B) and protein were analyzed (C). The concentrations of each inhibitor were 500 nM of STAT3 inhibitor V, 20 M of U0126 (MEK1/2 inhibitor), 25 M of AG490 (JAK2 inhibitor), 25 M of LY294002 (PI3K/AKT signaling inhibitor), and 5 nM of PDTC (NF-B inhibitor). (D) F-36P cells were cultured with the following cytokines for 2 days: 5 ng/ml of IL-8, 5 ng/ml of IL-6, 100 pg/ml of G-CSF, 10 ng/ml of GM-CSF, 10 ng/ml of MIP-1, 10 ng/ml of IL-10, 10 ng/ml of VEGF, 10 ng/ml of IL-1, and 2.5 ng/ml of TGF-1. (E, F) MDS cell lines were cultured with 2.5 ng/ml of TGF-1 for 48 h. (GCJ) F-36P cells were pretreated with SD208, a selective inhibitor of TGF-RI kinase, at optimal concentrations for 2 h, followed by incubation with 2.5 ng/ml of TGF-1 (G, H) or HS-5 sup. (I, J) for 24C48 h. After incubation with HS-5 sup. or TGF-1, the cell surface (A, C, F, H, J) and mRNA (B, D, E, G, I) expression of Tim-3 was analyzed by FCM and real-time qPCR, respectively. Data represent mean SD. *P 0.05, **P 0.005 compared with the results without HS-5 sup. or cytokines (A, E, F) and with the results without inhibitors (B, C, H, I, J). Next, we evaluated which major cytokines produced by HS-5, i.e., interleukin (IL)-6, IL-8, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-CSF (GM-CSF), macrophage inflammatory protein (MIP)-1, IL-1 [10], and MDS-associated cytokines, i.e., IL-10, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-1 [3, 11C13], could induce Tim-3 expression on F-36P cells. TGF-1 alone enhanced mRNA expression on F-36P cells (Figure ?(Figure2D).2D). mRNA expression was clearly upregulated by TGF-1 in all MDS cell lines (Figure ?(Figure2E).2E). However, while cell surface Tim-3 expression in F-36P cells was dramatically increased by TGF-1, it.
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