Wei Sang

MiR-150 impairs inflammatory cytokine production by targeting ARRB-2 after blocking CD28/B7 costimulatory pathway

MiR-150, a major modulator negatively regulating the development and differentiation of various immune cells, is widely involved in orchestrating inflammation. In transplantation immunity, miR-150 can effectively induce immune tolerance, although the underlying mechanisms have not been fully elucidated. In the current study, we found that miR-150 is elevated after blocking CD28/B7 co-stimulatory signaling pathway and impaired IL-2 production by targeting ARRB2. Further investigation suggested that miR-150 not only repressed the level of ARRB2/PDE4 directly but also prevented AKT/ARRB2/PDE4 trimer recruitment into the lipid raft by inhibiting the activities of PI3K and AKT through the cAMP-PKA-Csk signaling pathway. This leads to the interruption of cAMP degradation and subsequently results in inhibition of the NF-kB pathway and reduced production of both IL-2 and TNF. In conclusion, our study demonstrated that miR-150 can effectively prevent CD28/B7 co-stimulatory signaling transduction, decrease production of inflammatory cytokines, such as IL-2 and TNF, and elicit the induction of immune tolerance. Therefore, miR-150 could become a novel potential therapeutic target in transplantation immunology.

Control of mouse graft-versus-host disease following allogeneic bone marrow transplantation by blocking the CD28/B7 signaling pathway with lentiviral vector-mediated RNA interference.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective way to cure hematological malignancies. However, graft-versus-host disease (GVHD) following transplantation limits the clinical application to some extent. The donor T lymphocytes play a central role in the occurrence and development of GVHD. Control of GVHD by inhibition of T cell proliferation by blocking the CD28/B7 signaling pathway with RNA interference has not been examined. In this study, we constructed a lentiviral vector carrying CD28 shRNA and generated genetically engineered splenocytes through transduction in a murine allogeneic bone marrow transplantation model. The survival and the occurrence of GVHD in transplanted mice were monitored every day. Liver, intestine, skin, and other tissues from the mice in each group were used for histological examination. We also determined plasma concentrations of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, and interferon gamma (IFN-γ). Recipient bone marrow from mice that had survived for an extended period was examined to detect chimerism. We succeeded in suppressing the expression of CD28 gene and controlling mouse GVHD following allogeneic bone marrow transplantation in the engineered spleen cell group. These suggest that blocking the CD28/B7 signal transduction pathway with lentiviral vector-mediated RNA interference effectively controlled the occurrence of mouse GVHD following allogeneic bone marrow transplantation. Its mechanism could be due to the inhibition of T cell proliferation and, simultaneously, the promotion of the differentiation of TH0 to TH2 cells, thereby reducing GVHD in the mouse transplantation model.

Piperlongumine selectively suppresses ABC-DLBCL through inhibition of NF-κB p65 subunit nuclear import

Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys(38) to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has been reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumines mechanism of action and novel approach to ABC-DLBCL target therapy.

MicroRNA‐181a, a potential diagnosis marker, alleviates acute graft versus host disease by regulating IFN‐γ production

Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is a valuable therapeutic strategy for a wide variety of diseases. Acute graft‐versus‐host disease (aGVHD) is a major complication in up to 75% of allo‐HSCT. The absence of a reliable predicative marker for aGVHD onset prevents preemptive treatment and impedes widespread and successful application of this therapy. In this study we found that after allo‐HSCT, the levels of miR‐181a were reduced significantly prior to the onset of aGVHD. More importantly, the degree of its reduction correlated with the severity of aGVHD. Mechanistically, miR‐181a affects the function of T lymphocytes by down‐regulating IFN‐γ in a dose‐dependent manner. Meanwhile, we confirmed that miR‐181a can effectively preserve the anti‐leukemic effect in vitro. Using a murine allo‐HSCT model, we demonstrated that murine miR‐181b, the human miR‐181a homolog, served as an effective predictor of aGVHD. Moreover, expression of this microRNA ameliorated the severity of aGVHD. Collectively, these results show that the level of miR‐181a may serve as a reliable marker for the diagnosis and prognosis the onset of aGVHD. Am. J. Hematol. 90:998–1007, 2015.

MicroRNA-150 negatively regulates the function of CD4+ T cells through AKT3/Bim signaling pathway

Donor-derived CD4(+) T lymphocytes are the major effector cells directly involved in the development of graft-versus-host disease (GVHD). As a negative regulator of immune cell differentiation and development, microRNA-150 (miR-150) induces immunological tolerance in CD4(+) T cells after transplantation. However, the specific mechanisms have not been fully elucidated. In this study, we demonstrated that miR-150 is capable of not only inhibiting proliferation and activation of CD4(+) T cells but also promoting apoptosis. Mechanistically, miR-150 targets v-akt murine thymoma viral oncogene homolog 3 (AKT3), and subsequently downregulates B-cell lymphoma 2 (Bcl-2) interacting mediator of cell death (BIM). We have also demonstrated that re-expression of AKT3 reversed miR-150-mediated inhibition of CD4(+) T lymphocyte development. Therefore, we conclude that miR-150 negatively regulates CD4(+) T cell function by inhibiting the AKT3/BIM signaling pathway. These findings also suggest that manipulating the levels of miRNA-150 could be a valuable strategy in prevention and/or treatment of acute graft-versus-host disease.

Decreased level of cytotoxic T lymphocyte antigen-4 (CTLA-4) in patients with acute immune thrombocytopenia (ITP)

INTRODUCTION Previously, we demonstrated the importance of T-cell immune response cDNA 7 (TIRC7) in acute immune thrombocytopenia (ITP). As the downstream molecule of TIRC7, cytotoxic T lymphocyte antigen-4 (CTLA-4) has been verified its negative regulation of acute ITP. This study aimed to investigate the exact role of CTLA-4 and its relationship with TIRC7 in acute ITP. PATIENTS AND METHODS 37 patients with acute ITP were enrolled and received dexamethasone (40mg/day) for 4 consecutive days. Patients who had platelet counts more than 50×10(9)/L or less were defined as responders or non-responders after treatment. The plasma, protein and mRNA levels of CTLA-4 and TIRC7 were monitored by ELISA, western blot and q-PCR, respectively. RESULTS After high-dose dexamethasone therapy, CTLA-4 levels were significantly elevated not only in acute ITP patients (P<0.001; P<0.0001) but also in acute ITP responders (P<0.0001; P<0.0001). The levels of CTLA-4 were negatively correlated with the levels of TIRC7 before and after treatment; IFN-γ (Th1), IL-17 (Th17) and IL-22 (Th22) levels were all elevated, which were decreased after treatment not only in patients with acute ITP (P<0.01) but also in acute ITP responders (P<0.01). CONCLUSIONS CTLA-4 level might reflect treatment efficacy and it might be associated with the pathogenesis of acute ITP.

Increased expression of T cell immune response cDNA 7 in patients with acute graft-versus-host disease

Acute graft-versus-host disease (aGVHD) has become the important complication post-allogeneic hematopoietic stem cell transplantation. Abnormally activated T cells might play an important role in the pathogenesis of aGVHD. But its exact mechanism remains poorly understood. T cell immune response cDNA 7 (TIRC7) has been identified to be essential in T cell activation; however, the role of TIRC7 in aGVHD remains unclear. The purpose of this study was to measure the expression of TIRC7 and T helper (Th) cells in patients with aGVHD before and after treatment. We showed that TIRC7 levels in aGVHD patients were higher than those of healthy controls and markedly declined after treatment. The levels of IFN-γ (Th1), IL-17 (Th17), and IL-22 (Th22) were in accordance with the grade of aGVHD. In addition, TIRC7 levels were also associated with the severity of aGVHD. In conclusion, TIRC7 might be involved in the pathogenesis of aGVHD and TIRC7 level might be an indicator to evaluate the response of patients with aGVHD to treatment.

Potent anti-leukemia activities of humanized CD19-targeted Chimeric antigen receptor T (CAR-T) cells in patients with relapsed/refractory acute lymphoblastic leukemia

Chimeric antigen receptor T (CAR‐T) cell therapy has shown promising results for relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL). The immune response induced by murine single‐chain variable fragment (scFv) of the CAR may limit CAR‐T cell persistence and thus increases the risk of leukemia relapse. In this study, we developed a novel humanized scFv from the murine FMC63 antibody. A total of 18 R/R ALL patients with or without prior murine CD19 CAR‐T therapy were treated with humanized CD19‐targeted CAR‐T cells (hCART19s). After lymphodepletion chemotherapy with cyclophosphamide and fludarabine, the patients received a single dose (1 × 106/kg) of autologous hCART19s infusion. Among the 14 patients without previous CAR‐T therapy, 13 (92.9%) achieved complete remission (CR) or CR with incomplete count recovery (CRi) on day 30, whereas 1 of the 3 patients who failed a second murine CAR‐T infusion achieved CR after hCART19s infusion. At day 180, the overall and leukemia‐free survival rates were 65.8% and 71.4%, respectively. The cumulative incidence of relapse was 22.6%, and the nonrelapse mortality rate was 7.1%. During treatment, 13 patients developed grade 1‐2 cytokine release syndrome (CRS), 4 patients developed grade 3‐5 CRS, and 1 patient experienced reversible neurotoxicity. These results indicated that hCART19s could induce remission in patients with R/R B‐ALL, especially in patients who received a reinfusion of murine CAR‐T.

Effects of realgar (As 4 S 4 ) on degradation of PML-RARA harboring acquired arsenic-resistance mutations

Dear Editor, Acute promyelocytic leukemia (APL) is characterized by the accumulation of abnormal promyelocytes in blood and bone marrow and the specific chromosomal translocation t(15;17)(q24.1;q21.1). The t(15;17) fuses the retinoic acid receptor-alpha gene (RARA) on chromosome 17 with the promyelocytic leukemia gene (PML) on chromosome 15, resulting in PML-RARA fusion protein that is the key driver and therapy target of APL [1]. Arsenic trioxide has been shown to be the most active single agent in APL [2]. Arsenic trioxide cures APL by directly binding to the PML moiety and triggering the degradation of PML-RARA oncoprotein. However, it has been identified a panel of PML point mutations in arsenic-resistant APL patients, and the outcomes of these patients are extremely poor [3, 4]. Evaluation of whether the mutations contributes to clinical resistance should be performed. Recently, it has been demonstrated that an oral realgar (tetra-arsenic tetra-sulfide, As4S4) treatment alone can serve as a highly effective and safe remission therapy for APL [5]. Furthermore, the patients with oral realgar have outcomes similar to those with intravenous arsenic trioxide. The precise working mechanisms of realgar are still not clear. In this study, we investigated whether realgar could overcome mutationtriggered arsenic resistance. The schematic representations of the long and short isoforms of PML-RARA are shown in Fig. 1a. The short-form type of PML-RARA transcript lacks the nuclear localizing signal (NLS) in PML. Recently, seven arsenic-resistance mutations (L211P, C213R, S214 L, A216V, L217F, D219H and S220G) were identified in the PML B-box2 (B2) domain of PML-RARA (Fig. 1a). Furthermore, the new mutation D241G was firstly identified in the coiled-coil (CC) domain [3, 4]. As shown in Fig. 1b, growth inhibition by realgar was selective for PML-RARA positive cells (NB4), whereas the HL60 cells were resistant. For NB4 cells, the IC50 of realgar was 1.1 μM. To examine the effect of arsenic trioxide and realgar on apoptosis of NB4 cells, we evaluated the Caspase-3 activity using Caspase-Glo assay. As shown in Fig. 1c, realgar could induce activation of Caspase-3 with the same efficacy as it is induced by arsenic trioxide in NB4 cells. Immunoblot analysis revealed that realgar significantly induced degradation of both long and short isoforms PMLRARA protein (Fig. 1d). It has been demonstrated previously that the SUMOylation is required for arsenic-induced degradation of PML-RARA [6]. To investigate the role of SUMOylation in realgar-induced degradation of PMLRARA, we constructed the mutants which have a lysine residue substituted to arginine for the defined SUMO consensus motif. As shown in Fig. 1e, K65R and K490R had minor effect on realgar-induced degradation of PML-RARA. In contrast, K160R and 3KR mutants were apparently realgar resistant and failed to be degraded in the same experimental conditions. Realgar and arsenic trioxide had a similar mode of Mingshan Niu and Yangling Shen contributed equally to this work.

Fludarabine, idarubicin, and cytarabine regimen together with TKI followed by haploidentical hematopoietic stem cell transplantation, a success for relapsed Ph+ acute lymphoblastic leukemia

In this report, a case of relapsed Ph+ ALL was remedied by reinduction, and consolidation regimen of TKI and Flu+ Ara‐C+ IDA (FLAI) combination, followed by haploidentical SCT. Results suggest that FLAI together with TKI and subsequently with haploidentical SCT could be applied for relapsed Ph+ ALL.