Jiang Cao

Engineered regulatory T cells prevent graft-versus-host disease while sparing the graft-versus-leukemia effect after bone marrow transplantation

Regulatory T cells (Tregs) can prevent graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). Here we developed a lentivirus-based strategy to ectopically express Foxp3 in mouse CD4(+)CD25(-) T cells. These cells shared similar immunophenotypes and biological features of natural Tregs. Co-injection of engineered Tregs with donor bone marrow cells and splenocytes prevented recipients from lethal GVHD. Furthermore, we showed that graft-versus-leukemia (GVL) effect against EL4/DsRed leukemic cells was maximally preserved while GVHD was minimized during exposure to engineered Tregs in a mouse leukemia model. These findings provide a novel approach to preventing GVHD while maintaining GVL effect during BMT.

Bone Marrow–derived Endothelial Progenitor Cells Promote Hematopoietic Reconstitution After Hematopoietic Stem Cell Transplantation

OBJECTIVE A hematopoietic deficit is a serious complication after hematopoietic stem cell transplantation. It has been shown that fetal blood-derived endothelial progenitor cells (EPCs) can promote hematopoietic reconstitution after transplantation. This study investigated whether EPCs from bone marrow (BM) of adult mice could promote hematopoietic reconstitution. METHODS Lethally irradiated BALB/c mice were administered BM cells or BM cells plus EPCs. RESULTS The results showed that EPC-treated mice displayed accelerated recovery of peripheral blood white blood cells and reticulocytes. But the platelets were not significantly different with versus without EPCs. Accelerated recovery of BM sinusoidal vessels, promotion of stem cell implantation, and decreased adipocyte formation were associated with the mechanism. Systemic administration of anti-vascular endothelial cadherin antibody neutralized these effects significantly. CONCLUSION These data showed that BM-derived EPC infusions augmented hematopoiesis suggesting a new approach to promote hematopoiesis.

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.

The identification and characteristics of IL-22-producing T cells in acute graft-versus-host disease following allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) remains the major obstacle for allogeneic bone marrow transplantation, in which many proinflammatory cytokines secreted by alloreactive donor T cells are involved. Role of IL-22 as a member of IL-10 family in GVHD is still disputed and the properties of IL-22-producing cells are unclear. We demonstrated here that CD4⁺ T cells but not CD8⁺ T cells involved in GVHD were the main cellular source of donor-derived IL-22. Th1 and Th17 cells were detected not only express classical cytokine IFN-γ or IL-17, but also contributed to IL-22 secretion in GVHD. Th22 cells characterized by the independent secretion of IL-22 were identified and occupied almost half percentage of IL-22-producing CD4⁺ T cells. The frequency of IL-22-producing CD4⁺ T cells showed dynamic changes with the development of GVHD. Finally, we observed that IL-22-producing CD4⁺ T cells in GVHD mouse carried CD62L⁻CD44(high/low) surface markers. In conclusion, we illuminate the characteristics of donor-derived IL-22-producing CD4⁺ T cells, which may have potent implication for further study of pathogenesis of GVHD.

The effects of R683S (G) genetic mutations on the JAK2 activity, structure and stability

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in the hematopoietic and immune response. The acquired JAK2 R683S (G) mutations are presumed to be a biomarker for B-cell acute lymphoblastic leukemia (B-ALL). However, how these mutations leading to the B-ALL is still unclear. The crystal structure of JAK2 JH2 domain suggests that the residue R683 locating in the linker between the N and C lobes of JH2 domain is important for keeping the compact structure, activity and structural stability of this domain. Mutations R683S, R683G and R683E significantly increase JAK2 activity and decrease its structural stability. While the R683K and R683H mutations almost have no effects on the JAK2 activity and structural stability. Furthermore, the spectroscopic experiments imply that mutations R683S, R683G and R683E impair the structure of JAK2 JH2 domain, and lead JAK2 to partially unfolded state. It may be this partially unfolded state that caused JAK2 R683S (G) constitutive activation. This study provides clues in understanding the mechanism of JAK2 R683S (G) mutations caused B-ALL.

Amino acid residue E543 in JAK2 C618R is a potential therapeutic target for myeloproliferative disorders caused by JAK2 C618R mutation

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays a key role in the hematopoietic and immune responses. The acquired JAK2 C618R somatic mutation is detected in a subset of myeloproliferative disorders (MPDs) patients and presumed to be a biomarker for MPDs. However, how JAK2 C618R mutation causes MPDs is still unclear. Our results indicate that the amino acid residue E543 in JAK2 C618R is indispensable for its constitutive activation. When the glutamic acid at this position was mutated to alanine (E543A) in the JAK2 C618R, its activity significantly decreased. However when the glutamic acid was mutated to the acidic amino acid, aspartic acid, JAK2 C618R activity changed little. These results suggest that there is an interaction between the amino acid residue R618 and E543, and that this interaction is crucial to sustain the constitutive activation of JAK2 C618R. More importantly, the E543 single mutation had no effects on the function of wild type JAK2 (WT JAK2). This study suggests that the amino acid residue E543 might be a potential target for specific inhibitors to treat MPDs caused by the JAK2 C618R mutation.

Disrupting of E79 and K138 interaction is responsible for human muscle creatine kinase deficiency diseases

Creatine kinase (CK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphocreatine to ADP in vertebrates. Due to its important physiological functions, the acquired CK somatic mutations are closely correlated to diseases. In this study, the E79G point mutation was identified in two acute myocardial infarction patients with muscle CK activity deficiency. The crystal structure of CK indicates that the E79 and K138 interaction plays key roles in sustaining the recognition between N-terminal and C-terminal domains. Mutations of these residues caused pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Moreover, spectroscopic spectra experiments suggested that mutations disrupting this hydrogen bond impaired the secondary and tertiary structure of CK. Meanwhile, protein folding experiments implied that mutations lead them to the partially unfolded state which made them easier to be inactivated and unfolded under environmental stresses. Furthermore, this partially unfolded state upon environmental stresses might gradually decrease the CK level in the patients. Thus, these results might provide clues in the mechanism of CK deficiency diseases.

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.

Stromal cells attenuate the cytotoxicity of imatinib on Philadelphia chromosome-positive leukemia cells by up-regulating the VE-cadherin/β-catenin signal

β-Catenin is a key regulator of leukemia stem cell maintenance and drug resistance. Herein, we investigated the protective effects of the stromal cell-mediated VE-cadherin-β-catenin signal on Ph+ leukemia cells during imatinib treatment. We found stromal cells could desensitize imatinib and up-regulate VE-cadherin expression on Ph+ leukemia cells (K562 and SUP-B15 cells), which further stabilized and activated β-catenin. Knockdown of VE-cadherin with shRNA diminished the β-catenin protein and partly resensitized Ph+ leukemia cells to imatinib despite the presence of stromal cells, suggesting VE-cadherin is a potential target in the treatment of Ph+ leukemia.