ALBERT

Description: Abstract 3559 Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) is triggered by constitutively activated BCR-ABL and SRC family tyrosine kinases. They interact each other, then activate downstream growth-signaling pathways including Raf/MEK/ERK,Akt/mTOR and STAT5 pathways. The BCR-ABL tyrosine kinase inhibitor imatinib is the standard treatment for Ph+ leukemia. However, response rate of Ph+ ALL to imatinib is low, relapse is frequent and quick. Studies have documented the potential anti-tumor activities of curcumin, a yellow colored polyphenol from the perennial herb Curcuma longa. However, whether curcumin can be used in the therapy for Ph+ALL remains obscure. Here, we reported that curcumin induced autophagic cell death by activating RAF/MEK/ERK pathway in early stage of the 24-hour exposure course, later induced apoptosis by inhibiting AKT/mTOR, ABL/STAT5 signalings, down-regulating expression of bcr/abl gene and Bcl2 anti-apoptosis protein, and up-regulating the expression of pro-apoptosis protein BAX in Ph+ALL cell line SUP-B15. Furthermore, we found curcumin exerted synergetic anti-leukemia effect with imatinib by inhibiting imatinib-mediated up-regulation of the activation of AKT/mTOR signaling and down-regulating expression of bcr/abl gene. It is worth noting that curcumin provide advantages over dexmethasone as to synergetic anti-leukemia effect with imatinib because dexmethasone improved the imatinib-mediated up-regulation of the activation of AKT/mTOR/P70S6 signaling. In primary samples from Ph+ALL patients, curcumin inhibit growth signaling not only in newly-diagnosised patient but also in imatinib-resistant patient. Moreover, curcumin effectively exhibited anti-leukemia efficacy and synergetic anti-leukemia effect with imatinib in Ph+ALL mouse models. These results demonstrate that curcumin may be a promising agent for the treatment of patients with Ph+ ALL, and curcumin might be particularly effective when used with current induction regimens consisting of imatinib with or without chemotherapy for treating Ph+ ALL. [Grant Support:National Natural Science Foundation of China (No.30770912), Foundation of the Science & Technology Department of Sichuan Province (No.2008SZ0017)]. Disclosures: No relevant conflicts of interest to declare.

Description: Tumor necrosis factor-α (TNF-α) has been shown to sustain differentiation and proliferation of CD34+ cells toward dendritic cells (DCs). Granulocyte-colony stimulating-factor (G-CSF) sustains differentiation and proliferation of CD34+ cells toward neutrophils and has been shown to have immune-modulatory effects. We hypothesized that co-stimulation of G-CSF and TNF-α generate neutrophil progenitors and DCs together from human CD34+ cells and that interaction of these cells may provide physiological and/or a pathological roles in modulating immune response. Methods. Highly purified human CD34+ cells were cultured with G-CSF, with or without TNF-α and induced to undergo differentiation toward neutrophils. We enumerated neutrophil progenitors using the specific marker CD15, and DCs using CD4, CD11c, CD80, CD83, CD86, and CD123. The character and roles of co-developing DCs in the presence of TNF-α were analyzed by fluorescence-activated cell sorter, enzyme immunohistochemistry, confocal microscopy and mixed lymphocyte reaction (MLR). Cytokine production was assessed using a cytometric bead array system. T reguratory cells (Treg) were defined as CD4+CD25+ cells and the cells expressing Fox P3. Results. When CD34+ cells were cultured for 7 days in the presence of G-CSF, the generated cells predominantly expressed CD15 (71.8±0.6%), while rarely expressing CD11c (8.0±2.2%), CD80 (1.4±1.0%), CD83 (2.9±0.5%), or CD86 (5.6±2.9%). The addition of TNF-α significantly decreased the number of cells expressing CD15 (3.5±2.1%), but did not affect the number of total cells. In the presence of TNF-α, the generated cells expressed major histocompatibility complex (MHC) class I (99.5%) plus MHC class II (90.2%). A substantial number of cells became positive for CD11c (70.9±5.3%), and even co-stimulatory molecules such as CD80 (8.0±2.7%), CD83 (15.9±3.0%), and CD86 (39.6±3.2%). Immature CD11c+ DCs were physically associated with apoptotic and CD15+ cells, and capable of endocytosing CD15+ cells. Most of the CD11c+ cells did not co-express the G-CSF receptor, but expressed CD4 and CD123. About one half of CD11c+ cells co-expressed CD86. The DCs generated by TNF-α and G-CSF facilitated alloreactive T cell proliferation in the same extent, although cytokine production from activated T cells were low. Primary MLR facilitated the proliferation of CD4+CD25+ cells and Fox P3+ Treg. The CD4+ CD25+ T cells suppressed secondary MLR, whereas CD4+ CD25− T cells enhanced secondary MLR. Conclusions. This is the first report showing that. the non-neutrophilic cells with typical feature of DCs are co-generated from human CD34+ cells during neutrophil differentiation by G-CSF in the presence of TNF-α. The CD4+ CD11c+ CD123+ DCs physically associate with and phagocytoses developing or dying immature neutrophilic cells. The generated DCs promoted the proliferation of Treg that suppressed secondary MLR. Therefore, it may be conceivable that DCs with phagocytic activity during the development in bone marrow may play a crucial role in the maintenance of tolerance for self-substances derived from hematopoietic progenitor cells.

Description: Background. During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. Enucleation has been thought to be a process similar to cytokinesis. However, more concrete evidence has been difficult to obtain because of a lack of an ex vivo experimental system capable of confirming cytokinesis. Focusing on the mechanism of cell division, we investigated the redistribution of cytoplasmic proteins and integral membrane proteins during enucleation, using ex vivo generation system of mature human blood cells from hematopoietic stem cells. Materials and Methods. The highly purified human CD34+ cells were grown in the presence of interleukin-3, stem cell factor and erythropoietin (EPO) in a liquid phase. After 7 days of culture, the generated cells (day 7 cells) were replaced in a medium with EPO alone. The cells matured and terminally differentiated into reticulocytes during a 13–15-day culture period. We mainly used non-gravity and non-pipetting system to avoid physical stress that may disrupt the connection between the nucleus and reticulocyte. Day 9 cells, predominantly consisted of polychromatophilic erythroblasts and expressed glycophorin A (GPA) at a purity of 97%, were labeled with DNA-staining dye SYTO21 for the direct monitoring of the enucleation process, using differential interference contrast microscopy. We also cultured day 9 cells until day 14, on 4-well culture slides or on the membrane of a cell culture insert system, and removed culture medium by aspiration without centrifugation and pipetting. The day 14 cells on the slide were analyzed using immunohistochemical staining, whereas the cells on the membrane were embedded in O.C.T. compound for confocal microscopy. Results. Approximately a half of erythroblasts enucleated until day 14. The monitoring of the enucleation process at day 13 showed autonomous extrusion of SYTO21 positive nucleus from single erythroblast. Some of the expelled nuclei were still connected with reticulocyte through strings that were positive for antibody against tubulin, actin, GPA, band 3 and glycophorin C (GPC). The expelled nuclei were covered by lamin, a protein specific for nuclear membrane, which were surrounded by a substance positive for GPA, band 3, GPC, p55, 4.1R80, actin, tubulin, b-spectrin, calnexin and cytochrome C, although the distribution of each proteins were asymmetric between nuclei and reticulocytes. An intense area of GPA, GPC, band 3, 4.1R80, actin, tubulin, myosin and b-spectrin was found in the region of the constriction between the extruding nucleus and incipient reticulocyte in enucleating cells. In cells just before enucleation, tubulin and actin formed a radial array around the nucleus. The center of the radial array was positive for centrin and NuMA, indicating that the cenriole formation occurred during an enucleation process. Conclusion. Our investigations show that a part of human erythroblasts enucleate independent of an interaction with accessory cells. The appearance of cenriole and the asymmetric redistribution of cytoplasmic and integral membrane proteins during enucleation strongly suggest that enucleation of human erythroblasts is a process of asymmetric cytokinesis.

Description: Abstract 1351 Poster Board I-373 Introduction Hemophagocytic syndrome (HPS, also referred as hemophagocytic lymphohistiocytosis; HLH) is a life-threatening condition caused by hyperinflammatory response. It can be classified into familial and acquired forms. Familial HPS is a disorder inherited in an autosomal recessive or X-linked manner. Several loci and genes have been implicated in the familial HPS, including perforin, or cytotoxic granule exocytosis genes. While, acquired (secondary) HPS is a sporadic syndrome occurring in association with systemic infection (virus, bacteria, protozoa, and fungi), underlying malignancy, or immunodeficient disorder. The central pathophysiologic abnormality in HPS is cytokine dysfunction, resulting in uncontrolled accumulation of activated T-lymphocytes and activated histiocytes (macrophages) in many organs. Several cytokines including interferon-γ, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10, IL-12, and soluble IL-2 receptor (sCD25), found at extremely high levels in the plasma of HPS patients. Treatment of hemophagocytic lymphohistiocytosis is based on epipodophyllotoxin derivatives, etoposide, corticosteroids, cyclosporin A, antithymocyte globulin (ATG), and, in selected patients, intrathecal methotrexate. Recently, anti-TNF-α therapy has been reported to prove patients with refractory HPS, however, there is not enough evidence. In this study, we established murine HPS model by intravenous injection of CpG oligodeoxynucleotide (ODN)-1668, a ligand for Toll-like receptor 9 (TLR9) and combination of CpG and iE-DAP, which is a ligand for Nod1, and investigated the role of TNF-α and other cytokine in HPS model. Methods C57BL/6J mice were injected intravenously with 200 μg of CpG or combination of CpG and 100 μg of iE-DAP. The treated mice were assayed for temperature, spleen weight, and complete blood count. The serum cytokines levels (TNF-α and IL-6) and triglycerides were determined by ELISA and auto-analyzer, resectively. Hemophagocytosis was determined by flow cytometric (FCM) analysis in the peripheral blood and spleen. The spleen was subjected into immunohistochemistry staining and histological examination. For TNF-α and IL-6 blockade, the mice were treated with TNF receptor (TNFR)-Fc (etanercept) or combination of TNFR-Fc and anti-mouse IL-6 receptor (IL-6R) monoclonal antibody (MR16-1) by intravenous injection. Results CpG injected mouse displayed the typical HPS clinical criteria, such as, fever, cytopenia, splenomegaly, hemophagocytosis in the peripheral blood and spleen. Furthermore, elevations of inflammatory cytokines (TNF-α and IL-6) and triglyceride were detected in the serum of these mice. These clinical symptoms were not observed in CpG-injected TLR2−/−4−/−9−/− mice and LPS-injected wild type mice. Interestingly, combination challenge of CpG and iE-DAP led to synergistic induction of hemophagocytosis and enhanced production of TNF-α and IL-6 in the serum, however, single iE-DAP challenge did not induce HPS. FCM analysis revealed that monocyte-derived dendritic cells, which were CD11b, Ly6c, and F4/80, were mobilized and engulfed TER119+ erythrocyte in the peripheral blood and spleen after these challenge. Treatment of mice with TNFR-Fc partially prevented, and combination of TNFR-Fc and anti IL-6R mAb markedly suppressed hemophagocytosis in the peripheral blood and spleen. Conclusions: Based on these results, CpG or CpG + iE-DAP-induced mouse HPS model is valuable to study for cellular and molecular mechanism of HPS development, and potential system to apply preclinical research. Furthermore, prevention of both TNF-α and IL-6 might represent a novel therapy for HPS. Disclosures No relevant conflicts of interest to declare.

Description: The gene(s) responsible for natural killer (NK)–cell lymphoma/leukemia have not been identified. In the present study, we found that in NK-cell lymphoma lines (n = 10) and specimens of primary lymphoma (n = 10), levels of miR-21 and miR-155 expression were inversely related and were significantly greater than those found in normal natural killer (CD3−CD56+) cells (n = 8). To determine the functions of these microRNAs in lymphomagenesis, we examined the effects of antisense oligonucleotides (ASOs) targeting miR-21 (ASO-21) and/or miR-155 (ASO-155) in NK-cell lymphoma lines overexpressing one or both of these miRNAs. Conversely, cells showing little endogenous expression of miR-21 or miR-155 were transduced by the use of lentiviral vectors, leading to their overexpression. Reducing expression of miR-21 or miR-155 led to up-regulation of phosphatase and tensin homologue (PTEN), programmed cell death 4 (PDCD4), or Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1). ASO-21– and ASO-155–treated cell lines all showed down-regulation of phosphorylated AKTser473. Moreover, transduction with either miR-21 or miR-155 led to down-regulation of PTEN and PDCD4 or SHIP1 with up-regulation of phosphorylated AKTser473. Collectively, these results provide important new insight into the pathogenesis of NK-cell lymphoma/leukemia and suggest targeting miR-21 and/or miR-155 may represent a useful approach to treating NK-cell lymphoma/leukemia.

Description: Aberrant overexpression of the miR-17-92 polycistron is strongly associated with B-cell lymphomagenesis. Recent studies have shown that miR-17-92 downregulates the pro-apoptotic protein Bim, leading to overexpression of Bcl2, which likely plays a key role in lymphomagenesis. However, the fact that Jeko-1 cells derived from mantle cell lymphoma exhibit both homozygous deletion of BIM and overexpression of miR-17-92 suggests other targets are also involved in B-cell lymphomagenesis. To identify essential target(s) of miR-17-92 in lymphomagenesis, we first transfected miR-17-92 into genetically distinct B-cell lymphoma cell lines, including Raji cells, which overexpress c-Myc, and SUDHL4 cells, which overexpress Bcl2. Raji cells transfected with miR- 17-19b exhibited downregulation of Bim and reversible upregulation of Bcl2. On the other hand, SUDHL4 cells transfected with miR-17-19b showed aggressive cell growth reflecting facilitated cell cycle progression at the G1-S transition, and decreased expression of CDKN1A mRNA and p21 protein (CDKN1A/p21) that was independent of p53 expression. Conversely, transfection of antisense oligonucleotides against miR-17 and miR-20a into Jeko-1 cells led to upregulation of CDKN1A/p21, resulting in decreased cell growth with G1-S arrest. Thus, CDKN1A/p21 appears to be an essential target of miR-17-92 during B-cell lymphomagenesis, which suggests the polycistron has distinct targets in different B-cell lymphoma subtypes.

Description: Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening hyperinflammatory syndrome triggered by EBV infection. It often becomes relapsed or refractory (r/r), given that etoposide-based regimens cannot effectively clear the virus. r/r EBV-HLH is invariably lethal in adults without allogeneic hematopoietic stem cell transplantation. Here, we performed a retrospective analysis of 7 r/r EBV-HLH patients who were treated with nivolumab on a compassionate-use basis at West China Hospital. All 7 patients tolerated the treatment and 6 responded to it. Five of them achieved and remained in clinical complete remission with a median follow-up of 16 months (range, 11.4-18.9 months). Importantly, both plasma and cellular EBV-DNAs were completely eradicated in 4 patients. Single-cell RNA-sequencing analysis showed that HLH syndrome was associated with hyperactive monocytes/macrophages and ineffective CD8 T cells with a defective activation program. Nivolumab treatment expanded programmed death protein-1–positive T cells and restored the expression of HLH-associated degranulation and costimulatory genes in CD8 T cells. Our data suggest that nivolumab, as a monotherapy, provides a potential cure for r/r EBV-HLH, most likely by restoring a defective anti-EBV response.

Description: Recent studies have shown that anemia is commonly observed after exposure to pathogens or pathogen-derived products, which are recognized via Toll-like receptor 9 (TLR9). In the current study, we demonstrate that CpG oligodeoxynucleotide-2006, a TLR9 ligand with phosphodiester (PO; 2006-PO) but not with the phosphorothioate backbone, selectively inhibits the erythroid growth derived from human CD34+ cells. The 2006-PO was internalized by the erythroid progenitors within 30 minutes; however, expression of TLR9 mRNA was not detected in these cells. The 2006-PO directly inhibited burst-forming unit-erythroid growth, resulted in the accumulation of cells in S and G2/M phases, and increased cell size and frequency of apoptotic cells. These features were similar to those observed in erythroid progenitors infected with human parvovirus B19 that causes pure red cell aplasia. The consensus sequence of 2006-PO was defined as 5′-GTTTTGT-3′, which was located in the P6-promoter region of B19 and inhibited erythroid growth in a sequence-specific manner and down-regulated expression of erythropoietin receptor (EPOR) mRNA and EPOR. B19 genome extracted from serum also inhibited erythroid growth and down-regulated expression of EPOR on glycophorin A+ cells. These results provide a possible insight into our understanding of the mechanisms of human parvovirus B19-mediated inhibition of erythropoiesis.

Description: Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34+ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.

Description: Abstract 3429 Background: Idiopathic PRCA and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are major subtypes of adult-onset chronic PRCA. We have previously shown that these types of PRCA are responsive to immunosuppressive therapy but most patients require long-term maintenance immunosuppressive treatment. These results suggest that acquired chronic PRCA is an autoimmune disorder mediated by T lymphocytes and pathogenic T cell clones may be persistently present during remission. We have previously made an interesting observation that a thymoma-associated PRCA patient had an increase of Vd1 gd T cells in blood. We have also reported that recipients of allogeneic hematopoietic stem cell grafts had an oligoclonal expansion of Vd1 gd T cells and that Vd1 gd T clones had cytotoxicity against autologous EBV-transformed B cell line. Thus, gd T cell repertoires may be altered in PRCA patients in response to certain antigens. Objective: In order to clarify the role for gd T cells in the pathogenesis of chronic acquired PRCA, we have examined the gd T cell receptor repertoire in acquired chronic PRCA patients. Materials and Methods: Nineteen PRCA (8 idiopathic, 6 thymoma, 3 LGL-leukemia and 2 SLE) and 107 healthy volunteer donors were included in the study. This study was approved by the Institutional Review Board at Akita University and conducted in accordance with the Declaration of Helsinki. Blood lymphocyte subsets were analyzed by flow cytometry. Clonality of T cells was determined by complementarity-determining region 3 (CDR3) size distribution analysis and junctional sequence was determined by subcloning of PCR products and DNA sequencing. In some experiments, purified gd T cells from PRCA patients were co-cultured with allogeneic erythroid progenitor cells derived from CD34-positive cells in vitro in order to learn whether patient's gd T cells would exert cytotoxic or growth-inhibitory effect on erythroid progenitor cells. Results: The absolute numbers of ab T cells and gd T cells were normal in patients with PRCA, but there were an increase of Vd1 gd T cells and a decrease of Vd2 T cells (Table 1). More than 50% of Vd1 T cells from PRCA patients expressed HLA-DR, while 20 to 30% of those from healthy individuals expressed HLA-DR (Fig. 1). CDR3 size spectratyping revealed that CDR3 size distribution patterns were skewed in 9 out of 13 PRCA patients examined, although skewed CDR3 size distribution patterns were also observed in 7 out of 10 healthy individuals. In order to determine whether a particular Vd1-Jd rearrangement size was selected in PRCA patients, we performed statistical analysis comparing the CDR3 size distribution of 115 Vd1 TCR clones obtained by subcloning of PCR products in 7 PRCA patients versus 7 controls. No significant difference was found between the two groups (p=0.795 by Mann-Whitney test). Moreover, no apparent consensus amino acid motifs were identified in PRCA patients. Although the T cell clone carrying the -YWGIR- sequence in the CDR3d region was detected in 3 PRCA patients, the T cell clone carrying the -YWGIR- sequence was also detected in one healthy donor. Purified gdT lymphocytes from idiopathic PRCA neither showed an inhibitory effect on proliferation nor cytotoxicity against erythroid progenitor cells in vitro. Adjusted p value was calculated by Kruskal-Wallis ANOVA test. Conclusions: Expansion of Vd1 T cells and depletion of Vd2 T cells are unique features for chronic acquired PRCA. Expansion of Vd1 T cells does not seem to be the consequence of CDR3-dependent selection. Depletion of Vd2 T cells may be the result of chronic stimulation, because our previous study has revealed that the numbers of Vd2 T cells show an age-dependent decrease and Vd2 T cells are susceptible to activation-induced cell death (Int J Hematol, in press). Failure to demonstrate the cytotoxicity of gd T cells from a PRCA patient against erythroid progenitor cells suggests that expanded gd T cells are not effector T cells. Disclosures: No relevant conflicts of interest to declare.