ALBERT

Description: Key Points BMI1 overexpression is one of the second hit partner genes of RUNX1 mutations that contribute to the development of MDSs.

Description: Abstract 50 The Epstein-Barr virus (EBV) is one of the most major human pathogen that establish long-term latent, or chronic infections, which is associated with a heterogeneous group of lymphoma, including Burkitt's lymphoma, Hodgkin's lymphoma (HL), NK-T lymphomas and lymphoproliferative disease. These malignancies are subdivided into in terms of EBV latent infection pattern, with typical three types of latency: type I to type III. HL is characterized by a minority of neoplastic Hodgkin and Reed-Sternberg (HRS), which are embedded in non-neoplastic bystanders, mostly B and T cells, but also macrophages. Without these bystander cells, these HL cells are incapable of being engrafted in immunodeficient mice. In this context, the non-tumor immune cells are tumor-supportive “inflammatory niche”. Because of the complexity of interplay between tumor and tumor surrounding immune cells, the detailed mechanism and how tumor cells escape from the attack of host immune cells remains an open question. Small RNAs including miRNAs are well known intra-cellular regulatory elements of gene expression. Recently, it was reported that they are conjugated in exosomes and transferred to cells and are involved in tumor metastasis by educating tumor surrounding niche. Moreover, it was also reported that EBV-infected lymphocytes produce exosomes that contain viral encoded, EBV specific miRNAs (BART-miRNA) and that these could be transferred in host cells and decrease the levels of known cellular targets. Accordingly, we hypothesized that EBV+ tumor derived exosomal BART-miRNA might redirect tumor surrounding immune cells from tumor reactive into tumor- -supportive “inflammatory niche”, which ultimately leads to tumor progression. To this aim, first, we evaluated tumor derived viral encoded BART-miRNA in EBV+HL clinical specimens by using BART-miRNA specific probe in situ hybridization. As expected, these EBV specific BART-miRNA could be detected in HRS as well as in tumor surrounding inflammatory niche, especially macrophage. This result indicated that tumor derived EBV specific BART-miRNA could transfer to the non-tumor cells in the tumor inflammatory niche, supporting the in vivo relevance of secretary EBV specific miRNA. Next, we evaluated the properties of exosomes produced by EBV+ cells (EBV-Ex). To this aim, EBV-Ex was harvested either from the media of the type III or type I EBV-transformed lymphoid cell line. Then, by using transwell co-culture system, we tested the delivery and the effect of EBV-Ex on human peripheral blood mononuclear cells (PBMC) derived monocyte/macrophage (Mo/Mf). As a result, we detected uptake of fluorochrome dye-labeled EBV-Exo in Mo/Mf. We also confirmed exosomal BART miRNA transfer in Mo/Mf. Surprisingly, exosome from Type III latency (Type III-Ex) was relatively enriched in BART miRNA, and were potent on Mo/Mf in inducing surface CD69 expression (Fig.A). This is in contrast to that of exosome from Type I latency (Type I-Ex), in which BART miRNA were relatively vacant and were weak in inducing surface CD69 expression (Fig.A). Panels of cytokine analysis by Q-PCR revealed that type III-Ex treated Mo/Mf displayed an anti-inflammatory/immunosuppressive cytokine rich signature, especially IL-10, compared to type I-Ex treated Mo/Mf, suggesting the possibility that type III-Ex might polarize macrophage into immunosuppressive M2-like phenotype. Intriguingly, type III-Ex from BART miRNA deletion mutant derivative cell lines totally lack the type III -Ex signature. Moreover, ectopic expression of a part of BART in Type I cells changed the EBV-Ex signature from type III to type I (Fig.B), suggesting the importance of specific BART lesion in functional EBV-Ex production in terms of Mo/Mf polarization. Taken these together, secretary tumor derived miRNAs in EBV associated malignancy, specifically in EBV+HL, might play a certain role in tumor inflammation niche. EBV might utilize the exosomal machinery to secrete key viral-encoded miRNAs, through which a small number of neoplastic EBV+ cells could modulate the tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.

Description: Bone marrow is a complex organ system composed of two distinct lineages of cells: the hematopoietic cells and the supporting stromal cells, often referred as hematopoietic microenvironment (HME). Mesenchymal stem cells (MSCs) in bone marrow are shown to give rise to some of the components of HME, including osteoblasts, adipocytes and stromal fibroblasts in vitro, and to endothelial cells in vivo. It is a well accepted, but not definitely proven, concept that the HME provides structural niches, where dormant hematopoietic stem cells (HSCs) reside, and controls their renewal and differentiation. Although cotransplantation of human MSCs together with human HSCs resulted in increased chimerism of HSCs in animal models, existence of donor MSCs could only be detected using sensitive PCR-based analysis. Until this date, there is no physical evidence that transplanted MSCs have indeed engrafted in bone marrow and directly participated in that biological effect. In this study, we present the visual evidence for the sustained integration of human MSCs in murine bone marrow. Furthermore, we are able to delineate the physical interaction of injected human MSCs and cord blood derived CD34-positive HSCs (CBCD34). In order to assess the spatial distribution, lineage commitment and interaction of MSCs and HSCs in situ, we transplanted green fluorescent protein (GFP)-transduced MSCs and yellow fluorescent protein (YFP)-transduced CBCD34 into tibia of NOD/SCID mice. Ten weeks after intramedullary injection, longitudinal sections of mouse tibiae were made and stained with various antibodies for multicolor immunofluorescent analysis using a confocal microscope. We detected not only the existence of GFP-expressing MSCs in bone marrow, but also differentiation into several cell lineages. GFP-expressing cells exhibited phenotype and morphplogy of N-cadherin-positive bone lining osteoblasts, osteocalcin-positive osteocytes in bone, cells lining abluminal surface of vasculature, and in rare occasion, CD34 and CD31-positive endothelial cells. We then quantitatively evaluated the proportion of GFP-MSCs interacted with primitive YFP-CD34 and lineage committed YFP-CD15 and -Glycophorin-expressing cells as well as the proportion of above mentioned hematopoietic cells interacted with GFP-MSC. Approximately 50% of MSCs associated with CD34-posititive stem cells compared to only 2% and 3% of those with CD15 and Glycophorin-positive cells, respectively. It was also evident that the frequency of CD34-positive cells interacted with MSCs was significantly higher than those with CD15 and Glycophorin-positive cells. The results were consistent with a long appreciated notion that more primitive cells closely interact with hematopoietic supporting stromal cells. Furthermore, we quantitatively proved that the majority of YFP-CD34-positive HSCs were found close proximity to the bone. By transplanting GFP-MSCs together with YFP-HSCs, this study provided direct visual evidence that transplanted human MSCs engrafted in murine bone marrow and integrated into HME, which physically interacted with human HSC.

Description: Introduction EB virus (EBV) is associated with heterogeneous lymphomas. Hodgkin's lymphoma (HL) cells are embedded in non-neoplastic bystanders: B, T cells, and macrophages. Without these bystander cells, the lymphoma cells are incapable of being engrafted in immunodeficient mice. In this context, the bystanders are tumor-supportive “inflammatory niche”. Recently, EBV-infected cells produce exosomes that contain EBV specifically encoded miRNAs (EBV-miRNAs). The miRNAs are transferred to cells, and involved in tumor metastasis. However, the detailed mechanism is unknown. Accordingly, we hypothesized that exosomal EBV-miRNAs might redirect tumor surrounding immune cells from tumor reactive into tumor-supportive “inflammatory niche”. Methods We evaluated the expression of EBV-miRNAs in EBV+HL clinical specimens by in situ hybridization, their functional characterization in vitro, and their effects on persistent infection and tumor development in vivo humanized NOG mice model. Moreover, in order to clarify its sorting mechanism, trans factor and cis factor which determined secreted and non-secreted miRNAs was analyzed by use of mass-spectrograhy and next-generation sequencing. Results and Discussion The EBV-miRNAs effects were potent on monocyte/macrophage Mo/Mf in inducing CD69, IL-10, and TNF, suggesting that EBV-miRNAs might polarize Mo/Mf into tumor associated Mf (TAM). EBV-miRNAs suppress tumor cell proliferation in vitro, implying that it works as tumor-suppressor in the tumor cells, while they are required to develop LPD in vivo, which seems contradict to the result in vitro. These results suggest that EBV-miRNAs intra-cellularly regulate the tumor cells to adjust to the surrounding circumstances, for example, to escape from immune surveillance, and inter-cellularly regulate Mo/Mf to support the tumor survival or development. Most importantly, exosomal EBV-miRNAs derived from the tumor cells were transferred to Mf in human EBV+ HL samples. Interestingly, one EBV coded miRNA was not secreted at all, though it abundantly expresses in the cells. The miRNA has been reported to strongly promote cell proliferation in EBV infected tumor cells. It made us hypothesized that the sorting system of secretary and non-secretary miRNAs is critical in the formation of “inflammatory niche”. In order to clarify the mechanism of the sorting, the chimeric miRNA was constructed then, we determined the sequence, which regulates secretion and non-secretion, and purified the protein complex, which specifically bound to the sequence. Mass spectrography and successive knockdown assay, the trans factor which inhibits secretion was identified. Moreover, the next sequencing analysis for the small RNAs revealed that abundant EBV-coded small RNAs occupied RNA-induced silencing complex (RISC), and that non-secreted EBV-miRNA was specifically modified. It is now under investigation whether the modification is involved in the sort mechanism between secretary and non-secretary miRNAs. Taken together, EBV-miRNAs have critical roles in intra- and inter-cellular manner. Especially, the functions as an inter-cellular communicator might be important in the tumor formation and the mechanism needs further investigation. Disclosures: No relevant conflicts of interest to declare.

Description: CD34 negative hematopoietic stem cells (CD34− HSCs) were identified in mice and humans. Human HSCs are evaluated as severe combined immunodeficient mouse (SCID)-repopulating cells (SRCs), originally identified by the ability to reconstitute hematopoiesis in nonobese diabetic (NOD)/SCID mouse. CD34− cord blood (CB) cells have been hard to engraft in NOD/SCID mice until recent report of successlul engraftment by intra-bone marrow transplantation (iBMT). However, CD34− bone marrow (BM) cells have not been analyzed precisely. We prepared lineage negative CD34 negative (Lin-CD34−) cells by negative selection using CD2,3,7,14,16,19,20,33,34,36,41,56,127, and GlyA antibody. Lin-CD34− BM cells did not engraft in NOD/SCID mice even by using iBMT (0/6). In the previous study, we reported that Lin-CD34− BM cells were able to differentiate into CD34+ cells accompanied by the emergence of colony forming activity after 7 days of stroma-dependent culture, while SRC activity was not detected. (BMT 28, 587–595, 2001) Here we cultured Lin-CD34− BM cells on stroma cells transfected with human angiopoietin-1 cDNA (AHESS-5), since we detected Tie-2 expression on Lin-CD34− BM cells. AHESS-5 supported induction of CD34 much better than HESS-5 cells or empty vector transfected control cells (EVHESS-5), and the effect was blocked by anti-Tie-2 antibody (Fig.1). Furtheremore, CD34+ cells produced from CD34− BM cells engrafted in NOD/SCID mice (11/12). As previously reported, CD34− CB cells differentiate CD34+ cells and acquire SRC activity by stroma-dependent culture without angiopoietin-1. These results highlighted the characteristic differences of CD34− HSCs of BM from CB and the unique role of BM niche for CD34− HSCs. Fig. 1 CD34 expression on Lin − CD34 − BM cells after 7 days of culture Fig. 1. CD34 expression on Lin−CD34− BM cells after 7 days of culture

Description: The SCID-repopulating cell (SRC) pool is shown to be heterogeneous and is composed of at least two distinct subsets; short-term and long-term repopulating cells (STRCs and LTRCs), which appear in different time points following transplantation. However, the precise characteristics and their relationships regarding the stem cell function remain elusive. To clarify the specific stem cell activity of each SRC clones that contribute to various stages of hematopoietic reconstitution, we examined the functional aspects of individual SRCs. To determine the repopulating dynamics of individual SRC clones in vivo, we traced the kinetics of individual SRC clones by LAM-PCR based virus integration site analysis. Individual SRC clones which repopulate in each NOG mouse that received EGFP-transduced fractionated CD34+ populations were analyzed at two time points. At 3 weeks after transplantation, BM cells were aspirated from tibia of each recipient, and at 18 weeks recipients were sacrificed and BM cells were recovered from 4 long bones. At each time point, EGFP-expressing human hematopoietic lineage cells were sorted for integration site analysis by LAM-PCR, and the fate of individual SRC clones in the same recipient was examined by clone-tracking analysis. Using primers that were designated based on the genomic sequence information of the CD33+ myeloid cell integration site, we clonally traced distribution of each clone in lineage cells; CD34+ stem/progenitor, T-, and B-lymphoid cells. We found that the early phase of hematopoietic reconstitution was attributed to transient myeloid-restricted clones which rapidly exhausted from the CD34+ stem cell pool. Interestingly, the multilineage cell-producing clones that were responsible for the later phase of hematopoiesis were distinct from the transient myeloid-restricted clones, and these clones continuously self-replicated in the CD34+ stem cell pool. Next, CD34+ cells from the primary recipients were divided into two secondary recipients, and the fate of individual SRC clones in different phases was traced using the paired secondary mice. One recipient was sacrificed at 3 weeks, and the other recipient was sacrificed at 18 weeks after secondary transplantation. First, clones that were detected at the early phase in one recipient were also detected at the later phase in the other recipient (80%). This is clonal evidence that LTRC in the primary recipient produces STRC as well as self-replicating secondary transplantable LTRC. Second, all clones in the secondary recipients were also detected in the primary donor; however, most of clones (68.3%) found in the primary recipients did not contribute to the secondary recipient. In addition, LTRC clones detected in the CD34+ stem cell pool of secondary recipient demonstrated much larger clone size compared to primary recipient. These indicated that the quiescent LTRC clones in the primary recipients were stimulated by transplantation, there by expanded clonally in the secondary recipient and contributed to the later phase of hematopoiesis. Our clonal tracking study clearly demonstrated that the hierarchical structure of the human HSC pool composed of distinct clonal subsets which were heterogeneous in the self-renewal capacity and differentiation ability.

Description: Hematopoiesis is maintained by specific interactions between both hematopoietic and nonhematopoietic cells. Whereas hematopoietic stem cells (HSCs) have been extensively studied both in vitro and in vivo, little is known about the in vivo characteristics of stem cells of the nonhematopoietic component, known as mesenchymal stem cells (MSCs). Here we have visualized and characterized human MSCs in vivo following intramedullary transplantation of enhanced green fluorescent protein-marked human MSCs (eGFP-MSCs) into the bone marrow (BM) of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Between 4 to 10 weeks after transplantation, eGFP-MSCs that engrafted in murine BM integrated into the hematopoietic microenvironment (HME) of the host mouse. They differentiated into pericytes, myofibroblasts, BM stromal cells, osteocytes in bone, bone-lining osteoblasts, and endothelial cells, which constituted the functional components of the BM HME. The presence of human MSCs in murine BM resulted in an increase in functionally and phenotypically primitive human hematopoietic cells. Human MSC-derived cells that reconstituted the HME appeared to contribute to the maintenance of human hematopoiesis by actively interacting with primitive human hematopoietic cells.