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Background: Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective hematopoiesis, refractory anemia, and a tendency to transform to acute myeloid leukemia (AML). Ineffective hematopoiesis progression and immune deregulation are dominating pathophysiological process of MDS. Emerging evidences showed the role of bone marrow (BM) microenvironment in MDS. In MDS murine model, integral BM microenvironment contributes to inferior hematopoietic function and disease progression. As an important component of BM microenvironment, the relationship between endothelial cells (ECs) and MDS progression remains largely unknown. Although ECs from MDS patients have been identified to have decreased supporting ability to normal hematopoietic stem cells (HSCs), the supporting ability of ECs in different clinical stages of MDS remains to be elucidated. In addition, the role of BM ECs from MDS patients in supporting leukemia cells and their immunomodulatory ability remains unclear. Aims: To determine the number and functions of BM ECs in different subtypes of MDS patients. Moreover, to explore the correlation between BM ECs and MDS progression, which may represent a potential therapeutic target for MDS patients. Methods: In the prospective cohort study, patients with multilineage dysplasia (MDS-MLD, N=15), MDS with excess blasts (MDS-EB, N=15), or AML(N=15) and healthy donors (HD, N=15) were enrolled. BM ECs were analyzed in HD and patients by flow cytometry and in situ histological analyses. The functions of BM ECs were analyzed by migration, angiogenesis capacities, levels of apoptosis and reactive oxygen species (ROS). To evaluate the supporting abilities of BM ECs on HSCs, leukemia cells and T cells, in vitro co-culture strategies were used. The levels of apoptosis, ROS and colony-forming unit-plating (CFU) efficiency of CD34+ and HL-60 cells were investigated. T cell subsets were analyzed by flow cytometry as previously reported. To further investigate the underlying mechanism of dysfunctional ECs, RNA sequencing (RNA-Seq) analyses and real time-PCR (qRT-PCR) were performed in BM ECs from HD and MDS patients with different subtypes. Results: In the current study, gradually increased BM ECs were observed from MDS-MLD, MDS-EB to AML patients. Furthermore, dysfunctional BM ECs were found with MDS progression, characterized by increased levels of migration, angiogenesis capacities, apoptosis and ROS. More importantly, BM ECs from MDS patients exhibited decreased supporting ability of HSCs whereas increased supporting ability of leukemia cells in vitro with MDS progression. After coculture with ECs, levels of apoptosis and ROS in CD34+ cells were increased whereas their CFU efficiency reduced. On the other hand, levels of apoptosis and ROS of HL-60 cells were decreased. The proliferation capacity and leukemia CFU efficiency of HL-60 cells after co-cultured with ECs were enhanced with MDS progression. Furthermore, following coculture with BM ECs, deregulated differentiation was demonstrated in T cell subsets, characterized by elevating proportion of Th2 and Treg and decreasing proportion of Th1 and Th17 with MDS progression. RNA-Seq showed that the expression profile of BM ECs from MDS-EB was closer to MDS-MLD, whereas that of MDS-EB was closer to AML. Different gene expression profiles indicated the expression of hematopoiesis and immune related genes increased in BM ECs with MDS progression. Mechanistically, the mRNA levels of CX CL12, SCF and NFKB of ECs were increased with MDS progression. Summary/Conclusion: In summary, the number of BM ECs gradually increased, BM EC dysfunction more and more severe, and the supporting abilities of BM ECs on HSCs decreased, whereas on leukemia cells increased with MDS progression. Moreover, ECs regulated the differentiation of T cells into immune tolerant cells with MDS progression. Although further validation is required, these findings indicated that the improvement of BM ECs may represent a potential therapeutic approach for MDS patients. Keywords: Myelodysplastic syndromes, endothelial cells, disease progression, ineffective hematopoiesis, immune deregulation Disclosures No relevant conflicts of interest to declare.

Background Aplastic anemia (AA) is a life-threatening disease characterized by bone marrow (BM) failure and pancytopenia. Immunosuppressive therapy can rescue most patients with AA. However, the pathogenesis of AA is still not well elucidated and the new strategies need to be developed for AA patients. Increasing evidences suggested the dysfunctional BM microenvironment may be involved in the pathogenesis of AA. As important components of the BM microenvironment, endothelial cells (ECs) play a crucial role in supporting hematopoiesis and regulating immune. However, whether BM ECs are involved in the occurrence of AA and whether repairing BM ECs could improve the hematopoietic and immune status of AA patients remain to be elucidated. Aims To evaluate the quantity and function of BM ECs from AA patients. Moreover, to determine whether the dysfunctional BM ECs are involved in the occurrence of AA by affecting hematopoiesis and regulating immunity in vitro and in vivo. Finally, to uncover the therapeutic potential of repairing dysfunctional BM ECs to alter the hematopoietic and immunological status in AA patients. Methods This study enrolled 30 patients with AA and 30 healthy donors(HD). Flow cytometry and BM in situ immunofluorescence staining were used to analyze the proportion of ECs in BM of the two groups. The level of intracellular reactive oxygen species(ROS) and the proportion of apoptosis were detected by flow cytometry. The functions of BM ECs were evaluated by double-positive staining, migration and tube formation assays. To determine the effect of BM ECs on hematopoiesis and immunity, primary human BM ECs were separately cocultured with CD34 + and CD3 + cells. To further validate the role of BM ECs in the occurrence of AA, a classical AA mice model and VE-cadherin blocking antibody that could antagonize the function of BM ECs were used. Moreover, to explore potential approach of targeting the dysfunctional BM ECs, the exogenous EC infusion or N-acetyl-L-cysteine (NAC, a ROS scavenger) for repairing BM ECs were administrated to the AA mice. To further explore the repairing effect of NAC on BM ECs, the primary BM ECs from AA patients were treated by NAC in vitroand then the functions of BM ECs were evaluated. Results Compared with HD, BM ECs in AA patients were decreased and dysfunctional, which characterized by higher levels of ROS and apoptosis, impaired abilities of migration and angiogenesis. Furthermore, dysfunctional BM ECs from AA patients not only impaired their hematopoiesis-supporting ability but also promoted co-cultured T cells to polarize towards pro-inflammatory T cells in vitro, which resulted in an unbalanced T cell subsets. Consistently, AA mice demonstrated decreased BM ECs with increased level of intracellular ROS. Moreover, hematopoietic failure and immune imbalance in AA mice became more severe when the function of BM ECs was antagonized, whereas the administration of NAC or infusion of exogenous EC improved the hematopoietic and immunological status of AA mice via repairing BM ECs in vivo. In addition, we found the NAC treatment also restored the hematopoiesis-supporting ability and immunity-regulating ability of the primary ECs derived from AA patients in vitro. Summary/Conclusion Our study demonstrates for the first time that dysfunctional BM ECs with impaired hematopoiesis-supporting ability and abnormal immunomodulatory ability are involved in the pathogenesis of AA. Although further validation is required, restoring dysfunctional BM ECs via EC infusion or administration of ROS scavenger NAC might be a potential therapeutic approach for AA patients. Disclosures No relevant conflicts of interest to declare.

Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Macrophages (MΦs) are an important immune population that are essential for disease pathogenesis. MΦs are now commonly classified as either M1, which produce pro-inflammatory cytokines, or M2, which produce anti-inflammatory cytokines. Our recent study reported that patients who received an allograft with a higher M1/M2 ratio exhibited a higher incidence of grade 2-4 aGVHD(BMT, 2019). Moreover, an aberrant M1/M2 polarization was found in the colon of aGVHD mice, and regulating the polarization of MΦs cultured from aGVHD patients towards M2 phenotype modulated T cells favoring a type 2 response in vitro(Sci China Life Sci, 2020). However, the primary subsets and function of MΦs in aGVHD patients, and the precise roles of different MΦ subsets in the development of aGVHD remain to be elucidated. Aims: To determine the subsets and function of MΦs in primary peripheral blood(PB) of aGVHD patients. Moreover, to investigate whether M1 and M2 had different effects on the development of aGVHD, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT. Methods: In this prospective case-control study, a total of 20 patients with aGVHD and 20 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. MΦ subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. M1 and M2 were identified as CD14+CCR2+CD68+ and CD14+CX3CR1+CD163+, respectively. In order to determine the function of MΦs in patients with aGVHD and non-aGVHD, the phagocytosis was analyzed using a DiI-AcLDL assay. The protein expressions for the costimulatory molecules and the cytokine production of MΦs were measured by flow cytometry. To further investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of MΦs. Subsequently, to explore the role of different subsets of MΦs in the development of aGVHD, M1 and M2 were infused into aGVHD mice, respectively. Mice were monitored for survival, weight, and aGVHD score. Histological scores of tissues from aGVHD target organs (liver, intestine, spleen and skin) were evaluated by HE staining. Results: When compared with non-aGVHD patients, MΦs in primary PB of aGVHD patients were polarized towards pro-inflammatory M1, characterized by an elevated proportion of M1 and a reduced proportion of M2. Furthermore, MΦs isolated from aGVHD patients exhibited lower phagocytic function, higher expression of TNF-α and IL-6 and higher expression of costimulatory molecules CD80 and CD86. Consistent with the increased activated MΦs from aGVHD patients, the mRNA levels of genes involved in the pro-inflammatory M1 polarization, antigen presenting and promoting the activation of T cells pathway were substantially elevated in MΦs of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vivo infusion with pro-inflammatory M1 aggravated aGVHD response by modulating immune microenvironment of spleen and liver in mice, characterized by enhanced effector function of T cell, including elevated percentages of Tc1, Th1 and Th17 as well as increased proliferation of T cells from spleen and liver. By contrast, infusion with anti-inflammatory M2 alleviated aGVHD through down-regulating the activity of T cells derived from aGVHD mice, characterized by decreased proliferation and decreased percentages of Tc1, Th1 and Th17. Summary/Conclusion: The current study demonstrated that the primary MΦs in aGVHD patients preferentially polarize into pro-inflammatory M1. Moreover, M1 aggravate aGVHD whereas M2 ameliorate aGVHD by differently modulating immune microenvironment. Although further validation is required, modulating the polarization state of MΦs promises to be a novel therapeutic target for aGVHD patients after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Background Megakaryopoiesis and platelets production intensely depend on bone marrow(BM) microenvironment. Our previous studies found that impaired BM microenvironment and dysfunctional megakaryopoiesis are responsible for the occurrence of prolonged isolated thrombocytopenia (PT), which is defined as the engraftment of all peripheral blood cell lines other than a platelet count less than 20×109/L or a dependence on platelet transfusions for more than 60 days following allo-HSCT(BBMT 2014; BBMT 2017; Brit J Haematol 2018; Am J Hematol 2018). As an important component of the BM microenvironment, macrophages (MՓs) are heterogeneous and polarized into classically activated (M1) MՓs and alternatively activated (M2) MՓs with distinct phenotypes and function. Although inconsistent effect of BM MՓs was reported on megakaryopoiesis, the functional role of M1 and M2 MՓs and related pathway in regulating megakaryopoiesis and its effect on PT patients post-allotransplant remain to be elucidated. Aims To address the roles of M1 MФs and M2 MФs in regulating megakaryopoiesis as well as PI3K-AKT pathway in the process. Moreover, polarization status and the function of BM MФs in regulating megakaryopoiesis were evaluated in PT patients. Methods This prospective nested case-control study enrolled 12 patients with PT, 24 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 12 healthy donors (HD). BM standard monocyte subsets and M1/M2 MՓs polarization state were analyzed by flow cytometry. To generate M1 and M2 MՓs, both primary BM MՓs and THP1 cell lines were treated with LPS and IFN-γ or with IL-4 and IL-13. The functions of BM MՓs were evaluated by migration, phagocytosis and cytokine secretion assay. The sorted CD34+ cells from HD were co-cultured with BM MՓs from PT and GGF patients or M1 and M2 MՓs respectively for megakaryopoiesis. The quantification of the megakaryocytes(MKs), MKs apoptosis, MKs polyploidy distribution, colony-forming unit MK(CFU-MK) efficiency, and platelet production were analyzed in the coculture system. To understand the underlying mechanism of MՓs polarization in regulating MKs, RNA-seq analyses were performed in BM MՓs from PT and GGF patients. In addition, M1 and M2 MՓs were treated with the chemical inhibitors and lentivirus for PI3K-AKT pathway. Results Elevated intermediate and non-classical monocyte subsets were found in PT patients when compared with those in GGF patients. Moreover, PT patients displayed increased M1 and reduced M2 MՓs, resulting an unbalanced M1/M2 polarization, compared with GGF and HD. BM MՓs from PT patients, with high TNF-α levels and low TGF-β levels, showed decreased megakaryopoiesis-supporting ability. No significant differences in migration and phagocytosis function of MՓs among the three groups. RNA sequencing of BM MՓs showed down-regulated PI3K-AKT pathway in MՓs of PT patients compared with GGF. Consistently, the phosphorylation levels of AKT decreased significantly in MՓs of PT patients, suggesting that PI3K-AKT pathway may functionally regulate megakaryopoiesis-supporting ability of MՓs. Subsequently, BM-M2 and THP1-M2 showed superior effect on megakaryopoiesis-supporting ability compared with BM-M1 and THP1-M1. Specifically, the BM CD34+ cells cocultured with M2 MՓs demonstrated significant increased percentages of MKs and MK polyploidy, CFU-MK efficiency, and platelet count compared with those cocultured with M1 MՓs. Preventing PI3K-AKT pathway by PI3K inhibitor or Akt inhibitor significantly reduced the megakaryopoiesis-supporting ability of M2 MՓs. Moreover, knockdown of AKT1 induced the impairment of megakaryopoiesis-supporting ability via suppressing M2 MՓs polarization, which could be attenuated by AKT1 overexpression complementarily. Summary/Conclusion The current study demonstrated the polarization status of MՓs modulates their ability to support megakaryopoiesis. M2 MՓs, but not M1 MՓs, support megakaryopoiesis via up-regulating PI3K-AKT pathway. Defective M2 MՓs polarization via down-regulating PI3K-AKT pathway may be responsible for the pathogenesis of PT post-allotransplant, which provides a promising therapeutic target for PT patients. Disclosures No relevant conflicts of interest to declare.

Background: Bone marrow(BM) endothelial cells(ECs), a key component of BM microenvironment, is essential for the physiology and regeneration of hematopoietic stem cells (HSCs). The damage of ECs is recognized by us and other researchers as a mainstay in the pathophysiology of a serious of life-threatening complications after chemoradiotherapy and myeloablative hematopoietic cell transplantation(HSCT), including poor graft function (PGF) (2013BBMT, 2015BMT, 2016Blood, 2019Blood Advances). Despite numerous researches focused on the BM ECs contributing to HSC regeneration following myelotoxicity, the mechanisms underlying the injured BM ECs itself remain to be elucidated. Under physiological conditions, energy metabolism plays an instrumental role in maintaining EC function, and markedly perturbed of EC metabolism is linked to many pathologies, like cancer and diabetes. However, little is known about the metabolism state and its role in impaired BM ECs. Aims: The current study was performed to investigate the metabolism status in BM ECs after chemotherapy-induced injury. Moreover, we evaluated the metabolic state and its role in BM ECs of PGF patients post-allotransplant. Finally, we evaluated the therapeutical potential of anti-metabolic drugs to the dysfunctional BM ECs derived from PGF patients. Methods: Two EC injury models in vitro were established with the cultivated human BM ECs treated by 5-Fluorouracil (5-FU) and hydrogen peroxide. The findings from the above models were further validated by a prospective case-control study enrolled 15 patients with PGF, 30 matched patients with good graft function (GGF) and 15 healthy donors (HD). To determine the metabolic status of BM ECs, the expression of metabolism regulating genes was analyzed by qRT-PCR (mRNA level) and flow cytometry (protein level). Glucose metabolism levels were measured by glucose consumption and lactate production assays. To evaluate the functions of BM ECs, apoptosis, migration and tube formation assays were performed. To investigate the effect of anti-metabolic drugs to injured BM ECs, the glycolysis inhibitor 3PO and PPARd agonist GW501516 were administrated to the cultivated BM ECs treated by 5-FU , hydrogen peroxide or derived from PGF. Results: We demonstrated that the glycolysis in BM ECs could be induced by the treatment with either 5-FU or hydrogen peroxide in vitro, consistent with the dysfunction(impaired migration, angiogenesis, and higher level of apoptosis) of BM ECs, which could be attenuated by glycolysis restoration. Mechanistically, we revealed that the aberrant glycolysis and dysfunction of BM ECs could be triggered by PPARd knockdown in vitro, while the PPARd were down-regulated by either 5-FU or hydrogen peroxide treatment in vitro, Furthermore, PPARd agonist GW501516 treatment attenuated the perturbed function and number of injured BM ECs treated by either 5-FU or hydrogen peroxide. Subsequently, the prospective case-control study demonstrated elevated expressions of the glycolytic activator PFKFB3 and decreased PPARd were observed in BM ECs of PGF patients, when compared with those of GGF patients and HD, indicating that BM ECs of PGF patients have a hyper-glycolytic metabolism. Moreover, either glycolysis (PFKFB3) inhibitor 3PO or PPARd agonist GW501516 treatment reduced the aberrant glycolysis and improved the number and function of BM ECs derived from patients with PGF in vitro, revealing the critical role of defective glycolysis in the impaired BM ECs of PGF. Summary / Conclusions: These findings reveal that hyper-glycolysis mediated by PPARd inhibition is involved in the dysfunction of BM ECs after injury. Defective glycolysis may contribute to the pathobiology of BM ECs of PGF patients, which could be attenuated by glycolysis inhibitor 3PO or PPARd agonist GW501516 in vitro. Our findings might merit further consideration of targeting BM ECs glycolysis or PPARd as a promising therapeutic approach for PGF patients post-allotransplant in the future. Disclosures No relevant conflicts of interest to declare.

Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Metabolism not only provide energy and substrates for T cell growth and survival, but also instruct effector functions, differentiation, and gene expression of T cells. In this regard, the metabolic profile of T cells was reported to play a critical role in the occurrence and development of many immunological disorders such as systemic lupus erythematosus and rheumatoid arthritis. Murine studies found that alloreactive T cells use aerobic glycolysis as the predominant metabolic process to meet activation and proliferation demand after allo-HSCT. However, the metabolic profile of T cells and the approach for regulating T cell metabolism in aGVHD patients remains to be elucidated. Aims: To determine the metabolic state in T cells of patients with aGVHD. Moreover, to investigate the effect of the novel approach targeting the abnormal metabolism in T cells of aGVHD patients, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT. Methods: In this prospective case-control study, a total of 25 patients with aGVHD and 25 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. T cell subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. Th1, Th2, Th17, and Treg cells were identified as CD4+IFN-γ+, CD4+IL-4+, CD4+IL17A+, and CD4+CD25+Foxp3+, respectively. Tc1 and Tc2 cells were identified as CD8+IFN-γ+ and CD8+IL-4+, respectively. In order to determine the metabolic state in T cells of patients with aGVHD and non-aGVHD, the metabolic profile was determined using a Seahorse XF96 Analyzer. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. The mitochondrial mass, the mitochondrial membrane potential, the protein expressions for the lipid metabolism enzyme CTP1a and the glycolytic activator PFKFB3 were measured by flow cytometry. To further understand the metabolic state of T cells in aGVHD and non-aGVHD patients and investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of T cells. Subsequently, to explore the potential way of targeting the abnormal metabolism in T cells, the glycolysis inhibitor 3-PO was administrated to T cells from aGVHD patients. Results: When compared with T cells in non-aGVHD patients, T cells in aGVHD patients were polarized towards pro-inflammatory T cells, characterized by an elevated proportion of Tc1, Th1 and Th17. Furthermore, T cells isolated from aGVHD patients exhibited higher extracellular acidification rate, as well as the increased glucose consumption rate and lactate production rate compared to those in non-aGVHD patients. Moreover, elevated expression of PFKFB3 was observed in T cells, especially in naïve T cells of aGVHD patients, but oxygen consumption rate, CPT1A, mitochondrial mass or membrane potential showed no significant differences in T cells between aGVHD and non-aGVHD patients. These results implied higher glycolytic activity of T cells in aGVHD patients when compared with those in non-aGVHD patients. Consistent with the increased glycolytic activity observed in T cells from aGVHD patients, the mRNA levels of genes involved in the glycolytic pathway were substantially elevated in T cells of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vitro treatment with glycolysis inhibitor 3-PO improved the activity of T cells derived from aGVHD patients through down-regulating glycolytic activity of T cells. Summary/Conclusion: The current study demonstrated that T cells in aGVHD patients preferentially depend on glycolysis to meet activation and proliferation demands. Furthermore, the activity of T cells from aGVHD patients could be ameliorated by glycolysis inhibitor 3-PO in vitro. Although further validation is required, T cell glycolysis promises to be a novel therapeutic target for aGVHD patients after allo-HSCT. Disclosures No relevant conflicts of interest to declare.