Yuan Kong

Association of an Impaired Bone Marrow Microenvironment with Secondary Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

Poor graft function (PGF) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Whether abnormalities of the bone marrow (BM) microenvironment are involved in the pathogenesis of PGF is unclear. In the present prospective nested case-control study, 19 patients with secondary PGF, 38 matched patients with good graft function (GGF) after allo-HSCT, and 15 healthy donors (HDs) were enrolled. The cellular elements of the BM microenvironment, including endosteal cells, perivascular cells, and vascular cells, were analyzed by flow cytometry as well as hematoxylin and eosin and immunohistochemical staining in situ. The median time to occurrence of secondary PGF was 90 days post-transplantation (range, 58 to 264 days). The patients with PGF showed markedly hypocellular marrow (10% versus 45% versus 45%; P < .0001) with scattered hematopoietic cells and significantly lower CD34(+) cells (0.07% versus 0.26% versus 0.26%; P < .0001), endosteal cells (4 per high-power field [hpf] versus 16 per hpf versus 20 per hpf; P < .001), perivascular cells (0.008% versus 0.10% versus 0.12%; P < .0001), and endothelial progenitor cells (0.008% versus 0.16% versus 0.18%; P < .0001) compared with GGF allo-HSCT recipients and HDs, respectively. Multivariate analyses revealed that endothelial progenitor cells (odds ratio, 150.72; P = .001) and the underlying disease (odds ratio, 18.52; P = .007) were independent risk factors for secondary PGF. Our results suggest that the impaired BM microenvironment may contribute to the occurrence of secondary PGF post-HSCT.

Monocytic and promyelocytic myeloid-derived suppressor cells may contribute to G-CSF-induced immune tolerance in haplo-identical allogeneic hematopoietic stem cell transplantation

We investigated the effects of granulocyte colony‐stimulating factor (G‐CSF) on monocytic (M), promyelocytic (P), and granulocytic (G) myeloid‐derived suppressor cells (MDSCs) both in bone marrow and peripheral blood of 20 healthy donors and the association of MDSCs subgroups with acute and chronic graft‐versus‐host disease (aGvHD/cGvHD) in 62 patients who underwent haplo‐identical allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Patients who received a higher absolute counts of M‐MDSCs or P‐MDSCs exhibited lower incidence of grade II–IV aGvHD (P = 0.001; P = 0.031) and extensive cGvHD (P = 0.011; P = 0.021). In the multivariate analysis, absolute counts of MDSCs in allografts emerged as independent factors that reduced the occurrence of grade II–IV aGvHD (M‐MDSCs: HR = 0.087, 95% CI = 0.020–0.381, P = 0.001; P‐MDSCs: HR = 0.357, 95% CI = 0.139–0.922, P = 0.033) and extensive cGvHD (M‐MDSCs: HR = 0.196, 95% CI = 0.043–0.894, P = 0.035; P‐MDSCs: HR = 0.257, 95% CI = 0.070–0.942, P = 0.04). Delayed M‐MDSC reconstitution was associated with aGvHD onset. The 3‐year cumulative incidence of transplant related mortality and relapse, 3‐year probability of disease‐free survival, and overall survival did not differ significantly between these subgroups. Our results suggested that G‐CSF‐induced immune tolerance may be mediated by M/P‐MDSCs in allo‐HSCT. Am. J. Hematol. 90:E9–E16, 2015.

Atorvastatin enhances endothelial cell function in post transplant poor graft function

Poor graft function (PGF) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Murine studies suggest that endothelial progenitor cells (EPCs) are preferential supporting cells for hematopoietic stem cells in the bone marrow (BM) microenvironment. Our previous work found that a reduced number of BM EPCs was an independent risk factor for the occurrence of PGF after allo-HSCT. However, little is known about the functional role of BM EPCs and how to improve impaired BM EPCs in PGF. In the current study, we evaluated the function of BM EPCs in subjects with PGF postallotransplant. Moreover, we investigated whether atorvastatin could enhance the number and function of BM EPCs derived from subjects with PGF in vitro. Dysfunctional BM EPCs, which were characterized by impaired proliferation, migration, angiogenesis, and higher levels of reactive oxygen species and apoptosis, were revealed in subjects with PGF. Activation of p38 and its downstream transcription factor cyclic adenosine monophosphate-responsive element-binding protein were detected in BM EPCs from subjects with PGF. Furthermore, the number and function of BM EPCs derived from subjects with PGF were enhanced by atorvastatin treatment in vitro through downregulation of the p38 MAPK pathway. In summary, dysfunctional BM EPCs were observed in subjects with PGF. Atorvastatin treatment in vitro quantitatively and functionally improved BM EPCs derived from subjects with PGF through downregulation of the p38 MAPK pathway. These data indicate that atorvastatin represents a promising therapeutic approach for repairing impaired BM EPCs in subjects with PGF postallotransplant.

Aberrant T cell responses in the bone marrow microenvironment of patients with poor graft function after allogeneic hematopoietic stem cell transplantation

AbstractBackgroundPoor graft function (PGF) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, whether abnormalities of T cell subsets in the bone marrow (BM) immune microenvironment, including Th17, Tc17, Th1, Tc1, Th2, Tc2 cells and regulatory T cells (Tregs), are involved in the pathogenesis of PGF remains unclear.MethodsThis prospective nested case–control study enrolled 20 patients with PGF, 40 matched patients with good graft function (GGF) after allo-HSCT, and 20 healthy donors (HD). Th17, Tc17, Th1, Tc1, Th2, Tc2 cells, Tregs and their subsets were analyzed by flow cytometry.ResultsA significantly higher proportion of stimulated CD4+ and CD8+ T cells that produced IL-17 (Th17 and Tc17) was found in the BM of PGF patients than in the BM of GGF patients and HD, whereas the percentages of Tregs in PGF patients were comparable to those in GGF patients and HD, resulting in a dramatically elevated ratio of Th17 cells/Tregs in the BM of PGF patients relative to those in GGF patients. Moreover, both CD4+ and CD8+ T cells were polarized towards a type 1 immune response in the BM of PGF patients.ConclusionsThe present study revealed that aberrant T cell responses in the BM immune microenvironment may be involved in the pathogenesis of PGF after allo-HSCT. These findings will facilitate the optimization of immune regulation strategies and improve the outcome of PGF patients post-allotransplant.

Atorvastatin enhances bone marrow endothelial cell function in corticosteroid-resistant immune thrombocytopenia patients

The pathogenesis of corticosteroid-resistant immune thrombocytopenia (ITP), a clinically challenging condition in which patients exhibit either no response to corticosteroids or are corticosteroid-dependent, remains poorly understood. Murine studies suggest that bone marrow (BM) endothelial progenitor cells (EPCs) play a crucial role in regulating megakaryocytopoiesis. However, little is known regarding the number and function of BM EPCs or how to improve impaired BM EPCs in corticosteroid-resistant ITP patients. In the current case-control study, we evaluated whether the BM EPCs in corticosteroid-resistant ITP differed from those in corticosteroid-sensitive ITP. Moreover, whether atorvastatin could enhance the number and function of BM EPCs derived from corticosteroid-resistant ITP patients was investigated in vitro and in vivo. Reduced and dysfunctional BM EPCs, characterized by decreased capacities of migration and angiogenesis as well as higher levels of reactive oxygen species and apoptosis, were observed in corticosteroid-resistant ITP patients. In vitro treatment with atorvastatin quantitatively and functionally improved BM EPCs derived from corticosteroid-resistant ITP patients by downregulating the p38 MAPK pathway and upregulating the Akt pathway, and rescued the impaired BM EPCs to support megakaryocytopoiesis. Subsequently, a pilot cohort study showed that atorvastatin was safe and effective in corticosteroid-resistant ITP patients. Taken together, these results indicate that reduced and dysfunctional BM EPCs play a role in the pathogenesis of corticosteroid-resistant ITP, and the impaired BM EPCs could be improved by atorvastatin both in vitro and in vivo. Although requiring further validation, our data indicate that atorvastatin represents a promising therapeutic approach for repairing impaired BM EPCs in corticosteroid-resistant ITP patients.

miR-153-3p, a new bio-target, is involved in the pathogenesis of acute graft-versus-host disease via inhibition of indoleamine- 2,3-dioxygenase

Acute graft-versus-host disease (aGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Therefore, seeking reliable biomarkers and delineating the potential biological mechanism are important for optimizing treatment strategies and improving their curative effect. In this study, using a microRNA polymerase chain reaction (PCR)-based chip assay, microRNA-153-3p (miR-153-3p) was screened and selected as a potential biomarker of aGVHD. The elevated plasma miR-153-3p levels at +7 d after transplant could be used to predict the upcoming aGVHD. The area under the receiver operating characteristic curve for aGVHD+/aGVHD- patients receiving haploidentical transplant was 0.808 (95% confidence interval, 0.686-0.930) in a training set and 0.809 (95% confidence interval, 0.694-0.923) in a validation set. Interestingly, bioinformatics analysis indicated that indoleamine-2,3-dioxygenase (IDO) is a potential target of miR-153-3p. In vitro study confirmed that IDO could be directly inhibited by miR-153-3p. In a GVHD model, recipient mice injected with a miR-153-3p antagomir exhibited higher IDO expression levels at the early stage after transplantation, as well as delayed aGVHD and longer survival, indicating that the miR-153-3p level at +7 d post-transplant is a good predictor of aGVHD. miR-153-3p participates in aGVHD development by inhibiting IDO expression and might be a novel bio-target for aGVHD intervention.

Abnormalities of the bone marrow immune microenvironment in patients with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired autoimmune disease. Although antiplatelet antibodies are considered as the primary immunologic defect in these patients, dysfunctional cellular immunity is also important in the pathophysiology of ITP. Peripheral T cell abnormalities have been demonstrated in patients with ITP; however, whether the impaired bone marrow (BM) microenvironment, specifically the BM immune microenvironment, is involved in the pathogenesis of ITP remains unknown. In this study, the compartments of the BM immune microenvironment and BM vascular microenvironment were analyzed in 26 untreated patients with ITP and 26 healthy donors (HD). Subsets of T cells in the BM immune microenvironment, including Th1, Th2, Tc1, Tc2, Th17, and Treg cells, were analyzed via flow cytometry. BM endothelial cells and perivascular cells, which are key elements of the vascular microenvironment, were analyzed via flow cytometry as well as hematoxylin-eosin (H&E) and immunohistochemical (IHC) staining in situ. Elements of the BM vascular microenvironment were found to be normal in patients with ITP, but abnormal characteristics of the BM immune microenvironment, including excessive polarization in Th1, Tc1, and Th17 cells and a remarkable decrease in Treg cells, were observed in patients with ITP. Therefore, a deregulated T cell response in the BM microenvironment might play an important role in the pathogenesis of ITP.

Abnormalities of the Bone Marrow Immune Microenvironment in Patients with Prolonged Isolated Thrombocytopenia after Allogeneic Hematopoietic Stem Cell Transplantation

Prolonged isolated thrombocytopenia (PT) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Whether abnormalities of the bone marrow (BM) immune microenvironment are involved in the pathogenesis of PT remains unknown, however. Twenty patients with PT, 40 matched patients with good graft function (GGF) after allo-HSCT, and 20 healthy donors (HD) were enrolled in this nested case-control study. Th1, Th2, Tc1, Tc2, Th17, and Treg cells were analyzed by flow cytometry, and IFN-γ, IL-4, IL-17, IL-6, IL-21, and thrombopoietin levels in BM plasma were evaluated with a cytometric bead assay and ELISA. Relative to GGF patients and HD controls, PT patients had significantly higher proportions of Th1 and Tc1 cells, resulting in higher Th1/Th2 and Tc1/Tc2 ratios in the BM microenvironment. In addition, the excessive polarization of Th17 was observed in patients with PT. Changes in BM plasma cytokines were consistent with our cellular findings. These results suggest that dysregulated T cell responses in the BM microenvironment might play an important role in the pathogenesis of PT.

Lower incidence of acute GVHD is associated with the rapid recovery of CD4+CD25+CD45RA+ regulatory T cells in patients who received haploidentical allografts from NIMA-mismatched donors: A retrospective (development) and prospective (validation) cohort-based study

ABSTRACT To investigate the effects of non-inherited maternal antigen (NIMA) on clinical outcomes and immune recovery, especially of regulatory T cells (Tregs), in patients who underwent unmanipulated haploidentical transplantation. A retrospective cohort (n = 57) and a prospective cohort (n = 88) were included. All patients received haploidentical allografts from sibling donors. Reconstitution of immune subsets, including Tregs, was determined using multicolor flow cytometry. In the retrospective cohort, the cumulative incidence of grades II–IV acute GVHD in patients with NIMA-mismatched donors was significantly lower than that of cases with NIPA-mismatched donors (14.8% vs. 43.30%, p = 0.018). Patients with higher percentages of CD4+CD25+CD45RA+ T cells (naive Tregs) within CD4+ T cells recovered on day 30 (≥1.55%) experienced a significantly lower incidence of grades II–IV acute GVHD than that of cases with lower percentages of naive Tregs (<1.55%) (13.8% vs. 46.4%, p = 0.010). Multivariate analysis showed that NIMA mismatch and the percentages of naive Tregs were associated with the incidence of grades II–IV acute GVHD [p = 0.050, and 0.031, respectively]. In the prospective cohort, the association of NIMA mismatch [HR = 0.365, 95% CI, 0.169–0.786, p = 0.010] or higher percentages of naive Tregs recovered on day 30 (≥1.55%) [HR = 0.114, 95% CI, 0.027–0.479, p = 0.003] with a lower cumulative incidence of grades II–IV acute GVHD was further demonstrated. No effects of NIMA mismatch on chronic GVHD, transplant-related mortality, relapse, disease-free survival, or overall survival were found. Our results confirmed the role of NIMA mismatch in acute GVHD and provided the first demonstration, based on clinical data, that recovered Tregs may be involved in the effects of NIMA on acute GVHD in a haploidentical transplant setting.

IL-35 inhibits acute graft-versus-host disease in a mouse model.

Acute graft-versus-host disease (aGVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our previous study found that the novel anti-inflammatory cytokine IL-35 could suppress aGVHD in patients after allo-HSCT. In this study, we used C57BL/6 (B6, H-2b) mice as donors and (B6×DBA/2) F1 (BDF1, H-2b×d) mice as recipients to create a model of aGVHD and explore the relationship between IL-35 and aGVHD. The mice receiving IL-35 survived longer than did the control mice. We observed that treatment with IL-35 and RAPA could reduce the incidence of aGVHD. Additionally, this treatment inhibited intestinal and thymic epithelial cell apoptosis and liver infiltration by the donor T-cells, thereby ameliorating the enteropathy and liver injury caused by aGVHD. We found that IL-35 and RAPA also markedly suppressed TNF-α and IL-17A expression and enhanced IFN-γ expression in the intestine and liver. We measured Tregs in spleen and found that IL-35 and RAPA treatment expanded the number of Tregs in spleen. We found that the phosphorylation of STAT1 and STAT4 were inhibited in mice with aGVHD. In contrast, STAT1 and STAT4 were phosphorylated when the mice were treated with IL-35. IL-35 may have therapeutic potential in the treatment of aGVHD after allo-HSCT.