IL-35 and Rapamycin Reduce Acute Graft-Versus-Host Disease Associated with Decreased Platelet Aggregation in a Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3814-3814 ◽  
Author(s):  
Xiao-Hui Zhang ◽  
Yi Zhou ◽  
Shi-yuan Zhou ◽  
Fei-er Feng ◽  
Qian-ming Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Survival Rate ◽  
Platelet Aggregation ◽  
Inflammatory Cytokine ◽  
Control Group ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Thrombotic Complications ◽  
Ifn Γ ◽  
Acute Graft

Abstract Introduction: Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) caused by the activation of donor T lymphocytes by host antigen-presenting cells and the immune-mediated inflammatory response. Epithelial cells of the skin and mucous membranes, biliary ducts, and intestinal tract crypts are the primary tissue systems damaged during the pathobiological course of GVHD. IL-35, a member of the IL-12 family of cytokines, comprising an IL-12 p35 subunit and an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). It is an anti-inflammatory cytokine that suppresses the immune response through the expansion of regulatory T cells and suppression of Th17 cell development (Niedbala W, et al. European journal of immunology 2007). Rapamycin (Sirolimus; RAPA), a macrolide antibiotic produced by Streptomyces hygroscopicus, has been used for the prophylaxis and treatment of several immune reactions including GVHD and solid organ rejection (Ho-Jin Shin, et al. Blood 2011). We hypothesized that IL-35 has a protective effect in aGVHD, and that its function may be increased by RAPA. Methods: We used C57BL/6 (B6, H-2b) mice as donors and (B6×DBA/2)F1 (BDF1, H-2b×d) mice as recipients to create an aGVHD model (Kuroiwa T, et al. The Journal of clinical investigation 2001). Mice were divided into five groups, including a BMT control group, aGVHD control group, aGVHD treated with IL-35 group, aGVHD treated with RAPA group and aGVHD treated with IL-35 and RAPA group. Morbidity and mortality related to aGVHD were observed, and 2 weeks after BMT, tissues from the intestine and liver were stained with hematoxylin and eosin and examined by light microscopy. To detect apoptosis in intestinal sections, a modified terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) method was applied. CD4+CD25+Foxp3+ regulatory T cells were measured by flow cytometry. Quantitative RT-PCR was used to measure the production of IFN-γ, TNF-α and IL-17A in the spleen and intestine of each group of mice. We also measured platelet aggregation using a turbidimetric aggregation-monitoring device. Finally, western blotting was conducted to test the signaling pathways of IL-35. Results: Mice receivingIL-35 exhibited a higher survival rate compared with GVHD mice as well as those mice receiving RAPA. When the two drugs were given together, the survival rate was much higher than that in the other groups. The aGVHD control group had the highest morbidity rate of aGVHD, and IL-35 plus RAPA could prevent the occurrence of aGVHD. Additionally, this treatment inhibited apoptosis of intestinal epithelial cells as well as donor T-cell infiltration into the liver, thereby ameliorating the enteropathy and liver injury caused by aGVHD. The importance of the inflammatory cytokine cascade in the pathogenesis of both clinical and experimental GVHD is now well accepted. We found that IL-35 and RAPA also markedly suppressed IFN-γ, TNF-α and IL-17A expression in the intestine and liver. Because studies by other have showed that Tregs have the ability to inhibit aGVHD, we measured Tregs in serum and found that IL-35 and RAPA treatment expanded serum Tregs. We further explored the relationship between IL-35 and platelet aggregation. Platelet aggregation was high in aGVHD mice, and the ratio of platelet aggregation was inhibited by IL-35 and RAPA. Finally, we found that the phosphorylation of STAT1 and STAT4 were inhibited in GVHD mice, and thatSTAT1 and STAT4 were phosphorylated when mice were treated with IL-35. Conclusions: IL-35 may be useful for controlling aGVHD after allo-HSCT. IL-35 suppresses inflammatory cytokines and expands anti-inflammatory cells in aGVHD. IL-35 also prevents platelet aggregation in aGVHD mice, which could be helpful in treating thrombotic complications after HSCT. These results are readily translatable to the clinic in future clinical trials. IL-35 and RAPA may have potential clinical use for the prevention or treatment of aGVHD and thrombotic complications after HSCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mesenchymal stem cell exosome-derived miR-223 alleviates acute graft-versus-host disease via reducing the migration of donor T cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weijiang Liu ◽  
Na Zhou ◽  
Yuanlin Liu ◽  
Wei Zhang ◽  
Xue Li ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
Expression Profiling ◽  
High Throughput Sequencing ◽  
Graft Versus Host ◽  
T Cell Migration ◽  
Tnf Α ◽  
Ifn Γ ◽  
Acute Graft

Abstract Background Mesenchymal stem cells (MSCs) have been utilized in treating acute graft-versus-host disease (aGvHD) as they show strong immunosuppressive capacity through the release of various mediators, including immunosuppressive molecules, growth factors, chemokines, and exosomes. MicroRNAs (miRNAs) derived from MSC exosomes (MSCs-Exo) play a critical role in the regulation of immune responses. However, the function of miRNAs in treating aGvHD remains unknown. Here, we performed expression profiling of exosome-miRNAs from human umbilical cord MSCs (huc-MSCs) and murine compact bone MSCs (mb-MSCs) to investigate their immunoregulation effects in aGvHD. Methods Huc-MSCs-Exo and mb-MSCs-Exo were isolated and constructed MSCs-Exo-derived miRNA expression profiling using high-throughput sequencing. High expression of miR-223 was identified in both kinds of MSCs-Exo by bioinformatics analysis and quantitative real-time PCR (qPCR). In vitro cell crawling assay, transmigration assay and adhesion assay were subsequently applied to investigate the regulation of miR-223 on T cells. MiR-223 target gene was analyzed by western blot, luciferase analysis, and qPCR. Moreover, murine aGvHD model was established by infusing splenocytes and bone marrow nuclear cells from C57BL/6j mice (H-2Kb) into BALB/c recipient mice (H-2Kd). For therapeutic effect, MSCs or miR-223 Agomir were injected via tail vein. The general conditions of the mice in each group were monitored. Hematoxylin-eosin (H&E) staining was used to detect pathological changes of mice spleen, liver, and intestine. Mechanistically, immunofluorescence and flow cytometry were used to evaluate donor T cell migration, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of serum inflammatory cytokines IFN-γ, TNF-α, and IL-17. Results High-throughput sequencing revealed high expression of miR-223 in huc-MSCs-Exo and mb-MSCs-Exo. MiR-223 could restrain adhesion and migration of T cells by inhibiting ICAM-1 expression in mouse lymphatic endothelial cells. MiR-223Agomir infusion attenuated aGvHD clinical symptoms, reduced the donor T cell infiltration into the spleen, liver, and intestine, and decreased inflammatory cytokines IFN-γ, TNF-α, and IL-17. Conclusion MSCs-Exo-derived miR-223 could attenuate aGvHD in mice through decreasing donor T cell migration. Our results unveil a new role of MSCs-Exo containing miR-223 in the treatment of aGvHD.

scholarly journals Expression of Regulatory γδ T Cells in Patients with Acute Graft-Versus-Host Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5784-5784
Author(s):  
Li Xuan ◽  
Li Gao ◽  
Xiuli Wu ◽  
Zhiping Fan ◽  
Fen Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Effective Treatment ◽  
Research Funding ◽  
Γδ T Cells ◽  
Guangdong Province ◽  
Expression Levels ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Ifn Γ ◽  
Acute Graft

Abstract Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the most effective therapy for hematologic malignancies. Acute graft-versus-host disease (aGVHD) is a major complication after allo-HSCT and leads to high transplant-related mortality. Regulatory γδ T cells (γδ Tregs), as a novel subset of γδ T cells with Foxp3 expression and immunosuppressive effect, are found to effectively alleviate aGVHD in mice model. However, the effect of γδ Tregs on the occurrence and development of aGVHD are not fully understood. The aim of this study is to investigate the expression levels and clinical significance of γδ Tregs in patients with aGVHD. Methods The immunophenotyping of γδ Tregs was analyzed in peripheral blood from 15 patients with aGVHD at the following two time points (at the onset of aGVHD and two weeks after the treatment), using flow cytometry. Twelve patients responded effectively to the treatment within two weeks, and 3 did not respond within two weeks. The expression levels of immunoregulatory-associated molecules (Foxp3, CD25, CTLA-4, GITR, TLR8, RORc, STAT-1 and STAT-3) were analyzed in peripheral blood, using real-time RT-PCR with SYBR GreenⅠstaining. Liquichip technology was used to analyze the concentration of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10, IL-17 and tumour necrosis factor-α (TNF-α) in cultural supernatant. Results The proportions of γδ T, Vδ1 T and Vδ2 T cells did not differ significantly between the untreated patients and those with effective treatment (P=0.308, P=0.117 and P=0.099). However, the proportions of Foxp3+γδ T, Foxp3+Vδ1 T and Foxp3+Vδ2 T cells were significantly increased after effective treatment (P=0.023, P=0.008 and P=0.028). The mRNA expression levels of Foxp3, RORc and TLR8 genes were significantly increased after effective treatment (P=0.060, P=0.001 and P=0.041). The expression levels of CD25, CTLA-4, GITR, STAT-1 and STAT-3 genes were similar between the untreated patients and those with effective treatment (P=0.95, P=0.421, P=0.605, P=0.186 and P=0.809). In addition, the concentration of IL-10 was decreased after effective treatment (P=0.005), and the concentration of IFN-γ, IL-4, IL-17 and TNF-α was similar between the two groups (P=0.662, P=0.314, P=0.152 and P=0.254). At the same time, we found that the proportion of Foxp3+γδ T cells and the expression level of Foxp3 gene both had a decreased tendency after ineffective treatment. The concentration of IL-10 had a increased tendency after ineffective treatment. Conclusions γδ Tregs might influence in the occurrence and development of aGVHD. Disclosures Liu: Science and technology planning project of Guangdong Province (2014B020226004);: Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-1, 201400000003-4, 201508020254).: Research Funding; National Natural Science Foundation of China (81270647, 81300445, 81470349, 81400141, U1401221, 81401315).: Research Funding; Natural Science Foundation of Guangdong Province (2014A030310171, 2016A030310390) ;: Research Funding.

Idiopathic Pneumonia Syndrome in Mice After Allogeneic Bone Marrow Transplantation: Association Between Idiopathic Pneumonia Syndrome and Acute Graft-Versus-Host Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4502-4502
Author(s):  
Qifa Liu ◽  
Juan Ning ◽  
Yu Zhang ◽  
Xiaodan Luo ◽  
Zhiping Fan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Normal Lung ◽  
Idiopathic Pneumonia Syndrome ◽  
Host Disease ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Ifn Γ ◽  
Acute Graft

Abstract Abstract 4502 Objective To explore the association between idiopathic pneumonia syndrome (IPS) and acute graft-versus-host disease (aGVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods An aGVHD model was established using a mouse transplant (C57BL/6□BALB/c). Chest computed tomography (CT) scans were dynamically performed and histopathology and cytokine levels in lung tissues were kinetically detected using the Linchip system in three experimental groups: mice receiving simple irradiation, syngeneic transplants and allogeneic transplants. Results The incidence of aGVHD was 100% in allogeneic transplant mice. CT revealed bilateral diffuse infiltrates in the lungs of most mice with aGVHD vs normal lung tissue in syngeneic transplant mice. Histopathology confirmed mice with aGVHD had acute pneumonitis. Immunohistochemistry showed that during aGVHD onset the infiltrates were mainly CD4+ T cells whereas the domination of CD4+ T-cell was replaced by CD8+ T-cell during aGVHD progression. TNF-α and IFN-γ levels within lung tissues of the three groups were higher than in normal controls on day +3 and +7 post-transplant. On day +7, TNF-α levels were higher in allogeneic than in syngeneic transplant mice, but there was no statistical difference in IFN-γ levels. On day +12 and +16, TNF-α levels were significantly higher in allogeneic than in syngeneic transplant mice, but IFN-γ levels were lower in allogeneic than in syngeneic transplant mice. Conclusions The aGVHD is the underlying cause of IPS. T cells and TNF-α may play a role in the pathogenesis of aGVHD-induced IPS. IPS progression may be associated with decreasing levels of IFN-γ within the lung tissues. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reduced IL-35 Levels Are Associated with Increased Platelet Aggregation and Activation in Patients with Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1174-1174
Author(s):  
Xiao-Hui Zhang ◽  
Yi Zhou ◽  
Shi-yuan Zhou ◽  
Fei-er Feng ◽  
Qian-ming Wang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Inflammatory Cytokine ◽  
Thrombus Formation ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Thrombotic Complications ◽  
Anti Inflammatory ◽  
Anti Inflammatory Cytokine ◽  
Acute Graft

Abstract Introduction: Acute graft-versus-host disease (aGVHD) is a major limitation of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The mechanism of aGVHD has not been completely elucidated. aGVHD is primarily caused by immune responses to allogeneic disparities between the donor and recipient organs (Nakamura K, et al. Bone marrow transplantation 2005). aGVHD can also lead to thrombotic complications through endothelial damage. Following endothelial injury, inflammation triggers platelet activation and leads to enhanced expression of CD62P, which can then interact with the P-selectin glycoprotein ligand 1 receptor expressed on monocytes and mediate the rolling of monocytes on activated endothelium, facilitating platelet-leukocyte aggregation and inducing platelet thrombus formation. IL-35 is a novel anti-inflammatory cytokine that suppresses the immune response. Previous work has demonstrated that IL-35 is an anti-inflammatory cytokine that suppresses the immune response through the expansion of regulatory T cells and suppression of Th17 cell development that has a protective function in various autoimmune disorders. In this study, we hypothesized that patients with aGVHD may have increased platelet aggregation, which is associated with an increased risk of thrombus formation in aGVHD. Because the increased platelet aggregation is caused by inflammation during aGVHD, and IL-35 is a novel anti-inflammatory cytokine, IL-35 could also inhibit platelet activation and aggregation in aGVHD patients. Methods: In this study, we prospectively studied a total of 65 patients who received allo-HSCT. We measured plasma levels of IL-35 inpatients just at the onset of aGVHD. We collected time-matched samples from patients without aGVHD to serve as controls after HSCT. We also detected the levels of IL-35 in granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) and G-CSF-primed bone marrow (GBM) to explore the relationship between IL-35 levels and the occurrence of aGVHD. Then, we analyzed platelet aggregation in patients with and without aGVHD. We also added IL-35 to the plasma of these patients to test its function on platelet aggregation and platelet activation. Results: The plasma levels of IL-35 were reduced in the patients with grade III-IV aGVHD (23.46 ng/ml) compared with the patients with grade I-II aGVHD (40.26 ng/ml, p<0.01) and no aGVHD (41.40 ng/ml, p<0.05). Allografts from 38 patients were analyzed for IL-35 levels with respect to aGVHD. The patients were divided into either a high IL-35 group or a low IL-35 group according to the median levels of IL-35 in the patient’s GBM (28.0 pg/ml) and PBPC (53.1 pg/ml). We found that patients in the low IL-35 group demonstrated a higher cumulative incidence of aGVHD compared with patients in the high IL-35 group. In addition, patients with aGVHD have increased platelet aggregation compared with patients without aGVHD. In vitro, platelet aggregation was inhibited by IL-35 in a dose-dependent manner. The markers of platelet activation (CD62P/PAC-1) were also inhibited by IL-35. Conclusions: The results imply that IL-35 may predict the occurrence of aGVHD, as well as inhibit platelet activation and aggregation. These data suggest that IL-35 might represent a potential effective therapeutic agent against aGVHD and prevent thrombotic complications after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Increase of Regulatory γδ T Cells Reduces the Incidence of Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2230-2230 ◽  
Author(s):  
Li Gao ◽  
Li Xuan ◽  
Xiuli Wu ◽  
Zhiping Fan ◽  
Fen Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Γδ T Cells ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Ifn Γ ◽  
Acute Graft

Abstract Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary means for treatment of hematologic and non-hematologic malignancies. Acute graft-versus-host disease (aGVHD) is a major complication associated with high morbidity and mortality after transplantation. Regulatory γδ T cells (γδ Tregs), as a novel subset of γδ T cells with Foxp3 expression and immunosuppressive effect, are found to effectively alleviate aGVHD in mice model. However, whether γδ Tregs involve in the occurrence of aGVHD in recipients of allo-HSCT remains unclear. Therefore, the aim of this study is to investigate the effect of γδ Tregs in the occurrence of aGVHD in recipients of allo-HSCT. Methods The immunophenotyping of γδ Tregs was analyzed in peripheral blood from 24 aGVHD and 24 paired non-aGVHD patients (undergoing transplantation at the same time, basic data matched), using flow cytometry. The expression levels of immunoregulatory-associated molecules (Foxp3, CD25, CTLA-4, GITR, TLR8, RORc, STAT-1 and STAT-3) were analyzed in peripheral blood, using real-time RT-PCR with SYBR GreenⅠstaining. Liquichip technology was used to analyze the concentration of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10, IL-17 and tumour necrosis factor-α (TNF-α) in cultural supernatant. Results The proportions of total γδ T and Vδ2 T cells in non-aGVHD patients were significantly higher than that in aGVHD patients (P=0.044, P=0.000), and the proportion of Vδ1 T cells was similar between the two groups (P=0.061). Compared with aGVHD patients, the proportions of Foxp3+γδ T, Foxp3+Vδ1 T, Foxp3+Vδ2 T cells and Tregs were increased in non-aGVHD patients (P=0.005, P=0.014, P=0.026 and P=0.000). There was a negative correlation between the incidence of aGVHD and the percentages of Foxp3+γδ T, Foxp3+Vδ1 T and Foxp3+Vδ2 T cells (P=0.014, rs=-0.477; P=0.001, rs=-0.610; P=0.040, rs=-0.405). The mRNA expression levels of Foxp3 and TLR8 genes were significantly increased in non-aGVHD patients compared with aGVHD patients (P=0.042, P=0.002). There were no significant difference in the expression levels of CD25, RORc, CTLA-4, GITR, STAT-1 and STAT-3 genes (P=0.234, P=0.194, P=0.056, P=0.524, P=0.142 and P=0.775). In addition, the concentrations of IL-10 and TNF-α were significantly increased in aGVHD patients (P=0.002, P=0.036), and the concentrations of IL-4, IL-17 and IFN-γ were similar between the two groups (P=0.379, P=0.414, P=0.170). Conclusions Increase of γδ Tregs might reduce the incidence of aGVHD after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Apoptotic Cells Break Tolerance Induced by CD40-CD40L Blockade.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3221-3221
Author(s):  
Jian-Ming Li ◽  
John W. Gorechlad ◽  
Cindy Giver ◽  
Christian P. Larsen ◽  
Ned Waller
Keyword(s):  
T Cells ◽  
Inflammatory Cytokine ◽  
Low Dose ◽  
Apoptotic Cells ◽  
Myeloablative Conditioning ◽  
Post Transplant ◽  
Donor Chimerism ◽  
Donor Cells ◽  
Tnf Α ◽  
Ifn Γ

Abstract Background: Non-myeloablative conditioning can avoid early post-transplant toxicity associated with myeloablative conditioning prior to allogeneic hematopoietic progenitor cell transplantation (HPCT). In order to achieve bidirectional host vs. donor and donor vs. host tolerance, we developed a transplant model utilizing a pre-transplant dose of “tolerizing” donor cells in combination with anti-CD40L monoclonal antibody (mAb) treatment (Adams et al., JI167:1103, 2001). In the current study we tested whether pre-transplant administration of apoptotic donor cells would enhance immune tolerance in a non-myeloablative model of murine BMT utilizing a low dose busulfan and anti-CD40L mAb. Methods: we tested the effect of graded doses of ionizing gamma irradiation (7.5, 15, and 30 Gy) on donor splenocytes administered as a tolerizing dose of alloantigen on 6 days pre-BMT. Mixed lymphocyte reactions (MLR) were performed using host-type lymphocytes as responders and irradiated cells as stimulators in the presence or absence of anti-CD40L mAb. Secretion of inflammatory cytokine (IFN-γ and TNF-α) and anti-inflammatory cytokine (IL-10) and cell proliferation (CFSE dilution) were measured. Results: Graded doses of irradiation (0, 7.5, 15, and 30 Gy) produced increasing frequencies of apoptosis in murine splenocytes (4, 26, 41, 49, and 27 % apoptotic cells, respectively). Irradiation of MLR stimulators abrogated the immunosuppressive effect of anti-CD40L mAb. Using MLR with untreated (non-irradiated stimulators), proliferation of CFSE-stained responder T-cells (Figure 1A) and synthesis of IFN-γ and TNF-α were decreased, while production of IL-10 increased in the presence of anti-CD40L mAb (Figure 1B). Irradiated, apoptotic stimulators led to graded increases in IFN-γ and TNF-α synthesis (Figure 1B), increased proliferation of CFSE-stained responder T-cells (Figure 1A), and decreased production of IL-10 in MLR containing anti-CD40L mAb (Figure 1B). Contrary to our predictions, mixed-chimerism without GVHD was enhanced by pre-transplant administration of viable allogeneic splenocytes, and diminished in mice with prior exposure to apoptotic/necrotic donor splenocytes at test points up to 250 days post-transplant. Psoralen plus ultraviolet A treated splenocytes had a similar frequency of apoptotic cells and led to a similar level of donor chimerism as 7.5 Gy irradiation-treated cells. The highest level of donor chimerism was observed when viable donor CD11b+ splenocytes were administered pre-transplant as the “tolerizing” cell infusion. Conclusion: The pre-transplant administration of donor splenocytes rendered apoptotic/necrotic by irradiation doses of 15 Gy or higher led to lower levels of donor BM-derived chimerism compared to transplant regimens containing viable donor splenocyte administered on prior to BMT or day −6 regimens with no “ tolerizing ” pre-transplant donor cells in a non-myeloablative model of murine BMT utilizing low-dose busulfan and anti-CD40L mAb. Bidirectional tolerance induced by anti-CD40L mAb is an active process involving viable donor cells. The mechanisms whereby anti-CD40L mAb induces tolerance via participation of donor and host regulatory elements will be discussed. Figure 1 Figure 1.

Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4099-4099
Author(s):  
Zhenhua Qiao ◽  
Xiujuan Zhao
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Hematopoietic Cell ◽  
Control Group ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Tgf Β1

Abstract Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA). Methods: MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen. Results: There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality. Conclusions: morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.

scholarly journals Itacitinib, a JAK1 Selective Inhibitor Preserves Graft-Versus-Leukemia (GVL), Enhances Survival and Is Highly Efficacious in a MHC-Mismatched Mouse Model of Acute GvHD

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4522-4522 ◽  
Author(s):  
Ashish Juvekar ◽  
Bruce Ruggeri ◽  
Sindy Condon ◽  
Andrew Borkowski ◽  
Reid Huber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Inflammatory Cytokine ◽  
Acute Gvhd ◽  
Colon Tissue ◽  
Graft Versus Leukemia ◽  
Tnf Α ◽  
Dosing Regimens ◽  
Ifn Γ

Abstract Introduction: Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK1/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (GRAVITAS-301: NCT03139604; GRAVITAS-119: NCT03320642). Methods: Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2Kd) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells from allogeneic cell transfer from donor C57BL/6 (H-2Kb) mice. Animals were dosed orally with vehicle or the selective JAK1 inhibitor, itacitinib (60 mg/kg or 120 mg/kg twice daily). Engraftment was analyzed for the proportion of donor and host leukocytes (CD45+, H-2Kb, and H-2Kd). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analysis. Colon samples were sectioned and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3 and CD3+phosphoSTAT3 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of itacitinib on preservation of Graft-versus-Leukemia (GVL) were evaluated by injecting BALB/c mice with A20 lymphoma cells that are of H-2Kd phenotype along with combination of splenocytes and T cell depleted bone marrow from C57BL/6 (H-2Kb) mice. Results: Itacitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss and improving GvHD scores. Itacitinib did not significantly impact donor engraftment as determined by CD45+/H-2Kb quantification by flow cytometry. Similar efficacy was observed with 60 mg/kg versus 120 mg/kg twice daily dosing regimens. Oral itacitinib administration achieved JAK1 IC50 coverage for 4 h and 12 h at 60 mg/kg twice daily and 120 mg/kg twice daily, respectively. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Itacitinib (120 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4+ T cells and CD8+ T cells in blood and spleen with itacitinib treatment, but significant reductions were detected in CD4+ T cells and CD8+ T cells in the inflamed colon tissue along with significant JAK1/STAT3 inhibition as measured by reductions in normalized pSTAT3 in T cells and colonic epithelial cells. Itacitinib treatment did not negatively impact GVL responses, as evidence by T cell mediated reduction of tumor burden. Furthermore, itacitinib treatment enhanced the survival of the recipient BALB/c mice in comparison to the vehicle treated animals. Conclusions: Itacitinib, a selective JAK1 inhibitor ameliorated GvHD severity when administered prophylactically or therapeutically and had no detrimental effects on engraftment and preservation of GVL. Furthermore, itacitinib inhibited JAK1/STAT3 activation in diseased colon tissue and infiltrating T-cells, and reduced disease burden and improved survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD. Disclosures Juvekar: Incyte Corporation: Employment. Ruggeri:Incyte Corporation: Employment. Condon:Incyte Corporation: Employment. Borkowski:Biomodels LLC: Employment. Huber:Incyte Corporation: Employment. Smith:Incyte Corporation: Employment.

scholarly journals Regulation of Acute Graft-Versus-Host Disease by MicroRNA-155

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 245-245
Author(s):  
Nicole Stauffer ◽  
Sayed Mehdi Hamadani ◽  
Catherine Heaphy ◽  
Ramasamy Santhanam ◽  
Sukhinder Sandhu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Cd8 T Cells ◽  
T Test ◽  
Spleen Cells ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Donor T Cells ◽  
Acute Graft

Abstract Abstract 245 Acute Graft-versus-host disease (aGVHD) is a frequent and lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT) in which donor T cells destroy HLA mismatched host tissues by secreting soluble inflammatory cytokines (TNF-α, IFN-γ) and/or inducing direct cytotoxic cellular responses. Despite recent advances, GVHD still remains a major clinical problem, underscoring the need to elucidate further its mechanisms to then develop novel therapeutic strategies. Recent studies indicate that microRNAs (miR) play critical roles in the development and function of the immune system. In particular, miR-155 is required for normal function of B and T lymphocytes. MiR-155 has been shown to be up-regulated upon B and T cell activation and mice deficient for miR-155 are immunodeficient, exhibiting T cells with attenuated INF-γ and TNF-α release in response to antigen stimulation. Based on these observations, we hypothesized that miR-155 is up-regulated in donor T cells during aGVHD and is involved in the modulation of this process. To test this hypothesis, we initially measured miR-155 expression from activated CD8 T cells obtained from an experimental animal model of aGVHD. For this, a major histocompatibility complex (MHC) mismatched HSCT model was used in which spleen cells and BM from C57BL/6 (B6) donors were transferred i.v. into lethally irradiated B6D2F1 recipient mice (n=4). Two additional groups were included as controls with one group receiving no cell infusion (radiation only, n=4); and a second group receiving T cell depleted bone marrow only (t-BM) (5×106) (n=4). GVHD scores were performed daily according to Cooke et al, Blood 1996;8:3230-9. Recipient mice were sacrificed and tissues harvested when GVHD scores were ≥7 or at the end of the experiment. After an average of 3–4 weeks, mice receiving donor spleen cells but not BM alone developed severe GVHD (scores >7) which was confirmed by histology. Activated CD8 T cells from the spleen were isolated using CD8+/CD44+ antibodies and after obtaining total RNA, miR-155 expression was measured by RT-PCR. Mice receiving donor BM plus spleen cells developed severe aGVHD and exhibited increased miR-155 expression with respect to the BM only group (fold change 5.2, t-test p<0.001). To confirm that a causal relationship exists between miR-155 and aGVHD severity, we repeated the above MHC mismatched murine experiment using BL/6 mice deficient for miR-155 expression as donors (Rodriguez et al, Science 2007;316;608-611). The groups were as follows; radiation alone (n=4), t-BM (n=8), BM + wild type (WT) spleen cells (n=8), and t-BM+ miR-155 KO spleen cells (n=8). We found that mice receiving donor spleen cells from miR-155 KO mice exhibited dramatically lower mean GVHD scores (3 Vs. 5.5, t-test p=0.0007) and improved survival compared to those receiving WT spleen cells(87% vs. 13% of mice alive at 70 days, respectively; log-rank test p<0.001). Our results showed that recipients from miR-155 KO spleen cells did not exhibit high GVHD histological scores (III-IV) in the spleen, liver or gut, while 80%, 60% and 40% of the WT did. Overall survival, GVHD scores and histological GHVD findings were similar between miR-155 KO and WT t-BM only group. Since high levels of soluble TNF-α is characteristic for aGVHD, we measured serum TNF-α by ELISA assay in mice at the time of harvest. Mice receiving miR-155 KO spleen cells had significantly lower TNF-α levels (mean 14 pg/ml) than WT controls (mean 48 pg/ml, t-test, p=0.005). Finally, to establish the relevance of this finding to the human system, we measured miR-155 expression using Locked Nucleic Acid in situ hybridization from histologically confirmed gut biopsies of aGVHD patients (n=5), and healthy controls (n=3). We found a strong up-regulation of miR-155 expression in all patients with gut aGVHD while miR-155 expression was absent in normal gut. In summary, we have shown that miR-155 expression is up-regulated in CD8 T cells from mice with aGVHD and showed that mice receiving donor lymphocyte cells deficient for miR-155 exhibited less GVHD and improved survival as compared to mice receiving WT donor lymphocytes. Finally, up-regulation of miR-155 was also found in clinical specimens from patients with gut aGVHD. Altogether our data indicate a role for miR-155 in the modulation of aGVHD, and thus point to miR-155 as a novel target for therapeutic intervention in aGVHD. Disclosures: Off Label Use: Decitabine and bortezomib in AML.

Inhibition of Graft-Versus-Host Disease (GVHD) by Histone Deacetylase Inhibitor (HDAC) SAHA Leads to Downregulation of STAT1 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1311-1311
Author(s):  
Corinna Leng ◽  
Cuiling Li ◽  
Judy Ziegler ◽  
Anna Lokshin ◽  
Suzanne Lentzsch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Histone Deacetylase ◽  
Graft Versus Host Disease ◽  
Host Disease ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Ifn Γ

Abstract Histone deacetylase (HDAC) inhibitors have been shown to reduce development of graft versus host disease [GVHD] following allogeneic bone marrow transplantation [BMT]. Administration of the HDAC inhibitor suberonylanilide hydroxamic acid [SAHA] resulted in a significantly reduced GVHD-dependent mortality following fully MHC-mismatched allogeneic BMT. Median Survival Time (MST) for vehicle and SAHA-treated mice were 7.5 days and 38 days respectively. However, SAHA treatment did not affect T cell activation nor T cell expansion in vitro and in vivo as determined by MLR assays, phenotypic analysis of donor T cells with regard to expression of the CD25 activation antigen and calculation of donor CD4+ and CD8+ T cell numbers on days +3 and +6 post-BMT. Thus, SAHA treatment was not able to inhibit the strong upregulation of CD25 antigen on CD8+ T cells observed during induction of GVHD on days +3 and +6 post-BMT. We therefore focused on the effects of SAHA treatment on efferent immune effects including cytokine secretion and intracellular signaling events in vitro and in vivo following GVHD induction. SAHA treatment broadly inhibited lipopolysaccharide [LPS] and allo-antigen-induced cytokine/chemokine secretion in vitro like MIP-1-α, IP-10, IFN-γ, TNF-α and IL-6 and led also to a significant decrease in IFN-γ and TNF-α levels in vivo following induction of GVHD. Concomitantly, SAHA treatment inhibited phosphorylation of STAT1 and STAT3 in response to LPS and allo-activation in vitro. Furthermore, analysis of liver tissue and spleens from SAHA-treated animals with GVHD showed a significant decrease in phosphorylated STAT1. In contrast SAHA treatment had only moderate effects on p38 or ERK1,2 Mitogen-activated Protein Kinase (MAPK) pathway underscoring the relevance of the inhibition of the STAT1 pathway. In conclusion, GVHD is associated with a strong induction of phosphorylation of STAT1 in the liver and spleen and SAHA-dependent reduction of GVHD is associated with systemic and local inhibition of pSTAT1 and modulation of the inflammatory cytokine milieu during the efferent immune response.

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