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 Peritoneal Cavity Cells Ameliorate Graft-Versus-Host Disease of Mice after MHC Mismatched Bone Marrow Transplantation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5675-5675
Author(s):  
Zhigang Liu ◽  
Wanhua Zhang ◽  
Xinchuan Chen ◽  
Chuan He ◽  
Jie Ji ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Survival Time ◽  
Peritoneal Cavity ◽  
Inflammatory Factors ◽  
Cd8 Cells ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Ifn Γ

Abstract Background: Peritoneal cavity cells are a group of isolated cells with unique functions, they have great anti-inflammatory, immune modulation and tissue recovering abilities. Several studies have independently reported the therapeutic function of peritoneal cavity cells on mice models with ulcerative colitis or other inflammatory bowel diseases. In the current study, we further investigated the effect of peritoneal cells on treatment of GVHD on mice models. Methods: We selected a dose of 7.0Gy of TBI for BABL/c mice. And 4 hours after TBI, BABL/c mice were infused with 5*106BMCs cells or 1*106spleen cells of C57BL/6 mice through tail veins to construct MHC mismatched myeloablative allogeneic hematopoietic stem cell transplantation model. The mice were randomly separated into two groups, which were infused with normal saline and 5* 106peritoneal cavity cells on day 0, 3, and 5. Survival time, weight changes, GVHD score were evaluated. Peritoneal cavity cells of GFP C57BL/6 transgenic mice were injected into mice recipients at day 5 after transplantation to observe the distribution of GFP positive cells in mice recipients. Flow cytometry was used to test proportion of CD4/CD8 cells and proportions of effector T cells and Naïve T cells in CD4 and CD8 cells of spleen and bone marrow in BMT group and peritoneal cavity cells group at different time points after transplantation. TNF-α, IFN-γ, IL-2, IL-17, IL-15, IL-4, IL-10 and TGF-β in plasma were studied with the method of Luminex at different time points in two groups. Expression of IL-10 and TGF-β in colons, intestines, and livers were assessed with immunofluorescence staining. Results: Mice in peritoneal cavity cells group had significant longer survival time and rapidly weight loss recover. Mice in peritoneal group had better performance in activity, unhairing, and skin changes as well. Counts of blood cells and chimeric status at day 7, 14, 21, 28 after transplantation showed that blood count recovered and stable chimerism in both groups. Small living animal imaging technology found that peritoneal cavity cells concentrate in colons and intestines after injection of GFP C57BL/6 transgenic mice peritoneal cavity cells. Fluorescence microscope showed that large amounts of green fluorocyte distributed mostly in colons and intestines, with few in liver. Flow cytometry proved that many GFP positive cells in intestines and colons (30%, and 15%, respectively), and a few in livers and lungs (approximately 5%), while negative in control group. We analyzed the lymphocyte subsets of spleen and bone marrow in two groups with flow cytometry and found that peritoneal cells treatment could increase the proportion of CD4 cells and decrease CD8 cells. In CD4 subsets, proportion of effective T cell decreased apparently 3 weeks after transplantation, and count of naïve T cells increased, which is not found in BMT group. Flow cytometry also showed that proportion of Treg cells, Th2 cells and NK cells were significantly higher in peritoneal cavity cells group, while proportion of Th1 cells were lower. TGF-β, IL-10 and IL-4 were significantly higher in peritoneal cavity cell treatment group, while TNF-α, IFN-γ, IL-15, IL-2 were lower. Immunofluorescence staining also showed that TGF-β and IL-10 were strongly expressed in colons and intestines, but not in BMT group. Conclusion: These results demonstrate that peritoneal cavity cells could ameliorate graft-versus-host disease of mice after MHC mismatched bone marrow transplantation. Survival time was prolonged, and weight loss, GVHD score, and pathologic injuries to tissues improved after infusion of 5* 106peritoneal cavity cells into BMT mice. Peritoneal cavity cells injected to BMT mice concentrate mainly in colons and intestines, which functioned as anti-inflammation and tissue repairing cells. These cells could modulate differentiation of T lymphocytes of mice recipients, decrease proportion of CD8 cells and increase CD4 cells, increase proportion of Tn cells and decrease that of Te cells in CD4 subsets, and increase proportion of Tregs, Th2 and NK cells and decrease that of Th1 cells. Peritoneal cavity cells could influence levels of cytokines by increasing anti-inflammatory factors including TGF-β, IL-10, IL-4, and significantly decreasing inflammatory factors like IFN-γ, TNF-α, and IL-15. Figure. Figure. Disclosures Liu: West China Hospital of Sichuan University: Employment.

scholarly journals Macrophage TNF-α licenses donor T cells in murine bone marrow failure and can be implicated in human aplastic anemia

Blood ◽  
2018 ◽  
Vol 132 (26) ◽  
pp. 2730-2743 ◽  
Author(s):  
Wanling Sun ◽  
Zhijie Wu ◽  
Zenghua Lin ◽  
Maile Hollinger ◽  
Jichun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Bone Marrow Failure ◽  
Murine Bone Marrow ◽  
Immune Mediated ◽  
Tnf Α ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α−/− donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR−/− recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α−/− recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α−/− recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR−/− donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α–producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.

scholarly journals The Immunomodulatory Effect of Triptolide on Mesenchymal Stem Cells in Vitro

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5011-5011
Author(s):  
Haiping He ◽  
Atsuko Takahashi ◽  
Yuki Yamamoto ◽  
Akiko Hori ◽  
Yuta Miharu ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Complete Medium ◽  
Surface Markers ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ

Background: Mesenchymal stromal cells (MSC) are known to have the immunosuppressive ability and have been applied in clinic to treat acute graft-versus-host disease (GVHD), as one of severe complications after hematopoietic stem cells transplantation (HSCT) in Japan. However, MSC are activated to suppress the immune system only upon the stimulation of inflammatory cytokines and the clinical results of MSC therapies for acute GVHD are varied. It is ideal that MSC are primed to be activated and ready to suppress the immunity (=priming) before administration in vivo. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb - Tripterygium Wilfordii Hook F (TWHF). It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aim to use TPL as the activator for umbilical cord-derived MSC (UC-MSC) to entry stronger immunosuppressive status. Methods: The proliferation of UC-MSC with TPL at the indicated concentrations for different time of 24, 48, 72, and 96 hours. Cell counting kit-8(CCK-8) was added in the culture medium to detect cell toxicity and the absorbance was measured using microplate reader. Flow cytometry was used to identify the MSC surface markers expression. TPL-primed UC-MSC were once replaced with fresh medium and co-culture with mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and irradiated allogenic dendritic cell line (PMDC05) in RPMI 1640 medium supplemented with 10 % FBS (complete medium). IDO-1, SOD1, and TGF-β gene expression in TPL-primed UC-MSC and UC-MSC induced by 10 ng/ml IFN-γ and/or 15 ng/ml TNF-α were evaluated by RT-PCR. PDL1 and PDL2 expression in TPL-primed UC-MSC and UC-MSC in response to IFN-γ and/or TNF-α were checked by Flowjo. Results: Exposure of TPL for UC-MSC for 72hour at the concentration above 0.1 μM resulted in the cell damage significantly. Therefore, we added TPL in UC-MSC at 0.01μM of TPL for up to 48 hours, then washed thourouphly for the following culture for experiments. To evaluate the influence of TPL on the surface markers of UC-MSC, we cultured UC-MSC for 4 hours in complete medium following culture with 0.01μM of TPL for 20 hours (TPL-primed UC-MSC). TPL-primed UC-MSC revealed positive for CD105, CD73, and CD90, negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules as same as the non-primed UC-MSC. In MLR suppression by UC-MSC, the TPL-primed UC-MSC activity revealed stronger anti-proliferative effect on the CD4+ and CD8+ T cells activated by allogeneic DC than those of non-primed UC-MSC in MLR. Furthermore, the TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β in response to IFN-γ+/-TNF-α by RT-PCR and enhanced the expression of PD-L1 by FACS analysis. Discussion:In this study, we found the TPL-primed UC-MSC showed stronger antiproliferative potency on CD4+ and CD8+ T cells compared with non-primed UC-MSC. TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β stimulated by IFN-γ+/-TNF-α, although TPL alone did not induce these factors. Furthermore, we found that the PD1 ligand (PD-L1) was induced in TPL-primed UC-MSC, likely IFN-γ enhanced the PD-L1 expression, evaluated by flowcytometry. These results suggested that TPL-primed UC-MSC seemed more sensitive to be activated as the immunosuppressant. Here, we firstly report the new function of TPL to induce the upregulation of immunosuppressive effect, although the mechanisms of TPL inhibition to MSC need to be explore. Conclusively, TPL-primed UC-MSC might be applied for the immunosuppressive inducer of MSC. Figure Disclosures He: SASAGAWA Medical Scholarship: Research Funding; IMSUT Joint Research Project: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding. Nagamura-Inoue:AMED: Research Funding.

scholarly journals Heat stress combined with lipopolysaccharide alter the activity and superficial molecules of peripheral monocytes

2019 ◽  
Vol 33 ◽  
pp. 205873841982889
Author(s):  
Jiajing Luo ◽  
Yi Chen ◽  
Chengjia Ding ◽  
Jialing Qiu ◽  
Yulan Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Heat Stress ◽  
Western Blot ◽  
Inflammatory Mediators ◽  
Peripheral Blood ◽  
Heat Stroke ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Rt Pcr ◽  
Tnf Α

The purpose of this study was to focus on the underlying relationship between the hyperactivity for the peripheral monocytes and heat stroke by investigating the inflammatory oxidative activity of and the expression of superficial molecules. Peripheral blood samples were collected from 10 healthy adult volunteers. Human blood monocytes were isolated by density gradient centrifugation and sequent adherent culture. The objectives were divided into four groups: 43°C heat stress combined with lipopolysaccharide (LPS) group, 43°C heat stress group, LPS group, and control group. There were 10 cases in each group. An enzyme-linked immunosorbent assay (ELISA) test was used to measure the concentrations of supernatant inflammatory mediators (tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-10 (IL-10)). After loaded by 2,7-Dichlorodi-hydrofluorescein-diacetate (DCFHDA) fluorescent probe, intracellular reactive oxygen species (ROS) levels were determined by a flow cytometry. After fluorescent microspheres incubation, the phagocytosis of monocytes was observed under a fluorescent microscope. Respectively, the flow cytometry and Western blot were used to evaluate the level of triggering receptor expressed on myeloid cells-1 (TREM-1) and Toll-like receptor-4 (TLR-4) on the monocytes. Furthermore, the mRNA expression of TREM-1 and TLR-4 was detected by real-time polymerase chain reaction (RT-PCR). The heat stress combined with LPS stimulation promoted the peripheral monocytes to produce inflammatory mediators (TNF-α, IL-1β, and IL-10) and release ROS. Otherwise, such complex strike significantly suppressed the phagocytic activity of monocytes in peripheral blood. Moreover, the expression of TREM-1, TLR-4 and CD86 was measured by the flow cytometry on peripheral monocytes which were respectively promoted by the union of heat stress and LPS. The results of Western blot and RT-PCR demonstrated the similar kinetics on these superficial molecules (TREM-1, TLR-4, and CD86) stimulated by the combination of heat stress and LPS. The underlying mechanism of the dysfunction for the peripheral monocytes may be related to the abnormal expression of superficial molecules TREM-1, TLR-4, and CD86 on the monocytes induced by heat stress and LPS.

Prophylactic Amotosalen-Treated Donor T-Cells Prevent Late CMV Infection in Allogeneic Bone Marrow Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3024-3024
Author(s):  
Mohammad S. Hossain ◽  
John D. Robak ◽  
Edmund K. Waller
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Viral Infection ◽  
Cd8 T Cells ◽  
Immune Reconstitution ◽  
Chronic Gvhd ◽  
Mcmv Infection ◽  
Tnf Α ◽  
Donor T Cells ◽  
Ifn Γ

Abstract A major problem in allogeneic BMT is post transplant immunodeficiency leading to opportunistic infection and relapse. Previously we showed that amotosalen-treated allogeneic donor T cells given at the time of BMT and experimental murine cytomegalovirus (MCMV) infection could prevent lethal MCMV disease without producing GvHD. In this study we have focused on a more clinically applicable model where prophylactic amotosalen-treated allogeneic donor splenocytes are given at the time of BMT, followed by MCMV infection 100 days later. We observed that amotosalen-treated donor T-cells significantly expanded and responded well in presence of viral infection without inducing any GvHD, protected recipients against viral disease, and were associated with significantly improved hematopoietic engraftment and immune reconstitution. Methods: Using a parent to F1 mouse BMT model, splenocytes (3x106 untreated or 10x106 amotosalen-treated) from MCMV immunized C57BL/6 donors were transplanted along with 5x106 T-cell depleted bone marrow (TCD BM) from naïve congeneic mice into lethally irradiated (11Gy) CB6F1 recipients (C57BL/6 x Balb/C). Recipient mice were infected i.p. with a sublethal dose (5x104 pfu per mouse) of MCMV 100 days or more after transplant. Clinical chronic GvHD was monitored by weight loss, hair loss, ruffled fur, diarrhea, and decreased activity. Flow cytometry was used to quantitate T cell chimerism (in recipient PBMC, spleen, liver and thymus) and MCMV-peptide specific CD8+ T-cells (tetramer+ and IFN-γ producing). Serum IFN-γ and TNF-α were determined by ELISA. Liver and spleen viral loads were determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: Recipients of untreated control donor splenocytes suffered from chronic GvHD within 100 days of transplant, while those that received amotosalen-treated splenocytes experienced no GvHD. In response to MCMV infection at 100 days post transplant, residual amotosalen-treated donor T-cells rapidly expanded over 25-fold within 10 days, but did not cause lethality or detectable GvHD. Expanded amotosalen-treated T-cells showed activated anti-viral responses and developed a memory phenotype at late phases of viral infection. PBMC, spleen and liver showed elevated levels of MCMV specific tetramer+, IFN-γ+, and TNF-α+ CD8+ T-cells that were associated with accelerated viral clearance within day 3 after viral infection. While expansion and generation of amotosalen-treated donor T-cells mostly occurred in the liver, the generation of donor bone marrow-derived new T-cells occurred through both the thymus and the liver. In contrast, recipients of untreated donor splenocytes had reduced thymic function, resulting in severely impaired immune reconstitution and decreased anti-viral immunity. Conclusion: Prophylactically administered amotosalen-treated allogeneic donor T cells 1) were almost completely devoid of GvHD activity, 2) promoted hematopoietic engraftment and improved immune reconstitution, and 3) persisted long-term (&gt;100 days) and successfully protected recipients from sublethal MCMV infection. Thus, infusion of amotosalen-treated donor T-cells at the time of transplantation is a clinically-attractive approach to adoptive anti-viral immunotherapy without chronic GvHD following hematopoietic progenitor cell transplantation.

Transcription Factor BACH2 Inhibits AP1 Proteins JunB and FosL1 in Umbilical Cord Blood (UCB) CD4+ T-Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1743-1743
Author(s):  
Mathew L. Lesniewski ◽  
Laura R. Fanning ◽  
Margeret Kozik ◽  
Richard P. Weitzel ◽  
Yeal Hegerfeldt ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Sirna Knockdown

Abstract Introduction: Umbilical cord blood (UCB) CD4+ T-cells have been shown to express significant levels of BACH2 transcription factor protein compared to adult blood (AB) CD4+ T-cells. Previously, NFAT1 siRNA knockdown of UCB T-cells exhibited a significantly higher BACH2 mRNA expression, and IFN-γ, TNF-α. and CTLA-4 mRNA levels were significantly suppressed. BACH2, a member of the b-Zip family, has been shown to act as a heterodimer with the bZip protein MafK, as a transcriptional inhibitor via recruitment of a histone deacetylase class II complex (HDAC II) in differentiating B-cells, and neurons. Due to observed inverse expression of BACH2 and NFAT1 in UCB CD4+ T-cells, we hypothesized that BACH2 may regulate transcription factors known to bind with NFAT1 including AP-1 proteins JunB and FosL1. We tested this by siRNA knockdown of BACH2 in primary UCB-derived CD4+ T-cells. Key developmental transcription factors JUNB, FosL1, NFAT1 and downstream IFN-γ, and TNF-α were mRNA analyzed. Methods: UCB T-cells were purified using autoMACs system (Miltenyi). After overnight culture, T-cells were transfected with BACH2 siRNA (Dharmacon) using Amaxa Nucleofector system (Amaxa Inc). Both siRNA treated and control cells were incubated in media for 18 hours, and then stimulated using anti-CD3/anti-CD28 antibodies (BD BioScience). Aliquots of cells were collected at specified time points post-stimulation for protein and total RNA isolation. The relative change in mRNA levels for BACH2, JUNB, FosL1, IFN-γ, NFAT1, and TNF-α were determined by Lightcycler SybrGreen real time RT-PCR system (Roche). siRNA knockdown of BACH2 protein in transfected UCB T-cells was confirmed by western blot. Results: Real-time RT-PCR of BACH2 siRNA treated UCB CD4+ T-cells stimulated with anti-CD3/CD28 antibodies and analyzed after 6 hrs of stimulation showed a 4 log increase in FosL1 and NFAT1 mRNA, a 3 log increase in JunB mRNA, a 5 log increase in IFN-γ as compared to stimulated control UCB T-cells. TNF-α mRNA was decreased by 5 logs in BACH2 siRNA treated UCB T-cells as compared to control. CD3/CD28 stimulated untransfected UCB T-cells were previously shown to have decrease expression of NFAT1, JunB, FosL1, IFN-γ, and TNF-α, and in UCB T-cells compared to stimulated AB T-cells. Conclusions: BACH2 expression correlates with an inhibition of expression of AP1 transcription regulatory proteins in UCB T-cells during primary CD3/CD28 stimulation. The complete activation of the T-cell requires the activation of AP1 by CD28 pathway otherwise the antigen presenting cell signals the T-cell to enter anergy. In UCB CD4+ T-cells express BACH2, which acts as a transcriptional inhibitor of two critical AP1 genes, JUNB and FosL1, which mediate the CD28 co-stimulatory pathway. These results further suggests that expression of BACH2 in UCB T-cells may contribute to lower incidence of alloreactivity observed in leukemia patients receiving UCB stem cells compared to AB bone marrow stem cells and thus leads to low GVHD, and contribute to the weak Th1 response seen in stimulated UCB T-cells by reduced amounts of AP1 protein available for activating the T-cell.

SAP (SH2D1A), the Immunomodulator Deficient in X-Linked Lymphoproliferative Syndrome (XLP), Is Profoundly Decreased in Aplastic Anemia: An Immunologic Link between Constitutional and Acquired Bone Marrow Failure.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1141-1141
Author(s):  
Elena E. Solomou ◽  
Valeria Visconte ◽  
Federica Gibellini ◽  
Neal S. Young
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bone Marrow Failure ◽  
Protein Levels ◽  
Ifn Γ ◽  
Th1 Polarization

Abstract Ligation of the signaling lymphocyte activation molecule (SLAM), a member of the immunoglobulin superfamily expressed in T and B cells, results in T cell activation and Th1 cytokine production. SAP is a small cytoplasmic protein expressed in T- and NK cells that controls the activation signals mediated by SLAM. On T cell activation, SAP binds to Fyn kinase; Fyn is activated and phosphorylates tyrosine residues on SLAM; phosphorylation results in the formation of a complex that selectively down-regulates co-stimulatory signals in activated T cells, resulting in inhibition of IFN-γ production. Thus SAP acts as a natural suppressor of SLAM-mediated T cell activation, and, in the absence of SAP, T cells are constitutively activated and overproduce IFN-γ. Mutations in the SAP gene lead to abnormal T cell activation and enhanced Th1 cytokine production in mouse models and in humans: about half of patients with X-linked lympoproliferative disease (XLP) have functionally disabling SAP mutations. Acquired aplastic anemia (AA) is a bone marrow failure syndrome in which hematopoietic cell destruction is effected by cytotoxic T cells and type 1 cytokines. We have recently shown that T cells from patients with AA have increased protein levels of T-bet, resulting in IFN-γ overproduction (Solomou EE et al, Blood2006; 107:3983). IFN-γ inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. We examined SAP expression as an explanation for aberrant T cell activation and extreme Th1 polarization. SAP protein expression on immunoblot was very low to absent in unstimulated T cells from 16 of 20 AA patients examined, as compared to normal levels of expression in equivalent numbers of healthy donors (p&lt;0.001). No significant differences were detected in Fyn and SLAM protein levels between AA and controls. SAP mRNA levels were also significantly decreased in T cells from those AA patients with low SAP protein levels, as determined by RT-PCR. Peripheral blood DNA samples from 18 patients with AA were analyzed for SAP mutations: three novel intronic mutations, not present in controls, were identified among 7 unrelated patients: one mutation was in the promoter region of SAP (position 106, C to T; 3 patients), and two mutations in the intron-exon junction between exons 1 and 2 (position 38975, C toT; 3 patients) and 3 and 4 (position 62771, C to A; 1 patient). IFN-γ, as measured by ELISA, in three patients with undetectable SAP protein levels was significantly increased compared to healthy controls (n=5, p&lt;0.001). Increased IFN-γ levels and Th1 polarization in AA can in part be explained by functional SAP deficiency. SAP-deficient T cells in AA would be unable to block co-stimulatory signals, leading to an activated T cell phenotype and ultimately hematopoietic cell destruction and bone marrow failure. The SAP-deficient phenotype in T cells from patients with aplastic anemia may be secondary to subtle genetic alteration in the gene’s regulation (abnormal promoter binding sites or epigenetic modulation due to mutations in introns) or as yet unidentified aberrant upstream pathways (Ets-1 and Ets-2, the transcription factors that regulate SAP expression).

Impact of Cytokine Gene Polymorphisms on Risk and Treatment Outcomes of Aplastic Anemia In Korea

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4432-4432
Author(s):  
Yun-Gyoo Lee ◽  
Ji-Hyun Kwon ◽  
Dong-Yeop Shin ◽  
Eun-Young Song ◽  
Hyun Kyung Kim ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Conflicts Of Interest ◽  
Recessive Model ◽  
Multivariate Logistic Regression Model ◽  
Control Group ◽  
Dominant Model ◽  
Cytokine Genes ◽  
Hematologic Response ◽  
Tnf Α ◽  
Ifn Γ

Abstract Abstract 4432 Introduction The clinical evidence of responsiveness to immunosuppressive therapy (IST) supports immune pathophysiology for acquired aplastic anemia (AA). Recent studies suggested that auto-reactive cytotoxic T-cells against hematopoietic cells play a key role in the pathogenesis of AA with various cytokines. The purpose of this study is to investigate whether single nucleotide polymorphisms (SNP) of cytokine genes are related to the risk of AA and, furthermore, the response to IST. Methods We analyzed 80 adult patients diagnosed as acquired AA. The 84 unrelated, age and sex matched healthy subjects served as a control group. In 3 cytokine genes (IFN-γ, TNF-α, TGF-β) and one FAS gene, we selected 10 polymorphisms on the basis of allelic frequency and the assumption of clinical relevance from previously reported associations. To assess the association between polymorphisms and risk of AA, we calculated statistical differences in allele, genotype, and haplotype distributions between patients and controls using chi-square test in 3 genetic models (dominant, recessive, and additive). For the association between polymorphisms and response to initial course of IST, we analyzed 44 patients who were treated with IST using a multivariate logistic regression model. Results Among 10 SNPs in 4 genes, one SNP and one haplotype in IFN-γ gene were significantly associated with the development of AA: IFN-γ -2353A/T; the presence of the T allele in dominant model was protective and was related to a 2.3-fold reduction in the risk for AA (P =.012); the presence of the IFN-γ TCA haplotype was related to a two-fold reduced risk for AA (P =.038). The IFN-γ -2353 T allele was shown to be resistant to IST; the presence of T allele and TCA haplotype in IFN-γ gene (dominant model) was related to a 13.2-fold reduced hematologic response at 6 months following initial IST (P =.034). However, 4 SNPs and 2 haplotypes in TNF-α gene did not show any significant associations with the response at 3 and 6 months. In terms of 2 SNPs in TGF-β gene, TGF-β P10L T/C was independently related; the T allele (recessive model) was related to a 4.3-fold reduced response to IST at 3 months (P =.038). Accordingly, the response was related to the TGF-β haplotype; the TC haplotype homozygote (recessive model) was related to a 4.6-fold reduced response to IST at 6 months (P =.036); the presence of CT haplotype (dominant model) was favorable to IST and was related to a 5.7-fold higher response at 3 months than the absence of the CT haplotype (P =.038). Conclusion This exploratory study found that the genetic polymorphism of IFN-γ was susceptible to the development of AA and was related to the hematologic response following initial IST. In case of TGF-β, the polymorphisms were related to the response to IST, though they were not be related to the disease susceptibility. Large studies with a prospective design are needed to better understand the determinants of risk of AA and responsiveness to IST. Disclosures: No relevant conflicts of interest to declare.

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 Oligoclonal Expansion and T-Cells Subpopulations in Patients with Aplastic Anemia at Different Stages of the Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3864-3864
Author(s):  
Anastasia V. Abramova ◽  
Elena A. Mikhaylova ◽  
Zalina T. Fidarova ◽  
Yuliya O. Davydova ◽  
Nikolay M. Kapranov ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Memory T Cells ◽  
Clonal Expansion ◽  
Th Cells ◽  
Healthy Donors ◽  
Oligoclonal Expansion

Abstract Background. The main mechanism of the bone marrow (BM) failure in idiopathic aplastic anemia (AA) has an immunomediated character. Researching the T-cell clone's effect in the AA pathogenesis is very relevant at the present time. Oligoclonal expansion of T cells is frequent in AA and the identification of immunodominant T-cell clones can correlate with the disease activity and may possibly serve as response predictor to immunosuppressive therapy (IST). The aim. To identify T-cells subpopulations, expression of PD-1 and PD-L1 on T-cells and TCR-Vβ repertoires by flow cytometry in different groups of AA patients. Methods. Thirty AA patients (pts) with median age of 30.5 (19-71), m/f ratio 1:1,3 were divided in 3 groups: pts with newly diagnosed (ND) AA (n=13), pts with overall response to IST (OR) (n=10), non-response pts (NR) for 2 and more lines of IST (n=7). Flow cytometry was performed with BD FACS Canto II. We used commercial kit (IOTest® Beta Mark TCR Vb Repertoire) for evaluation of TCR-Vβ repertoire in the bone marrow (BM) of these patients. We performed analysis of BM samples from healthy donors as a control group (n=8). Due to low amount of donor samples the maximal value each of the 24 subclones (for CD4+ (T-helpers - Th) and CD8+ cells (T-cytotoxic cells - TCL)) was accepted as threshold. We concluded the presence of clonal expansion if TCR subclone exceeded this threshold. We identified different T-cell subpopulations in all 3 groups of AA and healthy donors by flow cytometry: double positive T-cells (CD3+CD4+CD8+), double negative T-cells (CD3+CD4- CD8-), Th (CD3+CD4+), TCL (CD3+CD8+), NK-T-cells (CD3+CD56+) out of CD3+ cells. Among Th and TCL cells was determined naive T-cells (CD28+CD95-), effector T-cells (CD28-CD95+), memory T-cells (CD28+CD95+), regulatory T-cells (CD4+CD127-CD25high) and subpopulations Th and TCL co-expressed PD-1 and PD-L1. Multiple comparisons were assessed by ANOVA or Kruskal Wallis test by GraphPad Prism software. Results. In our study all 30 AA patients had an immunodominant TCR-Vβ clones among Th and/or TCL cells. We identified the most common clonotypes in comparison with healthy donors - Vβ1, Vβ2, Vβ3 among the Th cells and Vβ3, Vβ9, Vβ13.1 among the TCL cells. In ND group Vβ1 was highly expanded in 5 (38.5%), Vβ3 - in 7 (53.8%) pts among Th, and Vβ3 - in 3 (23.1%) and Vβ9 - in 4 (30.8%) out of 13 pts among TCL. In OR group Vβ2 expansion was in 4 (40%) and Vβ3 - in 5 (50%) pts among Th; Vβ3 in 6 (60%) and Vβ9 in 6 (60%) out of 10 pts among TCL. In NR group the most frequent was Vβ13.1 clone in TCL - in 3 (42.9%) out of 7 pts. In NR group in overall clonal expansion was less frequent than in ND and OR groups. We also analyzed the previously mentioned subpopulations of T-cells in patients with AA in three groups (ND, OR, NR) compared to healthy donors (table 1). We obtained significant differences in the count of naive Th and TCL cells, memory T-cells in all three groups of AA patients compared to donors: proportion of naive Th and TCL cells was significantly higher and proportion of memory Th cells was lower in the donor group than in AA pts. The percent of TCL effectors was higher in ND AA pts compare to donors. We also found that cell count of activated Th (CD4+CD25+) was higher in the group of refractory pts. In OR pts proportion of PD-1-positive Th was higher than in donors. In NR pts Th and TCL co-expressed with PD-L1 were lower compare to donors (table 1). Conclusions. In our study we found immunodominant clonotypes in different AA pts and depletion of the pool of naive T cells. Dynamic observation of changes in the most common clonotypes in AA pts during treatment will provide suitable therapy tactics (allogenic bone marrow transplantation or IST). Disclosures No relevant conflicts of interest to declare.

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