Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3464-3464
Author(s):  
Yang Song ◽  
Yu-tong Wang ◽  
Xiao-jun Huang ◽  
Yuan Kong
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Endothelial Cells ◽  
Th17 Cells ◽  
Immune Thrombocytopenia ◽  
Perivascular Cells ◽  
Platelet Production ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5195-5195
Author(s):  
Lulu Lu ◽  
Yongping Song ◽  
Baogen Ma ◽  
Xiongpeng Zhu ◽  
Xudong Wei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Lymphocyte Subsets ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Abstract Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft. Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls. Results: The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P &lt;0.05). The difference between cord blood and mobilized peripheral blood was not significant (P&gt;0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P&lt;0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P&lt;0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P &lt;0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P&lt;0.01). Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.

Endothelial Cell Secretion Combined with Shear Stress Strongly Increase Platelet Production by Human Megakaryocytes,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3411-3411
Author(s):  
Yasmine Ouzegdouh ◽  
Laurence Momeux ◽  
Claude Capron ◽  
Elisabeth Cramer Bordé
Keyword(s):  
Electron Microscopy ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Conflicts Of Interest ◽  
Video Microscopy ◽  
Platelet Production ◽  
Platelet Size ◽  
Platelet Release

Abstract Abstract 3411 It is well established that megakaryocytes (MK), in order to achieve terminal maturation in the bone marrow, migrate from the osteoblastic niche to the vascular niche, close to medullary sinusoids. It is also admitted that, in order to release platelets, MK extend cytoplasmic extensions in the lumen of the sinusoid followed by platelet release. Therefore, we have investigated the role of endothelial cells combined with shear stress on MK late differentiation steps and platelet production. To do so, human MK were grown from umbilical cord blood or bone marrow CD34+ cells, in the presence of Stem Cell Factor (SCF) and thrombopoietin for 10 days. Then they were co-cultured with human endothelial cells (HUVEC) for 3 days and analysed by video-microscopy, flow cytometry, immunofluorescence and electron microscopy. In these conditions, most MK (>50%) extended numerous and prominent proplatelets: immunofluorescence showed virtually complete unwinding of their cytoplasm, which extended in the form of long proplatelets; this rarely occurred in control MK. Electron microscopy showed that these MK were formed by a coarse chromatin nucleus surrounded by a thin cytoplasmic ring and surrounded by platelet-size territories displaying alpha-granules, mitochondria and canalicular system which coincided to the sections of the proplatelet swellings and to the unwinding of the demarcation membrane system. Another type of experiment of co-culture MK/endothelial cells was conducted in transwells and led to similar results, ie, increase of MK cytoplasmic maturation and proplatelet formation indicating that cell/cell contact was not necessary for up-regulating proplatelet production, rather that (a) soluble product(s) was(ere) secreted from endothelial cells. Flow cytometry, at this step, failed to demonstrate significant changes in the platelet production rate. We then submitted MK co-cultured with endothelial cells to shear stress and examined them by flow cytometry and video microscopy. We could then demonstrate that platelet release was strongly increased (× 3.8 ± 0.9, n=3) when MK had been in contact with endothelial cells compared to control MK. The released platelet-size particles expressed CD41 and, when stimulated by thrombin, were also able to express CD62P. Video-microscopy confirmed that proplatelet and platelet shedding occurred after exposition of mature MK to shear stress. When examined by video-microscopy, live MK whose DNA had been stained with the fluorescent dye Hoechst 33342 showed that nuclear lobes separated under high shear stress: indeed they became located at opposite poles of the cell, while the cytoplasmic volume extended and elongated, becoming organized in proplatelets which exhibited a succession of platelet size subunits; eventually cytoplasmic scission occurred in parallel with MK nuclear lobe segregation in distinct cell fractions, each carrying proplatelets; proplatelets subsequently fractionated and were then released from the cell core containing the fluorescent nuclear lobe. Thus shear stress leads both to cytoplasm and nucleus fragmentation. This is a dynamic explanation to the fact that entire MK nuclei are rarely found in the human bone marrow. This observation also gives sense to the unique phenomenon of MK polyploidy. In conclusion, this study indicates that endothelial microenvironment combined with circulatory shear forces are determinant up-regulating factors which increase platelet production. It also shows that shear stress is able to induce nuclear as well as cytoplasmic MK fragmentation, leading to a new anatomical concept of circulating platelet shedding MK subunits. Disclosures: No relevant conflicts of interest to declare.

Increased Number of IL-17 Producing CD4+ T Cells in Low Risk Myelodysplastic Syndrome (MDS)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 637-637
Author(s):  
Shahram Y Kordasti ◽  
Behdad Afzali ◽  
Ziyi Lim ◽  
Wendy Ingram ◽  
Janet Hayden ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
High Risk ◽  
Autoimmune Diseases ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Absolute Number ◽  
Low Risk ◽  
Healthy Donors ◽  
Significant Difference

Abstract Immunological responses are increasingly recognized as important in the initiation and progression of MDS. Indeed, autoimmune diseases commonly occur in association with low risk MDS. Different studies, including our own, have shown that Foxp3+ Tregs play a role in the inhibition of tumour immune surveillance in high risk MDS; however, less clear are the potential factors which are inducing inflammatory response in low risk MDS that may also predispose to autoimmunity. CD4+IL 17 producing T cells (Th17) are considered as important pro-inflammatory T cells and contribute to the pathogenesis of a number of autoimmune diseases as well as some malignancies. The aim of this study was to investigate the potential role of Th17 cells in low risk MDS. We show for the first time that Th17 cells are markedly increased in low risk MDS in comparison to high risk disease and healthy age matched controls. We also describe an inverse relationship between numbers of Th17 cells and naturally occurring Tregs in MDS. 88 patients with newly diagnosed MDS, and 15 age matched healthy donors, were recruited. Analysis of Th17 cells was performed in a subset of 43 patients. WHO classifications were as follows: 19% had RA, 33% had RCMD, 32% had RAEB I & II and 16% had 5q- syndrome. A clear difference was observed in the median percentage and absolute number of Th17 cells between patients with low risk (based on IPSS) compared to high risk MDS (p&lt;0.01). In low risk, but not high risk MDS, CD4+ cells had greater median number of committed Th17 cells than healthy donors (p&lt;0.01). The absolute number of polyclonal Foxp3+ Tregs inversely correlated with Th17 cells, with higher Tregs in the high risk MDS when compared to low risk MDS or healthy donors (p&lt;0.001), consistent with our previous report. Analysis according to IPSS, BM blast percentage or WHO classification revealed the same patterns of Th17 and Tregs expression, with the higher risk phenotypes being associated with significantly higher Treg number and lower risk phenotypes with greater Th17 cells. This finding was specific for Th17 as there was no significant difference in the number of circulating Th1 and Th2 cells between MDS subtypes. Co-culture of Tregs from patients with MDS with their own T effectors lead to suppression of supernatant IFNγ levels. Importantly, Tregs did not have an inhibitory effect on IL17 production and indicates that reduced Th17 number in the presence of high Tregs does not indicate active suppression of the Th17 phenotype. To investigate differences in serum cytokines between low and high risk MDS and healthy donors, serum samples from patients were analyzed by Luminex for multiple cytokines. The cytokines IL12(p&lt;0.01) IL7(p&lt;0.005) IFNγ(p&lt;0.01) and RANTES (p&lt;0.005) were significantly higher in patients with low risk compared to high risk MDS and healthy controls. The inhibitory cytokine IL10 and soluble IL2 receptor were present at higher levels in the serum of patients with high risk MDS (p&lt;0.01 and p&lt;0.005 respectively). To confirm that the peripheral blood cytokines are a true reflection of cytokine concentration in the bone marrow, the concentration of 30 different cytokines was measured by Luminex in 10 matched bone marrow and peripheral blood samples from these patients and there was no significant difference between these two sets of samples. In addition, the level of apoptosis, assessed by TUNEL assay, was also higher in the bone marrow of low risk patients. In conclusion, the “unfavourable” Th17 to Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and bone marrow apoptosis. These findings may help identify subtype of low risk MDS patients likely to respond to immunosuppressive therapy.

Immune Derangements in Patients with Myelofibrosis- the Role of Treg, Th17, and sIL2α

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1763-1763
Author(s):  
Hemant Sindhu ◽  
Ajay Kundra ◽  
Chi Chen ◽  
Jen-Chin Wang
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
Th17 Cells ◽  
Interleukin 2 ◽  
Treg Cells ◽  
Cd4 Cells ◽  
Treg Function ◽  
Significant Difference ◽  
Normal Controls

Abstract Abstract 1763 Primary myelofibrosis (PMF), myelofibrosis post essential thrombocytosis (MF-ET), and myelofibrosis post polycythemia vera (MF-PV) have been reported to be associated with autoimmune phenomena, such as Coombs positive anemia, lupus anticoagulant, positive ANA and the presence of circulating immune complex, etc. Regulatory T cells (Treg) and IL-17 producing cells (Th17) have been known to play important roles in modulating immune responses. Hence we studied Treg cells in 38 patients with MF including PMF (25), MF-ET (8), MF-PV (5) and compared them to other MPD patients including ET (7), PV (14), and normal controls (16). Patients on lenalidomide or Pomalidomide were excluded. Blood ( 106 MNCs) were stained for flow cytometry analysis using the Treg Detection Kit (Miltenyi Biotec). The number of Treg cells was calculated as the percentage of positive CD4+ CD25+ FoxP3+ T cells from the number of gated CD4+ cells. Treg function was evaluated by XTT cell proliferation assay (Invitrogen) with ratios of Treg to T-effector cells at 1:1 in the presence of anti-CD3 and CD28 micro-beads (Invitrogen). The results (mean + SE) showed numbers of Treg cells in MF were 0.79 + 0.080, other MPD were 1.27 + 0.20, and normal controls were 1.21 + 0.30. No significant difference was found among the three groups. Treg function was evaluated in MF (18 patients). MPD (21) and controls (16). No significant difference was found among the three groups. Th17 cell assays were performed by culturing Blood CD4+ cells in IMDM medium and stimulated with PMA (25ng/ml), ionomycin (1 ug/ml ) and monensin ( 500ng/ml) for 4h at 37°C with 5% CO2. Then Th17 was stained with Th17 Flow Kit (Biolegend) and analyzed by flow cytometry. The results were expressed as % of isolated CD4+ cells (mean ± SE) as follows: MF (2.31 ± 0.65) (n=15), MPD (1.31 ± 0.32) (n=7), and controls (1.89 ± 0.44) (n=10). No significant difference was found among the three groups. We further studied the soluble interleukin 2 alpha subunit (sIL2α) levels. sIL2 α were measured in plasma by ELISA kits, results were expressed as (mean± SE ) ( pg/ml) as follows: MF ( 3534± 298) (n=18), MPD ( 2303 ± 171) (n=22) and controls ( 1734 ± 115) (n=16), P values were <0.05 in MF vs MPD and MF vs Controls, and P=NS in MPD Vs controls. These results confirm our previous observation (Br J Haematol,1994), that sIL2 α levels were significantly elevated in patients with MF, compared with MPD patients and controls. We further studied the effects of the sIL2α on the immune function in MF patients: A) Effects on the Th1, Th17, and Treg cells. CD4+ cells after isolation were cultured for 7 days in IMDM containing IL-2 (100ug/ml) and anti- CD3CD28 micro-beads, with or without recombinant sIL-2Rα (100ng/ml). The cells then were stimulated with PMA (20ng/ml) ionomycin (1ug/ml) and monensin (1uM) for 4 hours before harvest. Then Th1 Th17 and Treg were numerated by flow cytometry. The results showed no difference in Th1 and Th17 cells in cultures with and without sIL2α, but sIL2α significantly increased the numbers of T reg (1.71 +.28,P=0.02) ( fold increase). B) Effects on the Treg function. Viable CD4+CD25− (106) cells were labeled with CFSE ( Invitrogen), then added unlabeled Tregs at 1:1 (responder: Treg) ratio and stimulated with 50 μl of anti CD3/CD28 micro-beads with and without sIL2α for 4 –7days at 37 °C in a 5% CO2 incubator. Cell proliferation was measured by counting the percentage of CFSEdim cells. The results showed that sIL2α significantly increased the CFSE dim cells. Therefore sIL2α suppressd the T reg cell function and increase the T responsive cell proliferation. C) Identification of cells producing sIL2α. CD4+, CD14+, CD8+, CD20+, and Treg were isolated using isolation kits (Miltenyi ), then cultured either with PHA or anti CD3/CD28 micro-beads. ELISA assay for sIL2α were then performed on the cultured supernatant. The results (mean ± SE) ( pg/ml) showed that CD4+ cells produced 342 ±152.6; CD8 +,71.6 ± 19.8; Treg, 306.9 ± 53.5, while CD14+ and CD20+ cells produced negligible quantity of sIL2α. Thus Treg cells were the cells predominantly producing sIL2α in patients with MF. We conclude that in patients with MF, numbers of Treg and Th17 cells were not different from controls or other MPD patients, but Treg cells produce significantly increased amount of sIL2α which further inhibits Treg function and results in autoimmune phenomenon observed in patients with MF. Disclosures: No relevant conflicts of interest to declare.

Vascular Niche and Platelet Production: Stromal Cells Inhibit Proplatelet and Platelet Formation by Human Megakaryocytes (MK), Whereas Endothelial Cells Are Permissive

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 402-402
Author(s):  
Yasmine Ouzegdouh ◽  
Laurence Momeux ◽  
Elisabeth Cramer-Borde
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Growth Factors ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Types ◽  
Platelet Production ◽  
Proplatelet Formation ◽  
Platelet Formation

Abstract Abstract 402 The site of platelet production in the human body is still unclear, but several evidence, including the recent demonstration by our team that shear stress in vitro accelerates platelet formation, favour the hypothesis of an intravascular location of the platelet release process. Therefore we have undertaken the following study to compare the effect of two cell types from the human bone marrow microenvironment, stromal medullary cells and endothelial cells, on the final steps of megakaryocyte (MK) maturation and platelet production. Firstly, supporting the goal of our study, we show that entire mature MK can be encountered in the lumen of normal bone marrow sinusoids and therefore enter the circulation of flowing blood; Secondly, we have used a new microcapillary device coated with von Willebrand factor and were able to confirm by videomicroscopy that exposure of human mature MK to shear forces accelerates proplatelet extension and facilitates platelet liberation from the tips of proplatelets. During this process, the nuclear lobes of polyploid MK tend to separate and to form distinct proplatelet units ready to deliver platelets: this observation might explain why massive senescent MK nuclei are only rarely found in human tissues, bone marrow or lung. Then, we used flow cytometry, optical and electron microscopy, to document and visualise the interaction of human medullary stromal cells (HS5), murine medullary stromal cells (MS5), and human endothelial cells (HUVEC) with the final steps of human MK maturation, namely proplatelet formation. MK were grown from umbilical cord blood CD34+ cells in the presence of Stem Cell Factor (SCF) and thrombopoietin and co-cultured with stromal cells between day 10 and day 13 of culture. Stromal cells virtually completely inhibited proplatelet formation from MK, whereas endothelial cells showed no effect and allowed the emission of proplatelets. EM and flow cytometry confirmed that stromal cells blocked proplatelet and platelet formation and showed that this was accompanied with a considerable development of demarcation membranes which coincided with a 60% increase of CD41 expression by MK. In parallel, stromal cells induced a reduction of apoptosis signs with a reduction of annexin V fixation by maturing MK. Since the step of proplatelet formation is accompanied with cytoplasmic apoptotic signs, among which increased phosphatidyl serine exposure, this result is in accordance with the observed blockade of proplatelet extension. Stromal cell secretion include several growth factors, namely SCF and GMCSF. MK were cultured in the presence of stromal cell culture supernatants, and with these growth factors either individually or mixed together, but this had no effects on proplatelet production, suggesting that physical contacts between the two cell types are necessary. In conclusion, our data show that human bone marrow microenvironment (stromal cells) has an inhibitory effect on proplatelet and platelet formation whereas the vascular microenvironment (endothelial cells) is permissive. This may explain the absence of proplatelets in the bone marrow parenchyma and be an additional evidence that platelet formation and release has to mainly occur in the intravascular compartment. Disclosures: No relevant conflicts of interest to declare.

The CXCR4 Inhibitor Plerixafor Effectively Mobilizes Primary AML in NODscid IL2Rγc−/− Xenografts and Markedly Reduces but Does Not Eradicate Leukemia in Combination with Cytarabine +/− Clofarabine Chemotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1432-1432
Author(s):  
Sylvia Chien ◽  
Xin Zhao ◽  
Thalia Papayannopoulou ◽  
Frederick R. Appelbaum ◽  
Pamela S. Becker
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Fold Increase ◽  
White Blood Count ◽  
Saline Control ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Total Body

Abstract Abstract 1432 Background: The origin of relapse in AML is believed to be related to persistence of resistant “leukemia stem cells.” in the bone marrow microenvironment where adhesion confers drug resistance. Engraftment of human AML in immunodeficient mice is dependent on CXCR4 (Tavor et al 2004). CXCR4 inhibitors, such as AMD3100 (plexiglass,hereafter P), overcome adhesion mediated chemotherapy resistance (Zeng et al 2006) and mobilize human leukemia engrafted in immunodeficient mice (Zeng et al 2009). P also mobilized leukemia in an APL murine model and in combination with chemotherapy reduced tumor burden (Nervi et al 2009). Methods: We studied the combination of P 5mg/kg daily sc X 3, cytarabine (araC=A) 300mg/kg IP X 3 and clofarabine (C), 20mg/kg IP X 3 in the NODscid IL2R γc−/− mouse engrafted with primary patient AML CD34+ cells after 350 cGy total body irradiation. We could first detect circulating human CD45+ or human CD34+ cells, denoting engraftment, by flow cytometry as early as 5–13 weeks. We then injected plerixafor to assess mobilization capability at 8–16 weeks, followed by the combination of plerixafor and chemotherapy. Animals were sacrificed by 14–38 days after chemotherapy, and assessed for AML in blood, marrow, and spleen. Results: A single 5mg/kg dose of P, produced a 2.26 ± 0.94 (SD) fold increase in peak mobilization (at 2 hours) compared to saline control, p=0.026. P-induced mobilization was directly related to expression of CXCR4, with a patient exhibiting 10.3% CXCR4 showing 0.86× baseline, as compared to a patient with 24.7% CXCR4 exhibiting a 2.2-fold increase, and 84.9% CXCR4, a 3-fold increase. Chemotherapy,described above, was given 2 hours after plerixafor. For animals that received P/A vs. P/A/C, there was no statistically significant difference in leukemic burden (in millions of human CD34+ AML cells ± SD) of the animals sacrificed 14 days after initiation of treatment: bone marrow six bones 116.3 ± 33.7 vs. 111.7 ± 29.2 (p=0.86), spleen 50.8 ± 10 vs. 43.7 ± 19.1 (p=0.59), blood 10.9 ± 9.6 vs. 3.1 ± 1.4 (p=0.16), or estimated total body burden 178.0 ± 45.3 vs. 158.5 ± 30 (p=0.52). A comparison of 4 groups of animals, P/A/C vs. P/A vs. A/C vs. A demonstrated a statistically significant difference between certain groups, at certain time points. For example, on day 10, P/A/C treated animals had a lower human PB CD34+ count than P/A, 0.07 vs. 0.24 × 109/L (p=0.034). On day 14, P/A/C had a lower CD34+ count than A/C, 0.08 vs. 0.16 (p=0.047). But at day 38, the leukemia had already recurred, and there was no statistically significant difference in the organ or total body involvement by the leukemia. There was a very low white blood count days 5–24 post chemotherapy, with only minimal residual disease detectable by flow cytometry (analogous to a period of remission in humans), but by day 38, the leukemia had recurred (Figure 1), and there was no statistically significant difference in the organ or total body involvement by the leukemia amongst the groups (Figure 2). Conclusion: This model demonstrates the efficacy of chemotherapy in reducing circulating leukemia, but resistant cells appear to remain sheltered and give rise to relapse. Despite the beneficial effects of CXCR4 inhibitors in vitro and in vivo in a murine APL model, the combination of plerixafor with chemotherapy did not prevent or postpone leukemic relapse. These results could be attributed to either inefficient scheduling of plerixafor (for example, continuous infusion may have worked better), or non-cycling cells may be preferentially mobilized (Bonig H et al., 2009), and thus less susceptible to cytarabine treatment. These concepts are currently being explored. Alternatively, concomitant inhibition of other adhesion receptors may be necessary to prevent leukemia from homing back to the marrow. Precise timing and degree of mobilization in combination with chemotherapy may be required to optimize this approach in the clinic. Disclosures: Becker: Sanofi-Oncology (Genzyme): Research Funding.

scholarly journals Analysis of Interleukin-17 Levels in Patients with Thrombocytopenia

Biomedika ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 68-73
Author(s):  
Lidwina Septie Christyawardani ◽  
Mansyur Arief ◽  
Uleng Bahrun
Keyword(s):  
Blood Circulation ◽  
Immune Thrombocytopenia ◽  
Research Unit ◽  
Comparative Test ◽  
P Value ◽  
Interleukin 17 ◽  
Platelet Production ◽  
Immune Mediated ◽  
Significant Difference ◽  
Primary Immune Thrombocytopenia

Thrombocytopenia or platelet deficiency is a condition, in which platelet level in the blood circulation is below normal, which is less than 150,000 cells/µl. Thrombocytopenia is classified into some conditions, including decreased platelet production, increased need for platelets, and other thrombocytopenia. The need for increased platelets can be subdivided into primary immune thrombocytopenia, secondary immune thrombocytopenia, non-primary ITP, and thrombocytopenia that are not immune-mediated. Several cytokines play a role in the process of thrombocytopenia, one of which is Interleukin-17 (IL-17) that will be further discussed in this study. A previous study reported that IL-17 production increased in ITP and cITP patients. The objective of this study was to analyze the IL-17 levels and figure out the differences in IL-17 levels in the serums of patients with primary ITP and secondary ITP. The samples were taken from Wahidin Sudirohusodo Hospital and the specimens were examined in the Research Unit Laboratory of the Faculty of Medicine, Universitas Hasanuddin/Hospital of Universitas Hasanuddin. The comparative test resulted in p-value = 0.005, where p <α = 0.05; and therefore, there was a significant difference between IL 17 levels in ITP and non-primary ITP.

scholarly journals T Cell Subsets and Associated Cytokines in Chronic Graft-Versus-Host-Disease Using G-CSF Primed Bone Marrow in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4580-4580
Author(s):  
Monica M Rivera Franco ◽  
Eucario Leon Rodriguez ◽  
Diana Gomez Martin ◽  
Javier Merayo Chalico ◽  
Jorge Alcocer Varela
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Th17 Cells ◽  
Mononuclear Cells ◽  
Chronic Gvhd ◽  
The Other ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Ifn Γ

Abstract Background Graft versus host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation. It is characterized by an imbalance between the effector and regulatory arms of the immune system which results in the over production of inflammatory cytokines. Regulatory T (T regs) cells and T helper 17 (Th17) cells are two recently described lymphocyte subsets with opposing actions. Both can develop from naïve CD4+ T cell precursors under the influence of TGFβ1. Th17 lymphocytes, are key effector cells in rodent models of human diseases including GVHD. The other subset, T regs, is essential for dominant immunologic tolerance. At our institution, patients transplanted using G-CSF primed bone marrow (G-BM), have a lower incidence of acute and chronic GVHD when compared to those transplanted with peripheral blood and not primed bone marrow. Some microenvironment characteristics of this hematopoietic stem cells (HSC) source remain unknown, as well as the difference between Tregs, Th17 and cytokine levels in patients who develop GVHD and those who do not. Objective To analyze the characteristics of thirty-eight G-BM donor samples, identifying lymphocytes subsets and associated cytokines, and comparing patients who developed chronic GVHD (cGVHD) and those who did not. Materials and Methods A prospective analysis was performed in 38 G-BM samples from donors from 1999 to 2016. Mononuclear cells were defrosted, counted, and viability was evaluated. A 24 hour resting with RPMI, and posterior activation with PMA (50 ng/ml) for 48 hours was performed. Cells were harvested and cytokines were evaluated by flow cytometry (CBA assay). From each sample, one million mononuclear cells were permeabilized, fixed, and stained with CD4-FITC, IL17A-PE, IFN-γ APC, and IL-4 PECy7, for their posterior phenotipication by flow cytometry. The samples were obtained in a BD LSR Fortessa cytometry, and analyzed with the Flow-Jo software. Patients (recipients) information was analyzed using SPSS v.21. Results GVHD incidence was reported as following: Three (8%) patients developed acute GVHD (2 grade II, and 1 grade IV), 11 patients (29%) developed chronic GVHD (9% extensive, and 91% limited), and 24 patients did not present either. Mononuclear cells from G-BM from donors of patients who developed cGVHD showed a pro inflammatory response, characterized by an increased concentration of IL-17A (15.5 vs 0.71 pg/mL, p=0.013), TNF-α (80.27 vs 0.13 pg/mL, p=0.001), and IL-6 (4953.6 vs 11.75 pg/mL, p=0.025), after a mitogenic stimulation, compared to cells from donors of patients who did not developed GVHD. On the other hand, a decreased IL-10 production (2.62 vs 52.81 pg/mL, p=0.001) was documented in mononuclear cells from donors of patients who developed chronic GVHD, compared to donor cells of patients who did not. No significant difference in the production of IL-2, IL-4, and IFN-γ was observed. There was no difference in Th1 and Th2 between both groups, but mononuclear cells from donors of patients who developed chronic GVHD had a higher percentage of Th17 (1.02% vs 0.46%, p<0.001), and less Tregs (0.88% vs 1.95%, p<0.001), compared to those who did not developed GVHD. Conclusions Patients who develop cGVHD (29%) are characterized by a pro inflammatory response with an increased production of IL-17A, IL-6, and IFN-γ, and also a major percentage of Th17 cells. Also, a decreased suppressive response was documented with reduced IL-10 and Tregs levels. The low incidence of cGVHD show that G-CSF primed bone marrow is an excellent source for allogeneic HSC transplantations, and would be useful to compare these results with other HSC sources. Disclosures No relevant conflicts of interest to declare.

Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽  
2017 ◽  
Vol 27 (1) ◽  
pp. 49-59 ◽  
Author(s):  
X Yang ◽  
J Yang ◽  
X Li ◽  
W Ma ◽  
H Zou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Lupus Nephritis ◽  
Bone Marrow Cells ◽  
Tfh Cells ◽  
Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

Genotypic Representation of Myelodysplastic/Myeloproliferative Neoplasms in Nrg, Nrg-3GS and Srg-W41 Mice with Transgenic Expression of Human Cytokines

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2038-2038
Author(s):  
Hein Than ◽  
Naoto Nakamichi ◽  
Anthony D. Pomicter ◽  
John O'Shea ◽  
Orlando Antelope ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Myeloproliferative Neoplasms ◽  
Model Systems ◽  
Juvenile Myelomonocytic Leukemia ◽  
Functional Screening ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Myelomonocytic Leukemia

Abstract Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are complex clonal hematopoietic stem cell malignancies with overlapping dysplastic and proliferative features. Genomic analyses have charted the somatic mutation spectrum of MDS/MPN and revealed a major role for epigenetic dysregulation in their pathogenesis. No disease-modifying therapies are currently available, as progress has been hampered by a lack of genetically faithful in vivo model systems suitable for the preclinical development of new strategies. Yoshimi et al (Blood. 2017;130:397-407) recently showed that patients' chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) cells transplanted into NOD/SCID-IL2Rγ-/-mice expressing human IL3, GM-CSF and SCF transgenes (NSG-3GS mice) produced xenografts that had mutations characteristic of the input cells. Since we had demonstrated a superior level of chimerism achieved from transplants of normal human CD34+cord blood cells in SirpaNOD/Rag1-/-/IL2rγc-/-/W41/41mice with c-KIT deficiency (with an otherwise mixed NOD-C57Bl/6 background - SRG-W41 mice) compared to conventional NSG or NRG hosts (Miller et al. Exp Hematol. 2017;48:41-49), it was of interest to explore their use as hosts of samples from patients with MDS/MPN: CMML, atypical chronic myeloid leukemia (aCML) and secondary acute myeloid leukemia (sAML) progressed from CMML or aCML. Heparinized blood or bone marrow samples were obtained from patients treated at Huntsman Cancer Institute after informed consent. Diagnoses included CMML (n=5), aCML (n=2), and sAML (n=2). Unseparated cells were shipped by overnight courier to Vancouver and CD34+cells isolated on the same day were injected intravenously into sub-lethally irradiated female NRG mice or SRG-W41 mice, or in some cases the same sex and strains also carrying the human 3GS transgenes (NRG-3GS or SRG-W41-3GS mice) in accordance with British Columbia Cancer Agency institutional guidelines. Occasionally when mice were not immediately available, or large numbers of cells were available, cells were viably cryopreserved and transplanted later after thawing. Mice were observed for up to 36 weeks after xenotransplantation with .05 to 1.1x106 human CD34+cells. Engraftment of human CD45+cells in xenografts was evaluated by immunophenotyping, and a median of 90% human chimerism (range: 1% - 95%) was achieved at the time of bone marrow harvest from xenografts. Variant allele frequencies (VAF) were determined in genomic DNA extracted from both the patient samples (CD34+cells) and matching fluorescence-activated cells (FACS)-sorted human CD45+cells (hCD45+cells) purified from xenografts (1-5 xenografts per patient sample). DNA samples were subjected to PCR amplification with extension primers and analyzed using a MALDI-TOF mass spectrometer (MassArray, Agena Bioscience, San Diego, CA). Each mutation call was assigned by the software based on the molecular weight of the extended primer. Analysis of hCD45+cells from eight xenograft samples so far demonstrated a strong correlation of VAF between the patient samples and hCD45+cells from xenografts, in both SRG-W41-3GS (R2=0.94, p<0.01) and NRG-3GS (R2=0.97, p<0.01) models (Figure 1). This tight correlation of VAF was illustrated in hCD45+cells from xenografts transplanted with CMML, aCML or sAML cells. The majority of mutations detected were those in epigenetic regulator genes, such as ASXL1, EZH2 and TET2. No significant difference in VAF was observed between CD34+and CD34- compartments within the hCD45+cells. Additional samples, including specimens from patients with the related myeloproliferative neoplasm, chronic neutrophilic leukemia (CNL) are being analyzed and will be presented. These findings demonstrate the utility of SRG-W41-3GS as well as NRG-3GS as receptive hosts of primary human MDS/MPN cells with genetic evidence of their growth in these mice closely recapitulating the mutational profiles of the transplanted cells. These new strains may facilitate the development of functional screening and pre-clinical testing of novel therapeutic strategies for a range of human MDS/MPN and related myeloid disorders. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

Sign in / Sign up

Export Citation Format

Share Document