Acute Myeloid Leukemia Cells Export c-Myc in Extracellular Vesicles Driving a Proliferation of Immune-Suppressive Myeloid-Derived Suppressor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 703-703
Author(s):  
Athalia Rachel Pyzer ◽  
Dina Stroopinsky ◽  
Hasan Rajabi ◽  
Abigail J. Washington ◽  
Leandra Cole ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Research Funding ◽  
Healthy Donor ◽  
Myeloid Cells ◽  
Suppressor Cells ◽  
Negative Regulator ◽  
Myeloid Derived Suppressor Cells ◽  
Antigen Presenting ◽  
Donor Pbmcs

Abstract Myeloid-derived suppressor cells (MDSCs) play a critical role in promoting immune tolerance and disease growth. We have previously shown that MDSCs are expanded in patients with AML and can be induced from healthy donor PBMCs by co-culture with leukemic cells; a mechanism dependent on expression of the MUC1-C oncoprotein. We sought to elucidate the precise mechanism by which MUC1-C signaling mediates the expansion of this immune suppressive population of immature myeloid cells. We have previously demonstrated that AML cells release membrane bound extracellular vesicles, which traffic to co-cultured cells. We hypothesized that AML EVs may mediate the expansion of MDSCs. MOLM-14 and THP-1 AML EVs were isolated using the ExoQuick precipitation technique, and analyzed by flow cytometry, and compared to size standardized beads, demonstrating particles between 200-300nM in diameter. Furthermoreisolated AML EVs were visualised using Transmission Electron Microscopy demonstrating multiple rounded structures measuring 100-200nM in diameter and bound by darkly staining membrane. Subsequently, healthy donor PBMCs were cultured for three days with GFP tagged AML EVs and then quantified for CD33+/HLADR-/CD11b+ MDSCs and HLADR+/CD11c+ antigen presenting myeloid cells by flow cytometry. In the PBMCs co-cultured with EVs, the proportion of MDSCs increased 8-fold, whilst the proportion of HLADR+/CD11c+ antigen presenting myeloid cells decreased by 10 fold (n=3, p<0.05). We subsequently investigated how MUC1-C signaling, necessary for the expansion of MDSCs, might alter AML extracellular vesicles composition. We evaluated AML EVs for the presence of the pro-proliferative oncoprotein c-Myc by immune-blotting, demonstrating that AML cells secrete EVs containing c-Myc, which is abrogated by downregulation of MUC1-C. Furthermore, EVs containing MUC1 and c-myc led to an up-regulation of the c-Myc downstream targets cyclin D2 and cyclin E1 in co-cultured MDSCs, indicating that c-Myc containing EVs may drive MDSC proliferation. Critically, EVs from MUC1-C silenced AML cells failed to elicit this increase in c-Myc and cyclin D2 and E1 expression in EV exposed MDSCs. Interestingly, exposure of MDSCs to AML EVs lead to an increased expression of PD-L1, which was abrogated in EVs from MUC1-C silenced AML cells. We then sought to determine how MUC1 signaling promotes c-Myc signaling in AML. MUC1-C silencing did not alter c-Myc mRNA levels suggesting a post-transcriptional level of regulation. Micro RNAs are small non-encoding RNA molecules involved in post-translational regulation of gene expression. MiR34a, a known p53 inhibitor, has been implicated in regulating the expansion of MDSCs and it is known that tumor cells suppress MiR34a expression as part of their self-protective armoury. Furthermore, MiR34a is a predicted negative regulator of c-Myc, due to a complementary sequence for MiR34a in the c-Myc promoter region. Using qPCR, we have demonstrated that MUC1-C silencing results in increased expression of MiRNA34a. Furthermore, over-expression of MiR34a in AML cells led to a dramatic down-regulation of c-Myc protein expression, and conversely silencing of MiR34a led to a significant upregulation of c-Myc expression, confirming that MiR34a regulates c-Myc expression in AML. To confirm MiR34a as a critical negative regulator of MDSC expansion, MiR34a altered cells were interrogated for their ability to elicit an expansion of MDSCs in co-cultured PBMCs. Overexpression of MiR34a in AML cells partially abrogated their ability to induce MDSCs from co-cultured donor PBMCs. In concert, silencing of MiR34a in MUC1-C silenced AML cells, recapitulated their ability to induce MDSCs in this model. Taken together, this study illustrates a novel role of the MUC1-C and c-Myc oncoproteins in driving MDSC proliferation and MDSC PD-L1 expression. We have demonstrated that AML EVs alter the tumor microenvironment away from antigen presentation capable myeloid cells and towards immature immune suppressive MDSCs. Disclosures Arnason: Gilead: Consultancy. Küfe:Genus Oncology: Equity Ownership. Rosenblatt:Astex: Research Funding; BMS: Research Funding; DCPrime: Research Funding. Avigan:Astex: Research Funding; DCPrime: Research Funding.

scholarly journals Myeloid-Derived Suppressor Cells Are Expanded in Patients with AML and Are Dependent on MUC1 Expression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 226-226
Author(s):  
Athalia Rachel Pyzer ◽  
Dina Stroopinsky ◽  
Jacalyn Rosenblatt ◽  
Kristen Anna Palmer ◽  
Maxwell Douglas Coll ◽  
...  
Keyword(s):  
T Cells ◽  
Healthy Donor ◽  
Suppressor Cells ◽  
Fold Increase ◽  
Muc1 Expression ◽  
Healthy Controls ◽  
Myeloid Derived Suppressor Cells ◽  
Control Vector ◽  
Donor Pbmcs

Abstract Introduction: Myeloid-derived suppressor cells (MDSCs) are a critical component of the immunosuppressive milieu of the tumor microenvironment and play an important role in promoting immune tolerance and disease growth. They are comprised of granulocytic and monocytic compartments defined by a unique immunophenotypic signature. Importantly, the mechanism by which tumor cells evoke the expansion of MDSCs has not been well elucidated. In the present study, we examined the interaction of MDSCs with AML cells, a setting in which the presence and function of MDSC has not been well described. Methods and Results: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with active AML and granulocytic (CD33+/CD11b+/HLADR-/CD15+) and monocytic (CD33+/CD11b+/HLADR-/CD15-) MDSCs were quantified by multichannel flow cytometry. AML patients had a significantly higher mean granulocytic MDSC population of 17.2% (n=3) compared to healthy controls 1.9%, (n=10) p=0.0083 and a mean monocytic MDSC population of 6.5% (n=3), which was similar to healthy controls (monocytic MDSCs 4.1%, n=10). MDSCs isolated from an AML patient exhibited immunosuppressive effects as measured by the suppression of dendritic cell mediated stimulation of T cells. The addition of AML derived MDSCs resulted in a 40% reduction in CD4+T cell production of IFNϒ and an 11 fold increase IL-10 secretion by CD4 and CD8 T cells following coculture with allogenic DC stimulation. The ability of AML blasts to directly induce the expansion of MDSC was assessed in vitro. Healthy donor PBMCs were co-cultured for 6 days with or without the AML cell lines MOLM-14 and THP-1 at a ratio of 100:1. MDSCs were quantified after 6 days. Coculture with MOLM-14 and THP-1 induced a 2.35 and 8.2 fold increase in MDSCs respectively (n=4). MUC1 is a critical oncogene expressed on leukemic blasts and leukemia initiating cells and plays an important role in the tumor microenvironment promoting tumor growth and immune escape. In the present study, we demonstrated that silencing of MUC1 via shRNA significantly diminishes AML recruitment and expansion of MDSCs in vitro. MOLM-14 cells underwent lentiviral transfection to silence MUC1-C expression which was confirmed by Western Blot. MOLM-14 wild type, MUC1 silenced, and control vector treated cells were co-cultured with healthy PBMCs for 6 days in a ratio of 100:1. Of note, MUC1-C silenced MOLM-14 and THP-1 cells exhibited decreased capacity to expand MDSCs upon co-culture with healthy donor PBMCs, as compared to the control vector (2.4 fold higher expansion of MDSCs with control vector MOLM-14 compared to MUC1-C silenced MOLM-14, n=4, 1.92 fold higher expansion of MDSCs with control vector THP-1 compared to MUC-1C silenced THP-1, n=4). In an in vivo model, NSG mice were irradiated and inoculated with THP-1 control and THP-1 MUC1 silenced cells. Following establishment of disease, mice were sacrificed and spleens were FACS analysed for MDSC quantification. Mice inoculated with THP-1 MUC1 silenced cells had mean MDSCs of 7.5%, compared to 16.25% in mice innoculated with THP-1 Wildtype cells (n=4). In conclusion, the data demonstrates that MDSCs are increased in the circulation of patients with AML, and that leukemic blasts directly induce the expansion of MDSCs. MUC1 expression on AML blasts contributes to the immunosuppressive milieu, and notably, silencing of MUC1 in AML cells blunts their capacity to induce the expansion of MDSCs. Incorporating strategies to inhibit the expansion of MDSC in AML, and reverse their immunosuppressive phenotype has the potential to improve response to therapy in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mass Cytometry Identifies a Novel Signature for Myeloid-Derived Suppressor-Cells in Waldenstrom's Macroglobulinemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3976-3976
Author(s):  
Shahrzad Jalali ◽  
Jose Villasboas ◽  
Jie Shi ◽  
Cole Bothun ◽  
Hyojin Kim ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Clinical Trials ◽  
T Cells ◽  
Research Funding ◽  
Cell Function ◽  
Suppressor Cells ◽  
High Dimensional ◽  
Healthy Controls ◽  
Myeloid Derived Suppressor Cells ◽  
Activated T Cells

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of undifferentiated myeloid cells that are expanded and activated in pathological conditions and have the ability to potently suppress T-cell function and thereby contribute to immunosuppression and tumor progression. While there have been studies showing a role for MDSC in a variety of hematological malignancies, no data is available indicating that MDSCs contribute to the tumor progression in Waldenstrom's Macroglobulinemia (WM), an indolent lymphoma characterized by bone marrow (BM) infiltration of lymphoplasmacytic (LPL) cells and increased secretion of monoclonal IgM. In previous work, we have found increased GM-CSF and reduced arginine and cysteine in the BM microenvironment in WM. We hypothesized that this was due to the presence and activity of MDSCs in WM. BM aspirates from WM patients (n=17) were therefore processed to isolate LPL (CD19+/CD138+) cells from the rest of the BM cells (CD19-/CD138-). Sorted (CD19-/CD138-) cells from BM of patients with WM were studied with flow cytometry. Using a sequential gating strategy (lack of lineage markers, low levels of HLADR, CD33+, CD11b+) we identified a population of MDSCs that were then subdivided using CD14 and CD15 expression into total-, monocytic-, or granulocytic- MDSCs (m-MDSC, g-MDSC). We also analyzed unsorted BM cells using cytometry by time-of-flight (CyTOF) in order to further identify and phenotypically characterize the BM MDSC population in a group of WM patients with smoldering (asymptomatic) disease, symptomatic disease, or in remission post-treatment. BM samples from normal subjects were used as a control. Flow cytometry data showed significant higher numbers of MDSC subsets expressing PD-L1 and Arginase1 in WM patients when compared to the normal samples. BM cells from WM patients (n=18) then were compared to controls (n=11), and the absolute number of the total MDSC (p=0.05), m-MDSC (p=0.002), g-MDSC (p=0.02) was increased in WM specimens. When MDSCs from WM or normal monocytes from healthy controls were co-cultured with activated T-cells, the proliferation of activated T-cells in the presence of MDSCs from WM patients was impaired compared to controls, confirming the suppressive role of MDSCs. We then performed high dimensional analysis of the total BM MDSC cells using t-SNEand identified phenotypically distinct MDSC cell populations in the BM that were differentially present when healthy controls were compared to patients with smoldering WM or those with WM needing treatment. Specifically, WM patients needing treatment had increased numbers of a distinct MDSC population that was highly positive for CD163, and CD138. Moreover, conventional markers denoting m-MDSC and g-MDSC, such as CD14 and CD15, were highly expressed in all populations and their pattern of expression did not specifically define the MDSC subtypes, indicating that high dimensional phenotyping further details the MDSC sub-compartments beyond the conventional categorization of MDSC using conventional cytometry. In summary, we find that MDSCs are increased in the BM of WM patients compared to controls. MDSCs expressing CD163 and CD138 increase when WM patients become symptomatic and require therapy. Furthermore, MDSCs in the BM of WM patients suppress T-cell function and likely contribute to progression of the disease. MDSCs in the BM therefore present a therapeutic target that should be explored in WM patients. Disclosures Ansell: Bristol Myers Squibb: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; AI Therapeutics: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials.

scholarly journals Myeloid phenotypes in severe COVID-19 predict secondary infection and mortality: a pilot study

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Clémence Marais ◽  
Caroline Claude ◽  
Nada Semaan ◽  
Ramy Charbel ◽  
Simon Barreault ◽  
...  
Keyword(s):  
Critically Ill ◽  
Host Response ◽  
Secondary Infection ◽  
Myeloid Cells ◽  
Suppressor Cells ◽  
Human Leukocyte ◽  
Myeloid Derived Suppressor Cells ◽  
Phenotypic Characteristics ◽  
Leukocyte Antigen ◽  
Hla Dr

Abstract Background De-regulated host response to severe coronavirus disease 2019 (COVID-19), directly referring to the concept of sepsis-associated immunological dysregulation, seems to be a strong signature of severe COVID-19. Myeloid cells phenotyping is well recognized to diagnose critical illness-induced immunodepression in sepsis and has not been well characterized in COVID-19. The aim of this study is to review phenotypic characteristics of myeloid cells and evaluate their relations with the occurrence of secondary infection and mortality in patients with COVID-19 admitted in an intensive care unit. Methods Retrospective analysis of the circulating myeloid cells phenotypes of adult COVID-19 critically ill patients. Phenotyping circulating immune cells was performed by flow cytometry daily for routine analysis and twice weekly for lymphocytes and monocytes subpopulations analysis, as well as monocyte human leukocyte antigen (mHLA)-DR expression. Results Out of the 29 critically ill adult patients with severe COVID-19 analyzed, 12 (41.4%) developed secondary infection and six patients died during their stay. Monocyte HLA-DR kinetics was significantly different between patients developing secondary infection and those without, respectively, at day 5–7 and 8–10 following admission. The monocytes myeloid-derived suppressor cells to total monocytes ratio was associated with 28- and 60-day mortality. Those myeloid characteristics suggest three phenotypes: hyperactivated monocyte/macrophage is significantly associated with mortality, whereas persistent immunodepression is associated with secondary infection occurrence compared to transient immunodepression. Conclusions Myeloid phenotypes of critically ill COVID-19 patients may be associated with development of secondary infection, 28- and 60-day mortality.

Abstract 87: The Accumulation of Myeloid-derived Suppressor Cells Is a Compensatory Response to the Development of Hypertension

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Xiao Z Shen ◽  
Peng Shi ◽  
Jorge Giani ◽  
Ellen Bernstein ◽  
Kenneth E Bernstein
Keyword(s):  
Blood Pressure ◽  
T Cell ◽  
Angiotensin Ii ◽  
Critical Role ◽  
Myeloid Cells ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Compensatory Response ◽  
Immunosuppressive Cell ◽  
Induced Hypertension

The immune system plays a critical role in the development of hypertension. The immune response consists of pro-inflammatory cells, but also immunosuppressive cells that reduce T cell function. An important category of natural immunosuppressive cell is myeloid-derived suppressor cells (MDSC). We now show that blood and spleen CD11b+ Gr1+ myeloid cells are elevated 2-fold in both angiotensin II and L-NAME induced hypertension. These increased myeloid cells are MDSC in that they elevate IL-4R expression and suppress T cell proliferation. When hypertensive mice were depleted of MDSC, using either anti-Gr1 antibody or gemcitabine, there was a 15 mmHg rise in blood pressure and aggravation of T cells activation with increased production of IFN-γ, TNFα and IL-17 in both spleen and kidney. In contrast, adoptive transfer of MDSC reduced blood pressure in angiotensin-II induced hypertension by 25 mmHg (see Figure). These data suggest a new concept, that the accumulation of MDSC is a compensatory response to the inflammation induced by hypertension. They also indicate that MDSC play an important role in regulating blood pressure.

Myeloid-Derived Suppressor Cells Increase in Chronic Myeloid Leukemia and Exert Immune Suppressive Activity.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2779-2779
Author(s):  
Cesarina Giallongo ◽  
Nunziatina Parrinello ◽  
Daniele Tibullo ◽  
Piera La Cava ◽  
Alessandra Cupri ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Myeloid Leukemia ◽  
T Lymphocytes ◽  
Enzymatic Activity ◽  
Myeloid Leukemia ◽  
Myeloid Cells ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Arginase 1

Abstract Abstract 2779 Background: Tumor cells are able to develop immune evasion mechanisms which induce a state of immune tolerance and inactivate tumor-specific T cells. In this context, in some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. Aim: The aim of this study was to analyze MDSCs and investigate their involvement in T-cell anergy of CML patients. Methods: MDSCs were analyzed in peripheral blood (PB) of 13 CML patients (at diagnosis and during therapy) and healthy donors (HD; n=20) by cytofluorimetric analysis (CD14+DR- for monocytic MDSCs and CD11b+CD33+CD14-DR- for granulocytic MDSCs). Arginase 1 expression was assessed in PB of HD and CML patient using real time PCR. Purification of granulocytes, monocytes and lymphocytes from PB was performed by a positive magnetic separation kit (EasySep, STEMCELL Technologies). Arginase activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. To evaluate the activation of CD3+ T lymphocytes after incubation with phytoemagglutinin, we analyzed at 24, 48, 72 h the following markers: CD69+, CD71+, DR+. Microvesicles were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. Results: CML patients showed high levels of monocytic and granulocytic MDSCs at diagnosis in comparison to HD (63±8 and 83±12,2% respectively in CML vs 4,9±2,1 and 55,8±5,3% respectively in HD; p<0.001) while after 3–6 months of tyrosine kinase inhibitors (TKIs) therapy MDSC levels returned to normal values. Either in PB and in the purified granulocytes subpopulation, arginase1 expression showed a 30 fold increase in CML at diagnosis (CML vs HD: p<0.01) and decreased after therapy. We also evaluated arginase enzymatic activity in granulocytes and we found it increased in CML patients (n=4) compared to HD (n=5) (p<0.05). CML as well as HD T lymphocytes showed a normal activation in vitro which was significantly lost when they was incubated with CML serum (n=4). In addition, an increase of monocytic MDSCs in vitro was observed after incubation of HD monocytes with CML serum (39±6%; p<0.01) or microvescicles (9,2±1,2%; p<0.05) compared to control serum. Conclusions: Granulocytic and monocytic MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. Their levels also correlates with Arginase 1 expression and enzymatic activity in granulocytes. CML serum as well as CML microvesicles increase the percentage of HD monocytic MDSCs. Moreover, CML serum leads to anergy of T lymphocytes, probably by Arginase 1 secretion. Disclosures: Off Label Use: Eltrombopag is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura (ITP).

scholarly journals Hepatic myeloid-derived suppressor cells in tumor bearing mice exacerbate hepatitis and transform into pro-inflammatory myeloid cells

2013 ◽  
Vol 1 (S1) ◽  
Author(s):  
Jose Medina-Echeverz ◽  
Tamar Kapanadze ◽  
Chi Ma ◽  
Austin Duffy ◽  
Jaba Gamrekelashvili ◽  
...  
Keyword(s):  
Myeloid Cells ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Tumor Bearing
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