Absolute Monocytosis At Diagnosis Is Associated with Poor Survival in Diffuse Large B-Cell Lymphoma -Possibly Related to Increase in Monocytic-Myeloid Derived Suppressor Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2641-2641
Author(s):  
Tamar Tadmor ◽  
Rona Fell ◽  
Aaron Polliack ◽  
Dina Attias
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Immune Response ◽  
Multivariate Analysis ◽  
Prognostic Factors ◽  
Peripheral Blood ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Poor Survival

Abstract Abstract 2641 Background: monocytes and macrophages play a role in promoting angiogenesis and suppression of the anti-tumor immune response. Monocytic-myeloid derived suppressor cells (M-MDSCs) are a subpopulation of immature myeloid cells which have immune-suppressive function and play a role in cancer tolerance. Some patients with malignant lymphoma exhibit peripheral blood monocytosis (PBM) at diagnosis but its exact prevalence is not known. Aim: Recently we encountered PBM in a few patients with DLBCL who had a rapidly progressive fatal course. This observation coupled with emerging data on the role of M-MDSCs is in the tumor microenvironment, led us to undertake a retrospective study to determine prevalence of PBM and its possible prognostic significance in DLBCL. In a proportion of these patients we also quantitated the M-MDSCs pool in the peripheral blood using flow cytometry in an attempt to assess possible changes in this subpopulation of monocytic cells in DLBCL. Material and methods: Clinical and laboratory data from medical records of 91 newly diagnosed patients with DLBCL seen at our institute during 1996–2010, were evaluated for the presence of absolute PBM> 1000 cells/mm3, at diagnosis and for possible correlations with other prognostic factors including age, stage, gender, B symptoms, extra nodal involvement, serum LDH and CRP levels, bone marrow (BM) involvement and IPI score. A Cox proportional hazards regression model was used to determine significance of the prognostic factors in a multivariate analysis. In the last 23 consecutive patients flow-cytometry analysis peripheral blood cells was also performed using surface staining for CD45+CD14+ HLA DR−/LOW to define the proportion of M-MDSCs compared to 15 healthy volunteers. Statistical analysis of was done using student T-TEST. Results: The median age of the entire patient cohort was 66 years (21–87); median follow up was 30 months: (1–332 months) and 57 % were men. All patients received CHOP or R-CHOP. PBM was found in 18.3% at diagnosis. In the multivariate analysis (Cox model), only PBM, bone marrow involvement and IPI score were found to be independent prognostic factors for overall survival (OS) while age and LDH weren't (Table). Blood samples collected at diagnosis from 23 patients showed increased numbers of M- MDSCs compared to healthy volunteers (9.59% range: 5.6–19 % and, 5.44% range 4.8–7.7 % respectively p<0.05).Levels of MDSCs decreased to within normal limits when performed 3 month following chemotherapy in patient in CR (5.76% range: 5.1–7.2 %). In conclusion: These results show that PBM is an independent factor for predicting poor survival in patients with DLCL and was as significant a risk factor as IPI score. This simple routine laboratory test can be utilized in daily practice as another indicator of poor outcome in DLBCL. The findings provide further support for the functional role of monocytes in the immune response in DLBCL. Furthermore, the consistent finding of significantly increased numbers of M-MDSC in the peripheral blood in these patients suggests that this subpopulation of immunosuppressive monocytes may contribute to the decreased tumor surveillance seen in DLBCL and may help to explain why absolute PBM is associated with poor outcome in these patients. Studies on their biological role as a marker of disease activity in DLBCL are in progress. Disclosures: No relevant conflicts of interest to declare.

Metaherin Contributes To The Pathogenesis Of Chronic Lymohcytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4130-4130
Author(s):  
Peipei Li ◽  
Xin Wang ◽  
Chen Na ◽  
Lili Feng ◽  
Xueling Ge ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
Signaling Pathway ◽  
Peripheral Blood ◽  
Wnt Signaling Pathway ◽  
Beta Catenin ◽  
Proliferation And Apoptosis ◽  
Normal Controls

Abstract Introduction Dysregulation of proliferation and apoptosis is associated the pathogenesis of CLL. More recently, Metadherin (MTDH) involved in aberrant proliferation, survival, and increased migration, invasiveness, and metastasis of tumor cells, has been demonstrated as a potential crucial mediator of various types of huamn malignancies. MTDH promotes tumor progression by modulating multiple oncogenic signaling pathways (NF-kB, PI3K/Akt and Wnt/beta-catenin). However, there is no report about the role of MTDH in CLL. Since Wnt signaling pathway had been proven to be unusual activated in CLL, the objective of this study was to investigate the role of MTDH in CLL and the relationship between MTDH and Wnt/beta-catenin signaling pathway. Methods Peripheral blood mononuclear cells (PBMCs) came from samples of 31 CLL patients. The characteristics of CLL patients were shown in Table 1. CD19+B cells were selected from peripheral blood of age-matched heathy donor, cord blood, bone marrow and tonsil of normal controls using CD19+ magnetic selection kits and detected the purity with anti-CD19-PE antibody by flow cytometry. Qantitative PCR and Western blot were used to detect the expression of mRNA and protein for MTDH, and the key functional components of Wnt/beta-catenin signaling pathway (beta-catenin and LEF-1). We also measured MTDH level in B cells by flow cytometry after intracellular staining. CLL cell line(MEC-1) were infected by lentivirus to interfer MTDH and the infection efficiencies were determined by fluorescence microscope and flow cytometry. Both primary CLL cells and MEC-1 were exposed to 10ug/ml goat F(ab`)2 anti-human IgM for 48hours to mimic activation of BCR. The proliferation and apoptosis of these cells were evaluated by CCK-8 method and Annexin V kits. Results mRNA of MTDH in PBMCs of 31 CLL patients were overexpression compared with CD19+ B cells coming from 15 age-matched healthy donors (Figure 1A). 27 out of 31 CLL samples were detected MTDH expression in protein level but none in normal controls (Figure 1B). The expression of MTDH was associated with Rai staging of CLL. There were no MTDH detection in CD19+ B cells collected from bone marrow, peripheral blood, tonsil and cord blood, which stand for precursor, mature, germinal center, and lineage B cells, respectively. The transfection efficiency of MEC-1 cells by interfering MTDH expression with Lentivirus was shown in Figure 1C. The level of MTDH knockdown was accompanied with LEF-1 downregulation (Figure 1D, 1E), as well as the downregulation of c-myc and cyclinD1 expression (Figure 1F). siRNA targeting MTDH treatment in MEC-1 decreased the proliferation and increased the apoptosis(Figure 2A, 2B). We further observed that the proliferation and MTDH expression both in CLL cells and MEC-1 were upregulation after stimulation of anti-human IgM (Figure 2C, 2D, 2E). This effect in the proliferation was blocked by MTDH inteference (Figure 2F). Conclusions Our results demonstrated that MTDH is aberrant expression in B cells of CLL patients and correlated with clinical staging of CLL. MTDH was not expression in any subsets of normal B cells. MTDH may exert a preservative role through activation of Wnt signaling pathway. The CLL cell proliferation activation by BCR signaling pathway may be inhibited by MTDH interference. Our findings indicated that MTDH may be a potential therapeutic target of CLL. Disclosures: No relevant conflicts of interest to declare.

Role of Flow Cytometry of Peripheral Blood and Bone Marrow Aspirates in Early Myeloma

2011 ◽  
Vol 48 (1) ◽  
pp. 32-38 ◽  
Author(s):  
Constance M. Yuan ◽  
Maryalice Stetler-Stevenson
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood

scholarly journals IMPACT OF ENDOSTATIN GENE THERAPY ON MYELOID-DERIVED SUPPRESSOR CELLS FROM A METASTATIC RENAL CELL CARCINOMA

2018 ◽  
Vol 40 (1) ◽  
pp. 24-32
Author(s):  
K CB Chaves ◽  
E M Costa ◽  
L F Teixeira ◽  
M H Bellini
Keyword(s):  
Gene Therapy ◽  
Renal Cell Carcinoma ◽  
Flow Cytometry ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Metastatic Progression

Aim: To evaluate the role of endostatin (ES) gene therapy on myeloid-derived suppressor cells (MDSC) in a metastatic model of renal cell carcinoma (RCC). Materials and Methods: Balb/C mice bearing orthotopic Renca tumors were treated with NIH/3T3LendSN or, as a control, with NIH/3T3-LXSN cells. At the end of in vivo experiment, plasma and tissue lung samples were collected. Plasma ES and granulocyte colony stimulating factor (G-CSF) levels were measured by ELISA and Milliplex, respectively. Quantification of CD11b+Gr-1+ cells and their subsets was performed by flow cytometry. Reactive oxygen species (ROS) production was measured in CD11b+Gr-1+ MDSC using the DCFDA marker by flow cytometry. Results: Metastatic RCC (mRCC) induced expansions of CD11b+Gr-1+ MDSC and promoted accumulation of these cells and their subtypes in lymphoid organ and metastases. ES treatment promoted low G-CSF plasmatic levels which were produced by the tumor microenvironment, reflecting the reduced metastatic accumulation of CD11b+Gr-1+ MDSC in the lungs. However, the therapy was selective for granulocytic cells, thus reducing the production of ROS. Conclusion: These findings confirm the expansion of MDSC during metastatic progression of RCC and indicate the important role of ES in reducing MDSC and possible use of ES therapy in combined anticancer treatment.

scholarly journals Circulating Myeloid-Derived Suppressor Cells Reflect Mycosis Fungoides/Sezary Syndrome Disease Stage and Response to Treatment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4127-4127
Author(s):  
Christopher B. Hergott ◽  
Graham Dudley ◽  
David M. Dorfman
Keyword(s):  
Flow Cytometry ◽  
Disease Progression ◽  
Peripheral Blood ◽  
Suppressor Cells ◽  
Disease Spread ◽  
Sézary Syndrome ◽  
Sezary Syndrome ◽  
Myeloid Derived Suppressor Cells ◽  
Prognostic Information ◽  
Healthy Donors

Abstract Background: Despite mycosis fungoides (MF) and Sezary syndrome (SS) comprising the most common forms of cutaneous T cell lymphoma, the pathophysiology underlying these disorders remains poorly understood. Consequently, current prognostic guidelines based on disease spread exhibit wide variations in clinical outcome within each stage, underscoring an urgent need for novel approaches to MF/SS disease evaluation. A growing body of research suggests that systemic immune dysregulation represents an early, cardinal feature of MF/SS. We hypothesized that tracking this immune dysfunction in conjunction with disease spread may generate important pathophysiologic and prognostic information for patients. We focused on myeloid-derived suppressor cells (MDSCs), a recently discovered population of immunosuppressive innate immune cells related to neutrophils and monocytes, because their expansion in numerous solid tumor settings have correlated reliably with poor patient outcomes. Whether MF/SS augments circulating MDSC abundance remains unexplored, prompting us to evaluate whether this could serve as a marker for disease progression and treatment response. Methods: We used multiparametric flow cytometry to analyze the frequency and immunophenotype of MDSCs from the peripheral blood of 15 healthy donors and 30 patients with MF/SS. Patients at varying stages of MF/SS disease progression and treatment were included in the study. We defined granulocytic MDSCs (G-MDSCs) as cells positive for CD15, CD11b, and the recently discovered surface marker LOX-1, and negative for CD14. Monocytic MDSCs (M-MDSCs) were defined as cells positive for CD14 and CD11b, negative for CD15, and low/negative for HLA-DR. Each patient sample also underwent flow cytometry evaluating for circulating neoplastic T cells. These results were correlated with each participant's other hematologic parameters and clinical information through manual chart review. Results: We found that healthy donors harbored no quantifiable circulating MDSCs of either monocytic or granulocytic lineage, a result in keeping with previous studies. In contrast, MF/SS patients exhibited robust, statistically significant increases in the frequencies of both G-MDSCs and, to a lesser extent, M-MDSCs. G-MDSCs exceeded 20% of all CD15-positive cells in some patients. When patients were stratified by MF/SS clinical stage, those with more advanced disease displayed significantly higher G-MDSC abundance than early-stage patients. G-MDSC frequency was positively correlated with circulating CD4+ CD26- T cell counts often used in evaluating Sezary syndrome (R2 = 0.498; p < 0.0001). However, patients with early, skin-restricted disease also showed statistically significant increases in circulating G-MDSCs compared to healthy controls. This suggested that G-MDSC expansion may serve as a sensitive, blood-based disease marker even in the absence of systemic involvement by neoplasia. Patients who underwent recent treatment exhibited variable G-MDSC counts in the peripheral blood that were lower than in similar untreated patients on average. Serial measurements for two patients enrolled in a clinical trial for dual phosphoinositide 3-kinase and histone deacetylase inhibition revealed that G-MDSC frequencies markedly decreased over the course of treatment, mirroring the decrements of aberrant T cells circulating in the blood. Conclusion: These findings provide clear evidence of G-MDSC expansion in the peripheral blood of MF/SS patients that begins in early/locally restricted disease, grows with disease progression, and responds to systemic therapy. Such immunometric assays may illuminate a novel source of staging and prognostic information and may permit less invasive disease monitoring than current methods require. Disclosures No relevant conflicts of interest to declare.

Myeloid-Derived Suppressor Cells in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2794-2794
Author(s):  
Els Van Valckenborgh ◽  
Jo Van Ginderachter ◽  
Kiavash Movahedi ◽  
Eline Menu ◽  
Karin Vanderkerken
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cell ◽  
Suppressor Cells ◽  
T Cell Responses ◽  
Myeloid Derived Suppressor Cells ◽  
Cell Responses ◽  
T Cell Suppression ◽  
Cell Suppression

Abstract Abstract 2794 Poster Board II-770 Myeloid-derived suppressor cells (MDSCs) are a heterogeneous mix of myeloid cells in different maturation stages generated in the bone marrow. The role of MDSCs in cancer is to suppress T-cell responses, thereby likely regulating tumor progression. In mice, MDSCs are identified by the expression of the surface markers CD11b and Gr-1. Recently, Ly6G+ granulocytic (PMN-MDSC) and Ly6G− monocytic (MO-MDSC) subsets could be distinguished (Movahedi et al. Blood 2008, 111:4233-44). In multiple myeloma patients, the immune function is impaired and this is caused by an immunologically hostile microenvironment and cellular defects, such as decreased numbers of immune cells, and DC or T-cell dysfunction. However, the role of MDSCs in immune suppression in multiple myeloma is not yet described. In this study, we investigated the immunosuppressive activity and mechanism of MDSC subsets in the syngeneic and immunocompetent 5TMM mouse model (5T2 and 5T33 models). In first instance, CD11b+Ly6G− and CD11b+Ly6G+ lineage-committed myeloid MDSC subsets were detected in 5TMM-diseased bone marrow by flow cytometry. These subsets were purified via MACS from the bone marrow of naïve and 5TMM tumor-bearing mice, and analyzed for T-cell suppressive activity. Hereto, CD8+ TCR-transgenic OT-1 splenocytes were stimulated with ovalbumin protein in the presence of purified MDSC subsets, after which T-cell proliferation was measured via 3H-thymidine incorporation. Both MDSC subsets from 5TMM bone marrow were able to suppress antigen-specific T-cell responses at a higher level compared to purified MDSC subsets from normal bone marrow. On average, Ly6G− MDSCs were more suppressive than Ly6G+ MDSCs. The 5T2MM model has a tumor take of approximately 12 weeks. Three weeks after intravenous inoculation of the tumor cells, the suppressive effect of the myeloid subsets was already observed (while the plasmacytosis in the BM was very low and no detectable serum M spike was observed), indicating that T-cell suppression is an early event in MM development. To unravel the suppressive mechanism of the MDSC subsets, inhibitors were used in ovalbumin-stimulated cocultures. Ly6G− MDSC-mediated suppression was partially reversed by the iNOS inhibitor L-NMMA and the COX-2 inhibitor sc-791, both of which lower the NO concentration in culture. In contrast, superoxide dismutase and especially catalase enhance NO concentrations, resulting in enhanced T-cell suppression. None of these inhibitors had any impact on the Ly6G+ MDSC-mediated suppression. In conclusion, these data reveal the presence of MDSCs as a novel immune suppressive strategy employed by multiple myeloma cells in the bone marrow, already occurring early in the disease process. Disclosures: No relevant conflicts of interest to declare.

Myeloid-Derived Suppressor Cells in Patients with Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3662-3662
Author(s):  
Nunziatina Parrinello ◽  
Piera La Cava ◽  
Daniele Tibullo ◽  
Cesarina Giallongo ◽  
Orazio Di Bartolo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Hodgkin Lymphoma ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Suppressor Cells ◽  
Cell Number ◽  
Myeloid Derived Suppressor Cells ◽  
Peripheral Blood Mononuclear ◽  
Hla Dr

Abstract Abstract 3662 Poster Board III-598 Background Immune suppression and angiogenesis are mechanisms key to tumour growth and progression. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin and include immature macrophages, dendritic cells (DC) and other myeloid cells. In mice are phenotypically characterized as CD11b+Gr-1+ cells, while in human they have an immature phenotype, including lineage negative (Lin-), CD14-, HLA-DR-, CD15+, CD34+, CD11b+, CD33+, and CD13+ cells. MDSC reduce activated T-cell number and inhibit their function through different mechanisms including: L-arginine metabolism, nitric oxide (NO), up-regulation of reactive oxygen species (ROS), and secretion of immunosuppressive cytokines. MDSC also promote tumor-dependent angiogenesis as well as tumor metastasis. Their accumulation has been described in patients affected by some solid tumors but information on haematological neoplasms are lacking. Our study investigated by flow cytometry the presence of MSDC in the peripheral blood of patients affected by Hodgkin Lymphoma (HL). Methods We studied 14 patients with HL at diagnosis and 10 age-matched healthy controls (HC). Peripheral blood mononuclear cells were stained with the following monoclonal antibodies:CD11b, CD13, CD14, CD34, CD45, for 20 minutes at room temperature. After lysing red cells, cells were analyzed by flow cytometry. Results we observed a increased number of MDSC (CD11b+,CD13+,CD34+,CD14-, CD45+) in the peripheral blood of patients with HL compared to HC (13,37 ± 17,77 ×109/l vs 1,45± 0,98 ×109/l, p=0,0007). We also found that patients with advanced-stage Hodgkin disease (III and IV) have higher number of MDSC, compared to patients stage I and II (p= 0,04). Conclusion These data suggest a role for myeloid-derived suppressor cells in promoting tumor cell proliferation in hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

Administration Of An Oral PDE5 Inhibitor, Tadalafil In Conjunction With a Lenalidomide Containing Regimen In Patients With Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1959-1959 ◽  
Author(s):  
Nilanjan Ghosh ◽  
Lakshmi Rudraraju ◽  
Xiaobu Ye ◽  
Kimberly Noonan ◽  
Carol Ann Huff ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Pde5 Inhibitor ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Prior Therapy ◽  
Gi Bleed ◽  
Phosphodiesterase 5

Abstract Increasing tumor burden has been associated with an immunosuppressive network posing a significant barrier to anti-tumor immunity. Amongst these pathways, myeloid derived suppressor cells (MDSCs) play a critical role in suppressing immune function through upregulation of iNOS and arginase-1 (Arg1). There is evidence of increased MDSCs in patients with multiple myeloma compared to healthy donors [1]. Additionally, it has been shown that MDSCs regulate the growth of myeloma by inhibiting T cells in the bone marrow [2]. We therefore hypothesized that inhibiting MDSCs could augment the anti-tumor activity of the immunomodulatory drug lenalidomide. We have shown previously that phosphodiesterase 5 inhibitors such as tadalafil effectively inhibit MDSC function through downregulation of iNOS and Arg-1 production [3]. To prospectively study the effect of MDSC inhibition in myeloma, we initiated a clinical trial in patients who were refractory to lenalidomide-based regimens, with the oral PDE5 inhibitor, tadalafil, added to their lenalidomide-containing regimen. Refractory to lenalidomide containing regimen was defined as disease progression within 60 days of lenalidomide/dexamethasone (Rd) or Biaxin/lenalidomide/dexamethasone (BiRd). Responses were monitored by International Myeloma Working Group (IMWG) criteria. 13 patients were enrolled between April 2012 and March 2013. Median age was 63, 46.1% female, median number of prior therapies was 4 (range 3-10), 10 patients (80%) had BiRd as their immediate prior therapy, 3 (20%) patients had Rd as the immediate prior therapy, 4 (30.8%) patients had high risk cytogenetics/FISH, 4 (30.8%) patients had ISS III disease and 5 (38.4%) patients had a stem cell transplant in the past. 2 patients were not evaluable, 1 did not meet the eligibility criteria and another patient with a history of gastrointestinal (GI) bleed came off protocol in less than a week because of a recurrent GI bleed. 1 (9%) patient had a minor response (MR) lasting 3 months, 4 (36.4%) patients achieved stable disease (SD), 6 (54.5%) patients developed progressive disease (PD). For patients who achieved SD, the median duration was 66 days (range 48-161 days). Median PFS was 48 days (95% CI 25-71 days). 2 (18.1%) patients needed dose reduction of tadalafil for grade 3 back pain, which was the only toxicity attributable to the drug. There were no deaths on study. At a median follow up of 1 year, the OS is 81.8%. The trial met early stopping rule due to lack of response. Biologic correlates were performed pre and post treatment and included measurement of MDSCs numbers by flow cytometry using CD14+, CD33+, HLADRlow, IL4Rα+ or CD15+, CD33+, HLADRlow, IL4Rα+. Interestingly, MDSCs were not detected in any of the patients at baseline in both blood and marrow and this correlated with the lack of clinical response. In mice, lenalidomide can reduce MDSC numbers [4]. All patients on this trial were heavily pre-treated with lenalidomide for a median duration of 783 days (range 55-1741 days) which could explain the low numbers of MDSCs at enrollment. Strategies aimed at inhibiting MDSC function would be best tested in patients who have elevated levels of MDSCs by flow cytometry. References 1. Gorgun, G.T., et al., Tumor-promoting immune-suppressive myeloid-derived suppressor cells in the multiple myeloma microenvironment in humans. Blood, 2013. 121(15): p. 2975-87. 2. Ramachandran, I.R., et al., Myeloid-derived suppressor cells regulate growth of multiple myeloma by inhibiting T cells in bone marrow. J Immunol, 2013. 190(7): p. 3815-23. 3. Serafini, P., et al., Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function. J Exp Med, 2006. 203(12): p. 2691-702. 4. Sakamaki, I., et al., Lenalidomide enhances the protective effect of a therapeutic vaccine and reverses immune suppression in mice bearing established lymphomas. Leukemia, 2013. Disclosures: Off Label Use: Tadalafil for supression of myeloid derived suppressor cells.

MUC-1 Regulates MiR34a Expression in Acute Myeloid Leukemia Cells Resulting in an Accumulation of Granulocytic Myeloid-Derived Suppressor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 643-643
Author(s):  
Athalia Rachel Pyzer ◽  
Dina Stroopinsky ◽  
Hasan Rajabi ◽  
Jacalyn Rosenblatt ◽  
Maxwell Douglas Coll ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Suppressor Cells ◽  
Muc1 Expression ◽  
Myeloid Derived Suppressor Cells ◽  
Lentiviral Transduction ◽  
Downstream Signaling ◽  
C Terminus ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu that blunts effector cell function and the generation of tumor specific immunity. Myeloid-derived suppressor cells (MDSCs) are a critical component of the immunosuppressive tumor microenvironment that fosters immune tolerance and disease growth. The role of MDSCs in AML and the mechanism by which tumor cells evoke the expansion of MDSCs has not been well elucidated. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. The C-terminus of MUC1 (MUC1-C) forms dimers and translocates to the nucleus where it mediates downstream signaling. The effect of MUC1-C mediated signaling on immune modulation in AML has not been well characterized. In prior studies, we have demonstrated that in vitro expansion of MDSCs is correlated with MUC1 expression by the AML cells. In the present study, we sought to characterize the effect of MUC1 on in vivo recruitment of MDSCs and examine the mechanism by which this is accomplished. The murine AML cell line TIB-49 was transplanted in C57BL/6J mice. Following establishment of disease, the mice were euthanized, along with healthy controls, and bone marrow and spleen CD11b+ Gr1+ MDSCs were quantified by flow cytometry. Engrafted mice had an average MDSC burden of 47% in the marrow and 8.7% in the spleen, compared with 35% and 2% in control mice, respectively. The increase in MDSCs was granulocyte predominant, consistent with our findings in patients with AML. Gr1+CD11b+ cells derived from the marrow and spleens of engrafted mice showed higher levels of Arginase-1 compared to MDSCs from control mice, suggesting an increase in immune suppressive phenotype. To investigate the role of MUC1 on the expansion of MDSCs, expression of MUC-1C, the signaling C-terminus of MUC1, was silenced in TIB-49 cells by stable expression of a MUC1-C shRNA as determined by Western Blot analysis. MUC1 silenced or control vector transduced TIB-49 cells were transplanted into C57BL/6J mice. Following establishment of disease, the mice were euthanized, and bone marrow and spleens were quantified for MDSCs. Control AML engrafted mice had an average splenic MDSC burden 2-fold higher than MUC1 silenced AML engrafted mice (n=4). We have developed a cell-penetrating peptide (GO-203) that disrupts homodimerization of the MUC1-C subunit necessary for its downstream signaling. C57BL/6J mice were challenged with TIB-49 AML cells and after 24 hours were treated daily with GO-203. Mice treated with the MUC1 inhibitor had a 2-fold decrease in splenic MDSCs, compared to control mice (n=3) at time of analysis following disease establishment. Noncoding RNAs have emerged as a critical biologic effector of oncogenic pathways. MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. miR-34a has been implicated in regulating the expansion of MDSCs. In the present study we demonstrated that silencing of MUC1 expression via lentiviral transduction with a MUC1 specific shRNA resulted in a significant increase in miR-34a expression, as quantified by q-PCR. Consistent with the MUC1 mediated regulation of MDSC expansion by modulation of miR-34a levels, MDSCs induced by co-culture of healthy donor PBMCs with MUC1 silenced AML cells contained 4-fold higher levels of miR-34a, as compared to controls. AML cells were pre-incubated with SYTO® RNASelect™ Green Fluorescent cell stain. After 4 hours, co-cultures of PBMCs and AML cells were analyzed via flow cytometry. AML cells were excluded and cells positive for MDSC markers and containing green fluorescing exosome dye were quantified. AML derived exosomes were found in 18% of MDSCs expanded from AML cells (n=3), demonstrating exosome trafficking from tumor to MDSCs. Finally, miR-34a was over-expressed in MOLM-14 using lentiviral transduction. Over-expression of miR-34a in MUC1 expressing MOLM-14 cells resulted in a 30% reduction in MDSC expansion in co-cultured PBMCs (n=3). In conclusion, MUC1 regulates MDSC expansion in AML, via its effects on miR34a, acting as a critical mediator of tumor mediated immune suppression. Incorporating strategies to reverse the expansion of MDSCs in AML, potentially by targeting MUC1 and increasing miR-34a expression offers a novel therapeutic approach for cancer immunotherapy. Disclosures Küfe: Genus Oncology, LLC: Equity Ownership.

scholarly journals ROLE OF GLYCODELIN IN REGULATION OF MYELOIDDERIVED SUPPRESSOR CELL DIFFERENTIATION

2021 ◽  
Vol 23 (4) ◽  
pp. 641-646
Author(s):  
S. A. Zamorina ◽  
V. P. Timganova ◽  
M. S. Bochkova ◽  
K. Yu. Shardina ◽  
S. V. Uzhviyuk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Spontaneous Abortion ◽  
Peripheral Blood ◽  
Essential Role ◽  
Mononuclear Cells ◽  
Suppressor Cell ◽  
Complete Medium ◽  
In Pregnancy

Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with molecular weight of 42 to 56 kDa) is considered as a reproductive tissue receptivity marker. Despite that glycodelin immunosuppressive effects are well-known there still remains uncovered its role in myeloid suppressor cell (MDSC) regulation. MDSC represent the heterogeneous population of immature myeloid cells that acquire suppressor phenotype while inhibiting the immune response under the pathological states. MDSC are known to play an essential role in supporting the immune tolerance in pregnancy and at transplantation. Our hypothesis suggests that glycodelin is capable of inducing the MDSC formation as the level of these cells is elevated during the successful pregnancy, whereas the spontaneous abortion and progression of eclampsia are associated with low circulating glycodelin. Therefore, the aim of the work was to analyze the role of recombinant glycodelin in physiological concentrations in regulation of MDSC differentiation. Peripheral blood mononuclear cells of donor volunteers were separated via centrifugation on density gradient of 1,077 g/cm3 (Ficoll-Hypaque, Sigma-Aldrich) to obtain MDSC generation in vitro. Then cells obtained were cultured in 24-well plate at a concentration of 1 × 106 cell/ml in complete medium with cytokines IL-6 (20 ng/ml), GM-CSF (40 ng/ml) therein for 14 days at 37 °C and 5% CO2. Medium replacement was made by 7th day in culture followed by cytokine re-introduction, and on the 11th day recombinant glycodelin in physiological concentrations (0,2; 2 mkg/ml) was applied while the pharmacological concentration was 50 mkg/ml. The M-MDSC (LinHLA-DRCD33+CD11b+CD14+CD66b- ) and PMN-MDSC (LinHLA-DRCD33+CD11b+CD14- CD66b+) level was evaluated in cultures using flow cytometry (СytoFlexS (Beckman Coulter)) and “R&D Systems” antibodies according to standard protocol. Statistical data processing was realized with GraphPad Prizm software using Friedman test. It was found that glycodelin did not significantly affect cell viability being assessed with flow cytometry (PI). It was revealed that high GdA concentration (50 mkg/ml) being pharmacological did not render significant effect on MDSC differentiation. Meanwhile, glycodelin in concentrations correspanding the healthy pregnancy (0,2; 2 mkg/ml) was stated to increase the MDSC percentage in induced cultures of human mononuclear cells. When analyzing the subsets it was disclosed that this effect was conditioned by the increase in PMN-MDSC level while the M-MDSC level remained significantly unchanged. This result could be interpreted as glycodelin fetoprotective effect as the increase of the PMN-MDSC level is associated with the suppression of the immune response to paternal antigens. The PMN-MDSC level is known to be elevated in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to nonpregnant subjects whereas the M-MDSC amount remains unaltered. Meanwhile, patients with miscarriage demonstrated more that by 30% lowering in the MDSC amount in blood and endometrium and in I trimester, in particular. During the physiological pregnancy PMN-MDSC accumulate in placenta, but at spontaneous abortion their number is found to be declined. Placental PMN-MDSC efficiently suppress the T-cell response while concurrently polarizing the CD4+ lymphocytes in Th2 phenotype. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens that allows considering these as promising target of therapeutical manipulation in pregnancy complications. As a whole, we have originally demonstrated the GdA effect on MDSC differentiation. 

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