Identification Of Myeloid-derived Suppressor Cells Research Articles

P982: MYELOID-DERIVED SUPPRESSOR CELLS: CHARACTERIZATION IN PERIPHERAL BLOOD AND SPLEEN TISSUE OF PATIENTS WITH MYELOFIBROSIS

Background: Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that accumulate in patients with malignancies, sepsis or chronic inflammation. Features of MDSCs are: myeloid origin, immature state, strong ability to reduce cytotoxic functions of T/NK cells, potential to differentiate into endothelial cells favoring neoangiogenesis. MDSCs are conventionally divided into polymorphonuclear (PMN)- and monocytic (M)-MDSCs. PMN-MDSCs are characterized by the production of reactive oxygen species and arginase-1 (Arg-1), that directly inhibits T-cell proliferation and activation, while M-MDSCs predominantly express inducible nitric oxide synthase and produce nitric oxide. Myelofibrosis (MF) is characterized by clonal expansion of a hematopoietic stem cell, extramedullary hematopoiesis, bone marrow (BM) fibrosis, splenomegaly, BM and splenic neoangiogenesis and an acquired mutation of JAK2, CALR or MPL gene. Inflammation plays a relevant role in MF pathogenesis, as proven by high levels of inflammatory cytokines with prognostic significance and by a state of chronic oxidative stress. Aims: To characterize circulating MDSCs and assess their localization and quantification in spleen samples from MF patients. Methods: Peripheral blood mononuclear cells (PBMCs) from MF patients (n=15) and healthy subjects (HDs; n=10) were stained with surface antibodies (CD11b, CD14, HLA-DR, CD15, CD33) and intracellular Arg-1 and analysed by flow cytometry. Formalin-fixed paraffin-embedded samples of spleen specimens (MF n=41; HDs n=20) were immunostained with anti-Arg-1 antibody using the automated platform Dako Omnis Envision Flex with the EnVision FLEX, High pH, HRP Rabbit/Mouse High pH [GV800] revelation kit. Results: In PBMCs, the percentage of PMN-MDSCs (identified as CD11b+HLA-DRlow/-CD33dimCD14-CD15+) was higher (p=0) in patients (median 9.2, range 1-59) than HDs (median 1.7, range 0.1-4.5) and directly correlated with the allelic burden (R=0.86, p=0.006) in JAK2-mutated patients. Patients in pre-fibrotic phase (n=7) had levels of PMN-MDSCs (median 9.2, range 1-31.4) comparable to HDs; on the contrary, the percentage of PMN-MDSCs in patients with BM fibrosis (n=8; median 13.3, range 3.5-59) was higher (p=0) than in HDs. All PMN-MDSCs were Arg-1+, both in MF and in HDs. Gene expression studies are ongoing. The expression of the CXCR4 receptor on PMN-MDSCs was lower, although not statistically significant, in MF (median 1.1, range 0-9.3) than in HDs (median 2.5, range 0-11.9). PMN-MDSCs in MF inversely correlated (R=-0.89 p=0.007) with CXCR4 expressed on CD34+ cells. The percentage of M-MDSCs (identified as CD11b+HLA-DRlow/-CD33hiCD14+CD15-) was higher (p=0.015) in HDs (median 0.49, range 0.25-1.5) than in MF (median 0.29, range 0.02-0.76). Immunohistochemical staining of spleen sections showed a higher (p=0) percentage of Arg-1+ cells (calculated on total spleen cells) in MF (median 20, range 5-60) than in HDs (median 7, range 2-20); Arg-1+ cells were present in all patients and predominantly located in the red pulp in MF patients, while in HDs were detected in the white pulp. Summary/Conclusion: The identification of MDSCs and their relation with the mutational status suggest a new mechanism of MF pathogenesis, either related to the chronic inflammatory status of patients or as players of the neoangiogenesis that characterizes the disease. Moreover, the low expression of CXCR4 on PMN-MDSCs could be, on one hand, related to the low expression of this receptor on CD34+ cells or suggest a MF-specific recruitment mechanism, other than that observed in solid tumors.

Comments on the ambiguity of selected surface markers, signaling pathways and omics profiles hampering the identification of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSC) are important immune-regulatory cells but their identification remains difficult. Here, we provide a critical view on selected surface markers, transcriptional and translational pathways commonly used to identify MDSC by specific, their developmental origin and new possibilities by transcriptional or proteomic profiling. Discrimination of MDSC from their non-suppressive counterparts is a prerequisite for the development of successful therapies. Understanding the switch mechanisms that direct granulocytic and monocytic development into a pro-inflammatory or anti-inflammatory direction will be crucial for therapeutic strategies. Manipulation of these myeloid checkpoints are exploited by tumors and pathogens, such as M. tuberculosis (Mtb), HIV or SARS-CoV-2, that induce MDSC for immune evasion. Thus, specific markers for MDSC identification may reveal also novel molecular candidates for therapeutic intervention at the level of MDSC.

Identification of myeloid-derived suppressor cells that have an immunosuppressive function in NF2 patients.

There is no targeted drug therapy for NF2 patients, and surgery or radiosurgery is not always effective. Therefore, the exploration of new therapeutic pathways is urgently needed. We analyzed the expression of cytokines in the serum of NF2 patients and determined the percentage of HLA-DR-CD33+CD11b+ cells in blood and NF2-associated schwannomas. Furthermore, we analyzed the role of HLA-DR-CD33+CD11b+ cells in inhibiting T-cell proliferation, cytokine production, and transforming growth factor expression. NF2 patients are in an immunosuppressed state with elevated IL-10 and TGF-β expression in plasma and the lymphocytes from NF2 patients secrete less IFN-γ and CD3+ T cells proliferate slower than normal healthy donors. HLA-DR-CD33+CD11b+ cells frequency significantly increased in the PBMCs and infiltrated in the tumor, these cells express higher iNOS, NOX2 and TGF-β, and induce TGF-β secretion to inhibit CD8+ T-cell proliferation, and induce T-cell transformation to a CD4+CD25+Foxp3+ regulatory T cells phenotype. NF2-associated schwannoma cells induced monocytes transformation into an HLA-DR-CD33+CD11b+ phenotype, and surgical removal of the tumor reduced the percentage of these cells. HLA-DR-CD33+CD11b+ cells may represent a population of MDSCs in NF2 patients. Dissecting the mechanisms behind these suppressive mechanisms will be helpful for the design of effective immunotherapeutic protocols and likely provide a new effective treatment for NF2 patients.

Abstract LB-352: Identification of myeloid-derived suppressor cells in solid tumors by multiplex IHC

Abstract In healthy individuals, immature myeloid cells undergo differentiation into Macrophages, Dendritic Cells or Neutrophils. In individuals with several types of cancers, Tumor-Derived Factors promote a pathological differentiation of these immature cells into Myeloid-Derived Suppressor Cells (MDSCs) that phenotypically resemble either Macrophages (M-MDSCs) or Neutrophils (PMN-MDSCs). Rather than supporting antitumoral immunity like their regular counterparts, these abnormal, highly immunosuppressive cells, impair antitumoral immunity and promote tumor development. In addition to MDSCs, the tumor micro-environment is also infiltrated by Tumor-Associated Macrophages (TAMs), which can be roughly divided in two main subgroups, M1- and M2-polarized Macrophages, that exhibit anti- or pro-tumor activity, respectively. Over the last years, MDSCs have been recognized as valuable prognostic and/or predictive biomarkers, and there is a raising need for methods to phenotype these cells in clinical trials. Myeloid cell subpopulations are all closely related, and as a consequence, the phenotyping of each subset is complex, due to the high number of markers needed to discriminate them from the others. Recent advances have been made to define standard protocols to discriminate M- and PMN-MDSCs from TAMs and physiological Macrophages or Neutrophils combining flow cytometry and T-cell suppression assays. There are differences in the composition of subpopulations at the systemic level and those that infiltrate the tumor micro-environment, and a method for accurate phenotyping of MDSCs on tissue sections by ImmunoHistoChemistry (IHC) still need to be defined, but has been hampered by the limited number of antibodies that could be associated on a single slide. Using multiplex IHC and multispectral imaging, we have designed an approach to phenotype each myeloid cell population in the tumor micro-environment, in different solid tumor types. Antibodies to cell surface markers, cytoplasmic proteins contributing to the immune-regulatory activity and transcription factors, have first been validated as simplex IHC markers, tested as parts of multiplex panels, and evaluated in various combinations, to define a phenotyping strategy. A new Image Analysis Software has been designed to assess the co-expression of each of the markers for individual cells even for stains that do not strictly colocalize at the pixel level (e.g. nuclear and membrane proteins). Using a successive gating strategy, cells displaying MDSCs and M1/M2-polarized macrophages phenotypes have been numbered in sections from various tumor types. Our method is compatible with standard automated IHC platforms and an automated multispectral scanner, and provides a solution for the analysis of MDSCs in FFPE samples in clinical trials. Citation Format: Renaud BURRER, Océane ARIBO, Assil BENCHAABEN, Maroua TLIBA, Domenico LAZZARO, Jean-Philippe COTON. Identification of myeloid-derived suppressor cells in solid tumors by multiplex IHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-352.

Identification of myeloid-derived suppressor cells in peripheral blood of patients with non-Hodgkin lymphoma and its clinical significance

Objective To explore the presence of myeloid-derived suppressor cells (MDSC) in patients with non-Hodgkin lymphoma (NHL) and its clinical value. Methods Peripheral blood samples were collected from 69 NHL patients and 21 healthy controls admitted in Jilin Cancer Hospital from January 2014 to February 2015. Flow cytometry was conducted to identify unique cell surface markers of MDSC using antibodies against CD11b, CD33, CD14 or HLA-DR. MDSC were enriched by immunomagnetic beads, then arginase 1 (Arg-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in which were detected by real time-PCR. In vitro, cell proliferation assay was used to test T cell function. Statistical analysis was used to explore the correlation between MDSC and clinical features. Results There were a high level of CD11b+ CD14+ CD33+ cells in peripheral blood of NHL patients. The morphology of the cells belonged to mononuclear cells. The ratio of monocytic CD11b+ CD14+ CD33+ cells in NHL patients was higher than those in healthy controls [(42±10)%vs. (34±11)%, t= 0.300, P= 0.005]. The expressions of Arg-1, COX-2 and iNOS in CD11b+ CD14+ CD33+ and CD11b+ CD14+ CD33- cells were 0.12±0.04 vs. 1.00±0.25 (t= 6.095, P= 0.024), 3.03±0.45 vs. 1.00±0.78 (t= 7.766, P= 0.016) and 0.29±0.11 vs. 1.00±0.04 (t= 1.987, P= 0.209), respectively. In addition, the CD11b+ CD14+ CD33+ cells inhibited T cell proliferation. The levels of MDSC in patients with different international prognostic index (IPI) score were significantly different (F= 2.536, P= 0.049), but the levels of MDSC in patients with different sex, age, pathological type, stage, serum lactate dehydrogenase, physical status staging criteria and β2-microglobulin had no differences (all P < 0.05). Conclusions CD11b+ CD14+ CD33+ cells are characterized as MDSC in terms of higher level in NHL patients, expressing myeloid-specific proteins, and inhibiting T cell proliferation. The expression of MDSC is associated with IPI score, implying it might be a novel biomarker in clinical practice for NHL patients. Key words: Lymphoma, non-Hodgkin; Myeloid-derived suppressor cells; Antigens, differentiation; Biological assay

Myeloid-Derived Suppressor Cells: Critical Cells Driving Immune Suppression in the Tumor Microenvironment.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress innate and adaptive immunity. MDSCs are present in many disease settings; however, in cancer, they are a major obstacle for both natural antitumor immunity and immunotherapy. Tumor and host cells in the tumor microenvironment (TME) produce a myriad of pro-inflammatory mediators that activate MDSCs and drive their accumulation and suppressive activity. MDSCs utilize a variety of mechanisms to suppress T cell activation, induce other immune-suppressive cell populations, regulate inflammation in the TME, and promote the switching of the immune system to one that tolerates and enhances tumor growth. Because MDSCs are present in most cancer patients and are potent immune-suppressive cells, MDSCs have been the focus of intense research in recent years. This review describes the history and identification of MDSCs, the role of inflammation and intracellular signaling events governing MDSC accumulation and suppressive activity, immune-suppressive mechanisms utilized by MDSCs, and recent therapeutics that target MDSCs to enhance antitumor immunity.

Identification of myeloid-derived suppressor cells in the synovial fluid of patients with rheumatoid arthritis: a pilot study.

BackgroundMyeloid-derived suppressor cells (MDSCs) are a heterogeneous population of innate immune cells with a granulocyte-like or monocyte-like phenotype and a unique ability to suppress T-cell responses. MDSCs have been shown to accumulate in cancer patients, but recent studies suggest that these cells are also present in humans and animals suffering from autoimmune diseases. We previously identified MDSCs in the synovial fluid (SF) of mice with experimental autoimmune arthritis. The goal of the present study was to identify MDSCs in the SF of patients with rheumatoid arthritis (RA).MethodsRA SF cells were studied by flow cytometry using antibodies to MDSC cell surface markers as well as by analysis of cell morphology. The suppressor activity of RA SF cells toward autologous peripheral blood T cells was determined ex vivo. We employed both antigen-nonspecific (anti-CD3/CD28 antibodies) and antigen-specific (allogeneic cells) induction systems to test the effects of RA SF cells on the proliferation of autologous T cells.ResultsSF from RA patients contained MDSC-like cells, the majority of which showed granulocyte (neutrophil)-like phenotype and morphology. RA SF cells significantly suppressed the proliferation of anti-CD3/CD28-stimulated autologous T cells upon co-culture. When compared side by side, RA SF cells had a more profound inhibitory effect on the alloantigen-induced than the anti-CD3/CD28-induced proliferation of autologous T cells.ConclusionMDSCs are present among RA SF cells that are commonly regarded as inflammatory neutrophils. Our results suggest that the presence of neutrophil-like MDSCs in the SF is likely beneficial, as these cells have the ability to limit the expansion of joint-infiltrating T cells in RA.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-281) contains supplementary material, which is available to authorized users.

Novel therapeutic strategies targeting tumor-stromal interactions in pancreatic cancer

Therapy-resistance and postoperative recurrence are causes of the poor prognosis in pancreatic cancer. Conventional therapies have a limited impact on the control of pancreatic cancer, resulting in the rapid re-growth of the tumor. The indispensable role of tumor-stromal interaction, which acts as a defender of cancer cells and enhances malignant potential, is being uncovered now. For example, specific signaling pathways for desmoplasia induction have been identified, such as sonic hedgehog (Shh) or connective tissue growth factor (CTGF), whose inhibition causes desmoplasia depletion and therapeutic advantages at least in in vivo mouse models of pancreatic cancer. Revolutions in drug delivery methods have led to the establishment of novel chemotherapeutic regimens, with better patient survival. Furthermore, mechanisms of immunosuppression in the pancreatic cancer-bearing host were clarified by the identification of myeloid-derived suppressor cells (MDSCs), which also promote disease progression. Strategies to target these components of the tumor stroma revealed certain anticancer effects in vitro and in vivo, suggesting the possibility of stroma-targeting therapy. Suppression of the stromal cell function increases the sensitivity of pancreatic cancer cells to therapeutic intervention. Further study will clarify the complex nature of the tumor microenvironment, the targeting of which has the potential to improve clinical outcome.

Serum inhibits the immunosuppressive function of myeloid-derived suppressor cells isolated from 4T1 tumor-bearing mice.

As more groups investigate the role of myeloid-derived suppressor cells (MDSCs) in promoting the growth of primary tumors and distant tumor metastases, it is imperative to ensure the accurate detection and quantification of MDSC immunosuppression ex vivo. MDSCs are defined by their ability to suppress immune responses. Although different in vitro culture conditions have been used to study MDSCs, the effect of different culture conditions on MDSC immunosuppression is unknown. We therefore isolated MDSCs from the lungs and spleens of 4T1 murine mammary tumor-bearing mice and assayed MDSC-mediated suppression of T cell responses under different culture conditions. We found that 4T1-induced MDSCs effectively suppressed T cell proliferation under serum-free conditions, but not when fetal calf serum (FCS) was present. FCS neither altered the immunosuppressive activities of other myeloid cell types (i.e., peritoneal or tumor-associated macrophages) nor modified the susceptibility of T cells to myeloid cell-mediated suppression, but instead acted directly on 4T1-induced MDSCs to significantly reduce their immunosuppressive function. Importantly, we found that bovine serum albumin was a major contributor to the antagonistic effects of FCS on 4T1-induced MDSC immunosuppression by inhibiting reactive oxygen species production from MDSCs. This work reveals that in vitro culture conditions influence the immunosuppressive properties of MDSCs and highlights the importance of testing different culture conditions on MDSC phenotype to ensure that MDSC immunosuppression is not being masked. These data have important implications for the accurate detection and identification of MDSCs, as well as for determining the influence of MDSC-mediated immunosuppression on primary and metastatic tumor growth.

Identification of myeloid-derived suppressor cells in dogs (170.2)

Abstract Approximately six million dogs are diagnosed with cancer each year in the United States.Naturally occurring malignances in dogs share many features with human cancers that make dogs an excellent outbred spontaneous tumor model. The accumulation of myeloid derived suppressor cells (MDSCs) in tumor-bearing mice and in humans with cancer is known to be a key mechanism of tumor escape from immune surveillance and antitumor immunity. Due to their remarkable ability to downregulate the immune response against cancer cells in mice and in humans, we hypothesized that MDSCs would also play important role in tumor-induced immune suppression in dogs with cancer. Peripheral blood mononuclear cells of dogs with a variety of cancers and of age-matched healthy control dogs were analyzed by flow cytometry. Immunophenotyping revealed that dogs with cancer exhibited a significantly higher percentage of CD11b+ CD14- MHCII- cells compared with healthy dogs. More importantly, these cells have polymorphonuclear granulocyte morphology and demonstrated an ability to suppress T-cell proliferation in a dose dependent manner. Therefore, according to these results, canine MDSCs do exist and can be a potential target for therapeutic interventions in dogs with cancer. Furthermore, the study of MDSCs in dogs can be used to address underlying questions of resistance and evasion of malignant tumors that comprise an important target for translation cancer immunotherapy.

Identification of myeloid-derived suppressor cells and T regulatory cells in lung microenvironment after Urethane-induced lung tumor

Tumor growth occurs by the imbalance between cells with effector function and cells with suppressor/regulatory functions. To investigate this scenario we administered the chemical carcinogen Urethane in BALB/c mice and followed these animals during 120days to observe lung tumor development. In another set of experiments the same protocol was performed with the harvest of spleen, lung and blood at 20 and 30days after Urethane injection. The lung was used for histology, spleen cells were evaluated for IFN-γ production, and serum nitrite was measured as an indirect form of nitric oxide (NO) evaluation. The spleen and lung-infiltrating cells were evaluated by flow cytometry for CD11b+Gr-1+ myeloid suppressor cells and CD4+FoxP3+ T regulatory cells. Urethane led to lung nodules development after 120days and the time point evaluation showed that splenocytes stimulated ex vivo expressed higher levels of IFN-γ 20days after the chemical injection. Also, the level of nitric oxide in serum was higher after 20days of Urethane injection. There was no statistical difference in spleen cells percentages for CD11b+Gr-1+ and CD4+Foxp3+ in all groups. However, lung-infiltrating cells presented early (20days) a higher expression of CD11b+Gr-1+ suggesting suppression at this site. In conclusion, it was possible to observe two distinct events at the very early time point after Urethane injection. In periphery there was an increase at the effector immune response (as depicted by IFN-γ-producing cells) and in tumor development site there was an increase at the suppressor cell (CD11b+Gr-1+) phenotype. Suppressor/regulatory cells are targets for cancer therapy.