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Molecular and cellular characterization of Cord Blood derived, IDO expressing human fibrocystic Myeloid Derived Suppressor Cells

ABSTRACT: By restraining T cell activation and promoting regulatory T cell (Treg) expansion, myeloid-derived suppressor cells (MDSC) and tolerogenic dendritic cells (DC) (tDC) can control self-reactive and anti-graft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study we describe and characterize a novel population of myeloid suppressor cells, named fibrocytic MDSC (f-MDSC), that are differentiated from umbilical cord blood (UCB) precursors by a 4 day culture with FDA approved cytokines (rh-GM-CSF and rh-G-CSF). This MDSC subset, characterized by the expression of MDSC-, DC-, and fybrocyte-s associated markers, promotes Treg regulatory cells expansion and promote normoglycemia in a xenogeneic model of Type 1 Diabetes (T1D). In order to exert their pro-tolerogenic function, fibrocytic MDSC require direct contact with activated T cells, which leads to the induction and secretion of indoleamine 2,3 deoxygenase (IDO). This new myeloid subset may represent an important tool for the in vitro and in vivo production of Treg for the treatment of autoimmune diseases, and either prevention or control of allograft rejection. Keywords: expression profiling by array

ORGANISM(S): Homo sapiens

PROVIDER: GSE52376 | GEO | 2014/08/26

SECONDARY ACCESSION(S): PRJNA227735

REPOSITORIES: GEO

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Project description:By restraining T cell activation and promoting regulatory T cell (Treg) expansion, myeloid-derived suppressor cells (MDSC) and tolerogenic dendritic cells (DC) (tDC) can control self-reactive and anti-graft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study we describe and characterize a novel population of myeloid suppressor cells, named fibrocytic MDSC (f-MDSC), that are differentiated from umbilical cord blood (UCB) precursors by a 4 day culture with FDA approved cytokines (rh-GM-CSF and rh-G-CSF). This MDSC subset, characterized by the expression of MDSC-, DC-, and fybrocyte-s associated markers, promotes Treg regulatory cells expansion and promote normoglycemia in a xenogeneic model of Type 1 Diabetes (T1D). In order to exert their pro-tolerogenic function, fibrocytic MDSC require direct contact with activated T cells, which leads to the induction and secretion of indoleamine 2,3 deoxygenase (IDO). This new myeloid subset may represent an important tool for the in vitro and in vivo production of Treg for the treatment of autoimmune diseases, and either prevention or control of allograft rejection. Keywords: expression profiling by array Human Umbilical cord blood (UCB) samples were cultured for 4 days with GM-CSF and G-CSF after Ficoll-Paque gradient separation and red cell lysis. CD33+/IL-4RM-NM-1+ cells were sterilely sorted and part stored in Trizol for RNA extraction (BEFORE CONTACT), and part co-cultured with autologous PHA activated CD3+ T cells either in cell to cell contact (CONTACT) or using a transwell device to separate the two popyulations (TRANSWELL). Three days later, T cells were removed from the co-culture either by repeated washes with PBS or by simply removing the transwell device. Adherent cells were lysed with Trizol and stored at -80M-bM-^@M-^YC. RNA extraction was carry on with miRNAeasy mini kit from Quiagen; RNA QC was assessed by Agilent BioAnalyzer 6000 and samples were analyzed with Affymetrix Human 2.0 ST array

Project description:Tumor growth is associated with a profound alteration of myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). Analyzing the cytokines affecting myelo-monocytic differentiation produced by various experimental tumors, we found that GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse and human bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by the ability to impair the priming of IFN- -producing CD8+ T cells upon in vivo adoptive transfer. Moreover, adoptive transfer of syngeneic, GM-CSF+IL-6-conditioned MDSCs to diabetic mice transplanted with allogeneic pancreatic islets resulted in long term acceptance of the allograft and correction of the diabetic status. Cytokines inducing MDSCs acted on a common molecular pathway. Immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on C/EBP transcription factor, a key component of the emergency myelopoiesis triggered by stress and inflammation. Adoptive transfer of tumor antigen-specific CD8+ T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBP in myeloid compartment. These data unveil another link between inflammation and cancer and identify a novel molecular target to control tumor-induced immune suppression. We used gene expression analysis to identify those factors, secreted by tumor-infiltrating MDSC, which could drive emathopoiesis. Moreover we compare gene expression profile of tumor-induced MDSC, obtained from either the spleen and the tumor infiltrate of tumor bearing mice, and in vitro bone marrow-derived MDSC. CD11b+ cells were immunomagnetically enriched from various murine tissue and experimental conditions, and cRNA samples were prepared accordingly to Expression Analysis: Technical Manual. 701021 Rev. 5. Santa Clara, CA, Affymetrix; 2004, and hybridized to the Affymetrix GeneChip MOE430 2.0 array which contains more than 45,000 probe sets, representing more than 34,000 genes. CD11b+ cells obtained from the spleen of healthy BALB/c and C57BL/6 mice were used as reference sample for tumor induced CD11b+ MDSC, enriched from either the spleen and the tumor infiltrate of tumor-bearing mice. Moreover CD11b+ cells enriched from fresh bone marrow were used as reference sample for in vitro bone marrow-differentiated MDSC, obtained with either GM-CSF+IL-6 and GM-CSF+G-CSF 4 days cytokine cocktail treatment.

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Molecular and cellular characterization of Cord Blood derived, IDO expressing human fibrocystic Myeloid Derived Suppressor Cells

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