Project description:Myeloid-derived suppressor cells (MDSC) is a heterogeneous population of cells that can negatively regulate T-cell function. As opposed to murine MDSC, which are characterized as Gr-1+CD11b+ cells, human MDSC are not so clearly defined due to lack of specific markers. Our lab has previously identified a new subset of MDSC as CD14+HLA-DR-neg/low cells from PBMC. CD14+HLA-DR-neg/low MDSC not only suppress proliferation and IFN-gamma secretion of autologous T cells, but also induce CD25+Foxp3+ regulatory T cells that are suppressive in vitro, whereas the counterpart CD14+HLA-DR-high monocytes don’t have the effect. In this study, we compare the immune-related gene expression between CD14+HLA-DR-neg/low MDSC and CD14+HLA-DR-high monocytes to better characterize the difference between these two populations and to find new potential specific marker for human MDSC. PBMC were isolated from fresh blood healthy donor by density centrifugation. CD14+ cells were isolated by AutoMACS CD14 microbeads using a AutoMACS (Miltenyi), and then stained with CD14 and HLA-DR antibodies. MDSC and monocytes control cells were sorted as CD14+ HLA-DR-neg/low and CD14+HLA-DR-high cells respectively. The sorted two populations were immediately frozen in liquid nitrogen and shipped to the company on dry ice for RNA isolation and further microarray.
Project description:Myeloid-derived suppressor cells (MDSC) is a heterogeneous population of cells that can negatively regulate T-cell function. As opposed to murine MDSC, which are characterized as Gr-1+CD11b+ cells, human MDSC are not so clearly defined due to lack of specific markers. Our lab has previously identified a new subset of MDSC as CD14+HLA-DR-neg/low cells from PBMC. CD14+HLA-DR-neg/low MDSC not only suppress proliferation and IFN-gamma secretion of autologous T cells, but also induce CD25+Foxp3+ regulatory T cells that are suppressive in vitro, whereas the counterpart CD14+HLA-DR-high monocytes don’t have the effect. In this study, we compare the immune-related gene expression between CD14+HLA-DR-neg/low MDSC and CD14+HLA-DR-high monocytes to better characterize the difference between these two populations and to find new potential specific marker for human MDSC.
Project description:Study of human monocytic Myeloid-Derived Suppressor cells Mo-MDSC (CD14+ HLA-DRneg/low) has been hampered by the lack of positive cell-surface markers. In order to identify positive markers for Mo-MDSC, we performed microarray analysis comparing Mo-MDSC cells from healthy subjects versus CD14+ HLA-DRhigh monocytes. We have identified the surface ectoenzyme Vanin-2(VNN2) protein as a novel biomarker highly-enriched in healthy subjects Mo-MDSC. Indeed, healthy subjects Mo-MDSC cells expressed 68% VNN2, whereas only 9% VNN2 expression was observed on CD14+ HLA-DRhigh cells (n=4 p<0.01). The top 10 percent positive VNN2 monocytes expressed CD33 and CD11b while being negative for HLA-DR, CD3, CD15, CD19 and CD56, consistent with a Mo-MDSC phenotype. CD14+VNN2high monocytes were able to inhibit CD8 T cell proliferation comparably to traditional Mo-MDSC at 51% and 48% respectively. However, VNN2 expression on CD14+ monocytes from glioma patients was inversely correlated to their grade. CD14+VNN2high monocytes thus appear to mark a monocytic population similar to Mo-MDSC only in healthy subjects, which may be useful for tumor diagnoses. Myeloid-Derived Suppressor Cells (MDSC) are identified by upregulated expression of the cell surface ectoenzyme Vanin-2 in healthy subjects
Project description:Human left ventricular cardiac tissue was isolated from patients at the time of surgery. Tissue was digested into single cell suspension and labeled with CD45, CD14, HLA-DR and CCR2 antibodies. Macrophages were sorted into three groups (CCR2+ Monocytes: CD14+ CCR2+ HLA-DRNeg; CCR2+ Macrophages: CD14+CCR2+HLA-DR+; MHC-II hi Macrophages: CD14+CCR2-HLA-DR+)
Project description:Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) inhibit immune responses in cancer, limit the effects of therapies and promote tumor cell metastasis. Here, we have identified a new precursor that differentiates into granulocytes in vitro and in vivo yet belong to the monocytic lineage. We have termed these cells monocyte-like precursors of granulocytes (MLPG). Under steady state conditions MLPG were absent in the spleen and barely detectable in the bone marrow (BM). In contrast, these cells significantly expanded in tumor-bearing mice. Selective depletion of monocytic cells had no effect on the number of granulocytes present in naïve mice but decreased the population of PMN-MDSC in tumor-bearing mice by 50%. The expansion of MLPG was found to be controlled by the down-regulation of the protein Rb1 and not the IRF8 transcription factor that is known to regulate the expansion of PMN-MDSC from classical granulocytes precursors. In cancer patients, putative MLPG were found within the population of CD15 CD14+HLA-DR-/lo M-MDSC. CXCR1+CD15 CD14+HLA-DR-/lo cells lacked immune suppressive activity but had potential to differentiate to neutrophils in contrast to monocytes and CXCR1- suppressive M-MDSC. These findings describe a mechanism of abnormal myelopoiesis in cancer and suggest potential new approaches for selective targeting of MDSC.
Project description:Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) inhibit immune responses in cancer, limit the effects of therapies and promote tumor cell metastasis. Here, we have identified a new precursor that differentiates into granulocytes in vitro and in vivo yet belong to the monocytic lineage. We have termed these cells monocyte-like precursors of granulocytes (MLPG). Under steady state conditions MLPG were absent in the spleen and barely detectable in the bone marrow (BM). In contrast, these cells significantly expanded in tumor-bearing mice. Selective depletion of monocytic cells had no effect on the number of granulocytes present in naïve mice but decreased the population of PMN-MDSC in tumor-bearing mice by 50%. The expansion of MLPG was found to be controlled by the down-regulation of the protein Rb1 and not the IRF8 transcription factor that is known to regulate the expansion of PMN-MDSC from classical granulocytes precursors. In cancer patients, putative MLPG were found within the population of CD15 CD14+HLA-DR-/lo M-MDSC. CXCR1+CD15 CD14+HLA-DR-/lo cells lacked immune suppressive activity but had potential to differentiate to neutrophils in contrast to monocytes and CXCR1- suppressive M-MDSC. These findings describe a mechanism of abnormal myelopoiesis in cancer and suggest potential new approaches for selective targeting of MDSC.
Project description:In our study, MegaClust - an unsupervised, data-driven algorithm - barely described mDCs and pDC subsets were identified. To confirm these findings we performed RNA sequencing Facs sorted of mDCs (CD123-CD11c+CD4+HLA-DR+), pDCs (CD123+CD11c-CD4+HLA-DR+) and monocytes (CD14+) from healthy donors and compared these with publicly available data.
Project description:Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14++CD16-, intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes. However, CD14++CD16+ monocytes remain the most poorly characterized subset so far. Therefore we analyzed the transcriptomes of the three monocyte subsets using SuperSAGE in combination with high-throughput sequencing. Analysis of 5,487,603 tags revealed unique identifiers of CD14++CD16+ monocytes, delineating these cells from the two other monocyte subsets. CD14++CD16+ monocytes were linked to antigen processing and presentation (e.g. CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (e.g. TGFB1, AIF1, PTPN6), and to angiogenesis (e.g. TIE2, CD105). Therefore we provide genetic evidence for a distinct role of CD14++CD16+ monocytes in human immunity.
Project description:Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14++CD16-, intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes. However, CD14++CD16+ monocytes remain the most poorly characterized subset so far. Therefore we analyzed the transcriptomes of the three monocyte subsets using SuperSAGE in combination with high-throughput sequencing. Analysis of 5,487,603 tags revealed unique identifiers of CD14++CD16+ monocytes, delineating these cells from the two other monocyte subsets. CD14++CD16+ monocytes were linked to antigen processing and presentation (e.g. CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (e.g. TGFB1, AIF1, PTPN6), and to angiogenesis (e.g. TIE2, CD105). Therefore we provide genetic evidence for a distinct role of CD14++CD16+ monocytes in human immunity. Human monocyte subsets (CD14++CD16-, CD14++CD16+, CD14+CD16++) were isolated from 12 healthy volunteers based on MACS technology. Total RNA from monocyte subsets was isolated and same aliquots from each donor and monocyte subset were matched for SuperSAGE. Three SuperSAGE libraries (CD14++CD16-, CD14++CD16+ and CD14+CD16++) were generated.
Project description:Myeloid-derived suppressor cells (MDSCs) are highly immunosuppressive myeloid cells, which increase in cancer patients. The molecular mechanism behind their generation and function is unclear. Whereas granulocytic-MDSCs correlate with poor overall survival in breast cancer, the presence and relevance of monocytic-MDSCs (Mo-MDSCs) is unknown. Here we report for the first time an enrichment of functional blood Mo-MDSCs in breast cancer patients before they acquire a typical Mo-MDSC surface phenotype. A clear population of Mo-MDSCs with the typical cell surface phenotype (CD14+HLA-DRlow/-Co-receptorlow/-) increased significantly first during disease progression and correlated to metastasis to lymph nodes and visceral organs. Furthermore, monocytes, comprising the Mo-MDSC population, from patients with metastatic breast cancer resemble the reprogrammed immunosuppressive monocytes in patients with severe infections, both by their surface and functional phenotype but also at their molecular gene expression profile. Our data suggest that monitoring the Mo-MDSC levels in breast cancer patients may represent a novel and simple biomarker for assessing disease progression. Peripheral blood monocytes were isolated using magnetic cell sorting from 4 patients with metastatic breast cancer, 3 healthy controls, 3 patients with sepsis and 3 patients with active tuberculosis were immediately frozen at -80C in TRIZOL.