Population Of Immature Cells Research Articles

Effect of First-Line Treatment on Myeloid-Derived Suppressor Cells’ Subpopulations in the Peripheral Blood of Patients with Non–Small Cell Lung Cancer

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells of myeloid origin whose expression is induced by, among others things, vascular endothelial growth factor. We have previously identified two monocytic and one granulocytic MDSC subpopulations associated with the clinical outcome in patients with non-small cell lung cancer (NSCLC). The aim of the present study was to evaluate the effect of chemotherapy on these MDSC subpopulations. Circulating immune cells from 46 patients with unresectable NSCLC were analyzed by flow cytometry before the initiation of chemotherapy and after three cycles. Changes in the frequencies of the MDSC subpopulations were correlated with clinical outcome. Chemotherapy had no uniform effect on either the number or the functionality of monocytic and granulocytic MDSCs. However, three cycles of bevacizumab-containing regimens significantly reduced the percentage of the granulocytic-MDSCs compared with non-bevacizumab-based regimens (p= 0.0086). At the time of evaluation of response, disease progression was associated with significantly higher levels of all three MDSC subpopulations compared with in patients with disease control. Ιn patients with disease progression after three cycles of chemotherapy, the percentage of CD15-positive monocytic MDSCs was significantly increased compared with baseline. In the peripheral blood of patients with NSCLC, bevacizumab-based chemotherapy significantly reduced the levels of granulocytic MDSCs. An increase in the levels of CD15-positive monocytic MDSCs was associated with poor response to treatment and disease progression, providing evidence of their clinical relevance in patients with NSCLC.

Identification of B7-H4 as a functional surface marker for monocytic myeloid-derived suppressor cells

Abstract Myeloid-derived suppressor cells (MDSC), a heterogeneous population of immature myeloid cells, have a strong ability to suppress T cell proliferation and function. However, so far there is no universal surface marker of MDSC for mice and humans. Here we report that B7-H4 is expressed in monocytic myeloid-derived suppressor cells (M-MDSCs, CD11b+Ly-6G−Ly-6Chigh) but not granulocytic myeloid-derived suppressor cells (G-MDSCs, CD11b+Ly-6GhighLy-6Cmid). Both M-MDSCs and G-MDSCs are isolated from tumor-bearing mice. Cell sorting of B7-H4-positive and B7-H4-negative cells from the M-MDSC fraction isolated from tumor-bearing mice shows that B7-H4-positive M-MDSCs have significantly stronger T cell suppressive function than B7-H4-negative M-MDSCs independent of the expression level of iNOS and Arginase 1. Consistent with this, M-MDSCs from B7-H4-deficiency mice display a reduced immunosuppressive function compared to M-MDSCs from wild-type mice. Furthermore, by inducing B7-H4-positive M-MDSCs from mouse bone marrow-derived MDSCs or from human peripheral blood mononuclear cells, glucocorticoid treatment significantly suppresses T cell proliferation and function. In summary, our findings identify B7-H4 as a novel functional surface marker for both mouse and human M-MDSCs.

Different characteristics of Myeloid-derived suppressor cells from septic and tumor-bearing mice

Abstract Background Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells and immature myeloid cells. These cells have immunosuppressive properties and express myeloid differentiation markers Gr-1 and CD11b in mice. The characteristics of MDSCs in different pathological conditions remain unclear. Methods Polymicrobial sepsis was induced by cecal ligation and puncture. For tumor model, mice were injected s.c. in the flank with 5×105 3LL or B16 tumor cells. Splenic Gr-1+ CD11b+ MDSCs were harvested from 7-day septic mice and three-week tumor-bearing mice, which purified using magnetically assisted cell sorting. Results MDSCs from tumor (3LL and B16)-bearing and septic mice shared a common phenotype and morphology. In contrast to tumor-bearing MDSCs, septic MDSCs were mainly characterized as neutrophil like-MDSCs, which expressed lower levels of CD115. Tumor bearing MDSCs have a greater inhibitory effect on Ag-induced CD4+ T cell proliferation than those from septic mice, and expressed higher levels of ARG-1. In addition, septic MDSCs significantly induced macrophage to M1 (F4/80+ CD206−) and expressed proinflamatory cytokine IL-6, whereas tumor-bearing MDSCs induced macrophage to M2 (F4/80+ CD206+) and expressed anti-inflammatory cytokine TGF-beta. Conclusion Sepsis derived MDSCs are functionally different from tumor-bearing MDSCs, which suggests there different functions in the disease’s progress.

Correction: Yeast-Derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-Derived Suppressor Cells (MDSC) by Inducing Polymorphonuclear MDSC Apoptosis and Monocytic MDSC Differentiation to APC in Cancer.

Albeituni, S. H., C. Ding, M. Liu, X. Hu, F. Luo, G. Kloecker, M. Bousamra, II, H.-g. Zhang, and J. Yan. 2016. Yeast-derived particulate β-glucan treatment subverts the suppression of myeloid-derived suppressor cells (MDSC) by inducing polymorphonuclear MDSC apoptosis and monocytic MDSC

Reprogramming of myeloid compartments supporting tissue repair during DSS-induced colitis recovery

Abstract Myeloid-derived suppressor cells (MDSC) emerging during tumor growth or chronic inflammation play a critical role in regulating T cell function. However, mechanisms governing the generation of these cells remain unclear and need to be further defined. Using a DSS-induced colitis and recovery model, we characterized the dynamic changes within the myeloid compartments and the emerging of MDSC during active and resolution phases of inflammation. We show that the immature myeloid cell population expands and undergoes a functional reprogramming event in bone marrow (BM) and the spleen to form suppressive MDSC not at the active inflammation phase, but specifically at the resolution phase during colitis transition to recovery. In particular, two major functional changes occur when colitic mice were allowed to recover: 1) CD11b+Gr-1+myeloid cells in bone marrow and spleen acquire T cell suppressive functions, and 2) immature granulocytic myeloid cells increase the expression of chemokine receptor CXCR2, which enables their entry into circulation from BM. Additionally we determined enhanced activation of STAT3 and subsequent transcriptional enhancement of Arg1, iNOS, and gp91 in the MDSC isolated from resolution time points. We found that IL-17, the major cytokine associated with active colitis, remains elevated in the serum throughout the wound healing/resolution process. IL-17, together with the enhanced production of tissue repair cytokines, including IL-10, an activator of STAT3, produce a unique cytokine profile, resulting in a switch to induce reprograming and mobilization of the immature myeloid cells to suppress the inflammatory reaction in favor of tissue repair.

TNF Neutralization Results in the Delay of Transplantable Tumor Growth and Reduced MDSC Accumulation.

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells (IMCs) that, under normal conditions, may differentiate into mature macrophages, granulocytes, and dendritic cells. However, under pathological conditions associated with inflammation, cancer, or infection, such differentiation is inhibited leading to IMC expansion. Under the influence of inflammatory cytokines, these cells become MDSCs, acquire immunosuppressive phenotype, and accumulate in the affected tissue, as well as in the periphery. Immune suppressive activity of MDSCs is partly due to upregulation of arginase 1, inducible nitric oxide synthase, and anti-inflammatory cytokines, such as IL-10 and TGF-β. These suppressive factors can enhance tumor growth by repressing T-cell-mediated anti-tumor responses. TNF is a critical factor for the induction, expansion, and suppressive activity of MDSCs. In this study, we evaluated the effects of systemic TNF ablation on tumor-induced expansion of MDSCs in vivo using TNF humanized (hTNF KI) mice. Both etanercept and infliximab treatments resulted in a delayed growth of MCA 205 fibrosarcoma in hTNF KI mice, significantly reduced tumor volume, and also resulted in less accumulated MDSCs in the blood 3 weeks after tumor cell inoculation. Thus, our study uncovers anti-tumor effects of systemic TNF ablation in vivo.

Dickkopf-related protein 1 (Dkk1) regulates the accumulation and function of myeloid derived suppressor cells in cancer.

Tumor-stroma interactions contribute to tumorigenesis. Tumor cells can educate the stroma at primary and distant sites to facilitate the recruitment of heterogeneous populations of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs). MDSCs suppress T cell responses and promote tumor proliferation. One outstanding question is how the local and distant stroma modulate MDSCs during tumor progression. Down-regulation of β-catenin is critical for MDSC accumulation and immune suppressive functions in mice and humans. Here, we demonstrate that stroma-derived Dickkopf-1 (Dkk1) targets β-catenin in MDSCs, thus exerting immune suppressive effects during tumor progression. Mice bearing extraskeletal tumors show significantly elevated levels of Dkk1 in bone microenvironment relative to tumor site. Strikingly, Dkk1 neutralization decreases tumor growth and MDSC numbers by rescuing β-catenin in these cells and restores T cell recruitment at the tumor site. Recombinant Dkk1 suppresses β-catenin target genes in MDSCs from mice and humans and anti-Dkk1 loses its antitumor effects in mice lacking β-catenin in myeloid cells or after depletion of MDSCs, demonstrating that Dkk1 directly targets MDSCs. Furthermore, we find a correlation between CD15(+) myeloid cells and Dkk1 in pancreatic cancer patients. We establish a novel immunomodulatory role for Dkk1 in regulating tumor-induced immune suppression via targeting β-catenin in MDSCs.

Immune Dysfunction Induced by Myeloid-Derived Suppressor Cells in Lymphoma

Lymphoma cancer cells have strongly orchestrated interactions with immune cells and also other surrounding stromal cells in tumor microenvironment. Myeloid derived suppressor cells (MDSC) is believed to have major role in induction of immune suppressive effects in tumor microenvironment. This heterogeneous population of immature myeloid cells influence on immune cells function by indirectly Treg differentiation and directly oxidative stress induction and nutrient depletion in tumor milieu. Our understanding of the role of MDSCs in tumor microenvironment suggests novel immunotherapeutic approaches and need to be more explored. In this article we briefly discuss the main MDSCs immune dysfunction mechanisms with more focus in lymphoma.

Papillary Immature Metaplasia of the Anal Canal: A Low-grade Lesion That Can Mimic a High-grade Lesion.

In a natural history study of anal human papillomavirus (HPV) infection and HPV-related lesions among homosexual men in Sydney, Australia, we identified 15 examples of papillary immature metaplasia (PIM) in anal biopsy samples. PIM has previously been described in the cervix, but not in the anal canal. PIM is a form of exophytic low-grade squamous intraepithelial lesion (eLSIL) also known as condyloma. In contrast to the maturing keratinocytes and koilocytosis seen in conventional eLSIL, the slender papillary structures of PIM have a surface population of immature squamous cells. In our anal samples PIM was characterized by close proximity to conventional eLSIL, was negative for p16 (p16) expression, and revealed the presence of a single low-risk HPV genotype (either 6 or 11) in laser capture microdissected lesions. The clinical significance of recognizing PIM lies in preventing misdiagnosis as high-grade squamous intraepithelial lesion, (the presumed precursor to anal cancer), due to the morphologic immaturity of the cell population. In routine practice, awareness of anal canal PIM and p16 immunostaining will prevent this. Further study of the natural history of anal canal PIM is needed.

Invariant NKT cells are resistant to circulating CD15+ myeloid-derived suppressor cells in patients with head and neck cancer.

Myeloid‐derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with an immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). However, the effect on the host immune system, especially on invariant NKT (iNKT) cells with potent anti‐tumor activity, remains unclear. In this study, we investigated the effects of circulating MDSC subsets on the peripheral lymphocytes of patients with head and neck tumors. A significant accumulation of CD15+ granulocytic MDSC (G‐MDSC) and CD14+ monocytic MDSC (M‐MDSC) was demonstrated in HNSCC patients. The percentage of G‐MDSC showed an inverse correlation with the percentage of T cells in the peripheral blood. The increased G‐MDSC was significantly associated with advanced clinical stage and poor prognosis of HNSCC patients. The proliferation and viability of T cells were suppressed by CD15+ cells, and the suppression was reversed by adding the hydrogen peroxide scavenger catalase. However, iNKT cell activation upon α‐galactosylceramide (αGalCer) stimulation was not affected by the presence or absence of CD15+ G‐MDSC. These results indicate that increased G‐MDSC negatively affects peripheral T cell immunity, but not iNKT cells, in HNSCC patients, and that T cells are more sensitive to hydrogen peroxide produced by G‐MDSC than iNKT cells. Cancer immunotherapy designed to enhance the antitumor activity of iNKT cells by stimulation with αGalCer may remain effective in the presence of G‐MDSC.

Abstract B78: The influence of breast cancer on the metabolic status of myeloid cells

Abstract Breast cancer is the most common cancer and the second leading cause of cancer death in women. Except for surgery, radiotherapy, chemotherapy and targeting therapy, breast cancer vaccine is now actively developed. However, the outcome of most therapeutic cancer vaccines was not satisfying probably due to the induction of immunosuppression in cancer patients. Myeloid-derived suppressor cells (MDSCs) are one of the major immunoregulatory cell populations, which play important roles not only in T-cell suppression but also in angiogenesis, lymphangiogenesis and metastasis during tumor progression. MDSCs are heterogeneous populations of immature myeloid cells, which expand and developed in responses to growth factors, cytokines and chemokines derived from cancer cells and then are released from bone marrow to peripheral tissues including tumor site. It is well known as tumor cells could influence the activation and proliferation of myeloid cells through release of cytokines during tumor progression to result in the accumulation of large number of MDSCs in cancer patients. However little is known about the relevance of metabolic status and accumulation of MDSCs in cancer patients during tumor progression. We therefore try to elucidate the metabolic changes during differentiation of myeloid cells into MDSCs. To intensively study the transcriptional program of MDSCs in comparison to their healthy counterparts, we perform DNA microarray analysis using mRNAs from monocytic MDSCs, granulocytic MDSCs from primary tumor sites of 4T1 tumor-bearing mice, monocytes and neutrophils from bone marrow of healthy mice. MDSCs up-regulated mRNA levels of genes related to angiogenesis and lymphangiogenesis e.g. VEGFa, angiopoietin 1, PROK2 (BV8), MMP9, MMP12, MMP13 and MMP14, genes of pro-inflammatory cytokines e.g. IL-1α, IL-6 and TNFα, genes of immunosuppressive molecules e.g. arginase 1, iNOS, PD-L1, PD-L2, CD83 and CD155. We are currently examining the metabolomics of these MDSCs and their healthy counterparts and expect to prove the association of certain metabolic changes with the differentiation of MDSCs. Citation Format: Li-Rung Huang, Shiou-Ling Jian, Yi-Jyun Jhou. The influence of breast cancer on the metabolic status of myeloid cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B78.

Mesenchymal Stem Cells and Myeloid Derived Suppressor Cells: Common Traits in Immune Regulation.

To protect host against immune-mediated damage, immune responses are tightly regulated. The regulation of immune responses is mediated by various populations of mature immune cells, such as T regulatory cells and B regulatory cells, but also by immature cells of different origins. In this review, we discuss regulatory properties and mechanisms whereby two distinct populations of immature cells, mesenchymal stem cells, and myeloid derived suppressor cells mediate immune regulation, focusing on their similarities, discrepancies, and potential clinical applications.

Myeloid-derived suppressor cells (MDSC) facilitate distant metastasis of malignancies by shielding circulating tumor cells (CTC) from immune surveillance

The mechanisms of distant metastasis of malignancies largely remain unknown. Circulating tumor cells (CTC) derived from the primary cancer initiate distant metastasis by entering and traversing the bloodstream. Current methods to detect CTC are based on the notion that CTC do not express the common leukocyte antigen CD45. However, these methods neglect the fact that CTC can directly adhere to platelets and immune cells and therefore appear to be CD45-positive. The potential effects of interactions between CTC and adhesive immune cells have been largely overlooked, despite the fact that most CTC are killed by immune effector cells and only those that evade immune surveillance result in clonal expansion and metastatic lesions. It is crucial to define the characteristics that allow a select CTC population to escape immune surveillance; particularly, it must be determined whether interactions between CTC and adhesive immune cells provide a protective effect on CTC survival. If interactions between CTC and adhesive immune cells offer a selective advantage to those CTC cells, the next consideration is which characteristics of a CTC-immune cell population allow sufficient protection to facilitate immune evasion. Myeloid-derived suppressor cells (MDSC) are a large heterogeneous population of immature myeloid cells that accumulate during cancer progression to induce extensively systemic and local immunosuppression, a phenomenon that has been demonstrated to facilitate cancer distant metastasis. We hypothesize, therefore, that CTC populations interacting with adhesive immune cells will have different biological behavior than CTC populations alone. Further, we hypothesize that CTC can create a defensive shield consisting of adhesive MDSC, which allows evasion of immune surveillance and therefore facilitates distant metastatic lesions. This possibility highlights the importance of direct interactions between CTC and adhesive immune cells and suggests the potential target that the CTC-MDSC cluster represents for prevention and treatment of distant metastasis of malignancies.

Physiologic Hematopoiesis Is Dependent on Stromal Expression of Laminin-γ1

Abstract OBJECTIVE: The microenvironment of the bone marrow hematopoietic niche includes: 1) blood vessels; 2) adjacent stromal cells; 3) hematopoietic cells; and 4) cytokines, growth factors, and structural support molecules. Injury to the bone marrow niche affects hematopoietic cells through loss of either physical contact with stromal cells or niche-derived growth factors. Extracellular matrix factors required to maintain the steady-state bone marrow perivascular niche and hematopoiesis have not been fully identified. Here, we examine the role of the extracellular matrix protein laminin-γ1 in adult bone marrow for maintaining the perivascular hematopoietic niche and hematopoiesis. METHODS: A global and inducible laminin-γ1 deficient mouse, hereafter referred to as mutant, was generated in which LAMC1 gene recombination could be monitored by a fluorescent reporter transgene. Tamoxifen was used to induce LAMC1 gene recombination and knock-down of laminin-γ1 protein expression in 8-12 week old mice. Tamoxifen-treated mice lacking the inducible Cre transgene were used as controls for all experiments. Analysis of mutant mice was performed 17-24 days following the first dose of tamoxifen. Bone marrow samples were examined by immunohistochemistry for the presence of laminin-1 protein, and hematopoietic tissues, including bone marrow, peripheral blood, spleen, thymus and lymph node were analyzed by flow cytometry for hematopoietic stem and progenitor cells, and mature hematopoietic cell populations, and by ex vivo hematopoietic progenitor cell colony forming assays. To examine the stromal cell contribution to laminin-γ1 mediated hematopoietic dysfunction, control and mutant mice that had not yet undergone LAMC1 gene recombination were then transplanted with wild type bone marrow. After full hematopoietic reconstitution, chimeric mice were induced with tamoxifen and hematopoietic stem and progenitor cells and mature hematopoietic cell populations were examined. RESULTS: LAMC1 gene recombination in the bone marrow of tamoxifen-treated mutant animals varied between 20-45%. With LAMC1 gene recombination, laminin-protein in the bone marrow is rapidly depleted. Bone marrow blood vessels dilate, and the bone marrow becomes hypocellular. Hematopoietic stem and progenitor cells are reduced in number, as are bone marrow B-cell progenitors, and thymic double-positive T-cells. Erythrocyte and thrombocyte numbers are not changed, nor were NK cell populations, the majority of myeloid cell subtypes, or mature B cells in the bone marrow and blood. CD8+ T-cells were increased within the bone marrow, as were CD11b+, Ly6Clo Ly6G+ myeloid cells in the peripheral blood. Examination of gene recombination within each of these cell types was not consistent with cell-intrinsic mechanism of hematopoietic alterations. Analysis of bone marrow chimeric animals identified loss of stromal cell-produced laminin-γ1 as a significant mediator of these hematopoietic alterations. CONCLUSIONS: The rapid depletion of laminin-γ1 from the bone marrow indicates a high basal turnover rate for the extracellular matrix in the bone marrow. The attendant hematopoietic dysfunction that follows loss of laminin-γ1 production in the bone marrow is specific for immature hematopoietic cell populations, and is dependent on stromal cell production of laminin-γ1. This study identifies laminin-γ1 as a niche-dependent regulator of hematopoietic stem and progenitor cell populations, and suggests that that regulation of its production and degradation may be new targets for the study of the hematopoietic niche. Disclosures No relevant conflicts of interest to declare.

RUNX1-Evi-1 fusion gene inhibited differentiation and apoptosis in myelopoiesis: an in vivo study.

BackgroundAcute myeloid leukemia (AML) 1-Evi-1 is a chimeric gene generated by the t (3; 21) (q26; q22) translocation, which leads into malignant transformation of hematopoietic stem cells by unclear mechanisms. This in vivo study aimed to establish a stable line of zebrafish expressing the human RUNX1-Evi-1 fusion gene under the control of a heat stress-inducible bidirectional promoter, and investigate its roles in hematopoiesis and hematologic malignancies.MethodsWe introduced human RUNX1-Evi-1 fusion gene into embryonic zebrafish through a heat-shock promoter to establish Tg(RE:HSE:EGFP) zebrafish. Two males and one female mosaic F0 zebrafish embryos (2.1 %) were identified as stable positive germline transgenic zebrafish.ResultsThe population of immature myeloid cells and hematopoietic blast cells were accumulated in peripheral blood and single cell suspension from kidney of adult Tg(RE:HSE:EGFP) zebrafish. RUNX1-Evi-1 presented an intensive influence on hematopoietic regulatory factors. Consequently, primitive hematopoiesis was enhanced by upregulation of gata2 and scl, while erythropoiesis was downregulated due to the suppression of gata1. Early stage of myelopoiesis was flourishing with the high expression of pu.1, but it was inhibited along with the low expression of mpo. Microarray analysis demonstrated that RUNX1-Evi-1 not only upregulated proteasome, cell cycle, glycolysis/gluconeogenesis, tyrosine metabolism, drug metabolism, and PPAR pathway, but also suppressed transforming growth factor β, Jak-STAT, DNA replication, mismatch repair, p53 pathway, JNK signaling pathway, and nucleotide excision repair. Interestingly, histone deacetylase 4 was significantly up-regulated. Factors in cell proliferation were obviously suppressed after 3-day treatment with histone deacetylase inhibitor, valproic acid. Accordingly, higher proportion of G1 arrest and apoptosis were manifested by the propidium iodide staining.ConclusionRUNX1-Evi-1 may promote proliferation and apoptosis resistance of primitive hematopoietic cell, and inhibit the differentiation of myeloid cells with the synergy of different pathways and factors. VPA may be a promising choice in the molecular targeting therapy of RUNX1-Evi-1-related leukemia.

Expression of Cationic Amino Acid Transporter 2 Is Required for Myeloid-Derived Suppressor Cell-Mediated Control of T Cell Immunity.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells that expand during benign and cancer-associated inflammation and are characterized by their ability to inhibit T cell immunity. Increased metabolism of l-Arginine (l-Arg), through the enzymes arginase 1 and NO synthase 2 (NOS2), is well documented as a major MDSC suppressive mechanism. Therefore, we hypothesized that restricting MDSC uptake of l-Arg is a critical control point to modulate their suppressor activity. Using murine models of prostate-specific inflammation and cancer, we have identified the mechanisms by which extracellular l-Arg is transported into MDSCs. We have shown that MDSCs recruited to localized inflammation and tumor sites upregulate cationic amino acid transporter 2 (Cat2), coordinately with Arg1 and Nos2. Cat2 expression is not induced in MDSCs in peripheral organs. CAT2 contributes to the transport of l-Arg in MDSCs and is an important regulator of MDSC suppressive function. MDSCs that lack CAT2 have significantly reduced suppressive ability ex vivo and display impaired capacity for regulating T cell responses in vivo as evidenced by increased T cell expansion and decreased tumor growth in Cat2(-/-) mice. The abrogation of suppressive function is due to low intracellular l-Arg levels, which leads to the impaired ability of NOS2 to catalyze l-Arg-dependent metabolic processes. Together, these findings demonstrate that CAT2 modulates MDSC function. In the absence of CAT2, MDSCs display diminished capacity for controlling T cell immunity in prostate inflammation and cancer models, where the loss of CAT2 results in enhanced antitumor activity.

Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells.

Targeting YAP-dependent MDSC infiltration impairs tumor progression

Meeting abstracts Myeloid-derived suppressor cells (MDSCs) represent a phenotypically heterogeneous population of immature myeloid cells that play a tumor-promoting role by maintaining a state of immunological anergy and tolerance. Similar to other solid tumor types, Prostate cancer (PCa) is

CD38-Expressing Myeloid-Derived Suppressor Cells Promote Tumor Growth in a Murine Model of Esophageal Cancer.

Myeloid-derived suppressor cells (MDSC) are an immunosuppressive population of immature myeloid cells found in advanced-stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allows MDSCs to suppress T-cell-mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctn(f/f)), a model of oral-esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38(high) MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38(high) MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38(low) MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor-derived factors, specifically IL6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38(+) MDSCs in peripheral blood of advanced-stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy.

Abstract P183: Myeloid-Derived Suppressor Cells Prevent Cyclosporine A-Induced Hypertension, Endothelial Dysfunction, and Renal Injury in Mice

Cyclosporine A (CsA) is an immunosuppressive drug used to treat focal segmental glomerulosclerosis, reduce autoimmune diseases, and prevent allograft rejection; however a limitation of CsA is that it can induce vascular and renal injury as well as hypertension. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature granulocytes, macrophages, and dendritic cells that suppress pro-inflammatory immune responses by secreting anti-inflammatory cytokines, inhibiting innate and adaptive immune cells, and inducing regulatory T cells (Tregs). We hypothesized that adoptive transfer of MDSCs would prevent CsA-induced vascular and renal injury and hypertension. Daily treatment of male C57BL6/J mice for 1 week with CsA (50 mg/kg/day, i.p. injection) significantly increased systolic blood pressure (Day 7 SBP in mmHg: Con=97±2 vs. CsA=145±3, p<0.05 vs. Con), decreased aortic endothelium-dependent relaxation responses, increased aortic fibronectin levels, increased renal glomerular mesangial expansion, increased renal fibronectin levels, and decreased splenic Treg levels. Adoptive transfer of 1 million MDSCs by i.p. injection on days 1, 4, and 7 partially prevented the CsA-induced rise in systolic blood pressure (CsA+1M MDSCs=120±3 mmHg) and the detrimental vascular and renal effects. However, adoptive transfer of 2 million MDSCs fully prevented the CsA-induced hypertension (CsA+2M MDSCs=105±1 mmHg) and vascular and renal effects. The anti-hypertensive and vascular and renal protective effects of 2 million MDSCs were independent of Treg induction as splenic Treg levels remained significantly decreased. These data suggest that MDSCs can prevent the hypertension and toxicity caused by CsA independent of Tregs and may be a therapeutic target for CsA-treated patients.