CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer and various pathological conditions. However, the phenotypic and functional heterogeneity of human MDSCs represents a major hurdle for the development of therapeutic strategies targeting or regulating MDSCs in tumor progression, inflammation, and graft-versus-host disease (GVHD). We previously shown that circulating HLA-DR^-CD14⁺ monocytic MDSCs are a major contributor to clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we identified, using high-throughput screening, a set of surface markers that are strongly expressed in HLA-DR^-CD14⁺ monocytic MDSCs isolated from the peripheral blood (PB) of patients receiving allo-HSCT. Subsequent experiments showed the consistent dominant expression of CD1d in monocytic MDSCs of allo-HSCT PB in comparison with granulocytic MDSCs. In addition, CD1d-expressing cells isolated from PB of allo-HSCT patients showed the suppressive activity of T cell proliferation and higher expression of MyD88 and IDO compared with CD1d^? cells. Our results suggest that CD1d could be a valuable marker for further therapeutic evaluation of human monocytic MDSCs for immune-related diseases, including GVHD.     

Expression of the B-Cell Receptor Component CD79a on Immature Myeloid Cells Contributes to Their Tumor Promoting Effects

The role of myeloid derived suppressor cells (MDSCs) in promoting tumorigenesis is well-established, and significant effort is being made to further characterize surface markers on MDSCs both for better diagnosis and as potential targets for therapy. Here we show that the B cell receptor adaptor molecule CD79a is unexpectedly expressed on immature bone marrow myeloid cells, and is upregulated on MDSCs generated in multiple different mouse models of metastatic but not non-metastatic cancer. CD79a on MDSCs is upregulated and activated in response to soluble factors secreted by tumor cells. Activation of CD79a on mouse MDSCs, by crosslinking with a specific antibody, maintained their immature phenotype (CD11b+Gr1+), enhanced their migration, increased their suppressive effect on T cell proliferation, and increased secretion of pro-tumorigenic cytokines such as IL-6 and CCL22. Furthermore, crosslinking CD79a on myeloid cells activated signaling through Syk, BLNK, ERK and STAT3 phosphorylation. In vivo, CD79+ myeloid cells showed enhanced ability to promote primary tumor growth and metastasis. Finally we demonstrate that CD79a is upregulated on circulating myeloid cells from lung cancer patients, and that CD79a+ myeloid cells infiltrate human breast tumors. We propose that CD79a plays a functional role in the tumor promoting effects of myeloid cells, and may represent a novel target for cancer therapy.     

Chronic intake of high fish oil diet induces myeloid-derived suppressor cells to promote tumor growth

Omega-3 polyunsaturated fatty acids enriched fish oil exerts beneficial anti-inflammatory effects in animal models with acute and chronic inflammatory diseases. Myeloid-derived suppressor cells (MDSCs), comprised of myeloid progenitors and precursors of myeloid cells, play vital roles in cancer. How fish oil affects the generation of MDSCs and the tumor development remains largely unexplored. Here, we show that dietary intake of high fish oil diet suppresses CD8⁺ T cells activation and proliferation in vivo via elevated levels of MDSCs. Mechanistically, high fish oil diet induces the expression of immunosuppressive cytokine IL-10 and promotes myelopoiesis in the spleen as well as other peripheral tissues. The immature myeloid cells in the spleen exhibit morphological and functional characteristics of MDSCs with the capability to downregulate CD8⁺ T cells activation. Depletion of MDSCs using anti-Gr-1 antibody decreases the growth of subcutaneously transferred B16 melanoma in mice on high fish oil diet. Interestingly, diet-induced production of MDSCs is not solely dependent of the spleen, as splenectomy has no effect on the tumor progress. Our data show that the liver functions as an alternative extramedullary hematopoiesis organ to support MDSCs differentiation and maintain tumor growth. Taken together, our study provides a novel insight into the physiological effects of fish oil and points to MDSCs as a possible mediator linking dietary fish oil intake and immunosuppression in cancer immunosurveillance.     

Identification of myeloid derived suppressor cells in dogs with naturally occurring cancer

Dogs with naturally occurring cancer represent an important large animal model for drug development and testing novel immunotherapies. However, poorly defined immunophenotypes of canine leukocytes have limited the study of tumor immunology in dogs. The accumulation of myeloid derived suppressor cells (MDSCs) is known to be a key mechanism of immune suppression in tumor-bearing mice and in human patients. We sought to identify MDSCs in the blood of dogs with cancer. Peripheral blood mononuclear cells (PBMCs) from dogs with advanced or early stage cancer and from age-matched healthy controls were analyzed by flow cytometry and microscopy. Suppressive function was tested in T cell proliferation and cytokine elaboration assays. Semi-quantitative RT-PCR was used to identify potential mechanisms responsible for immunosuppression. PBMCs from dogs with advanced or metastatic cancer exhibited a significantly higher percentage of CD11b(+)CD14(-)MHCII(-) cells compared to dogs diagnosed with early stage non-metastatic tumors and healthy dogs. These CD11b(+) CD14(-)MHCII(-) cells constitute a subpopulation of activated granulocytes that co-purify with PBMCs, display polymorphonuclear granulocyte morphology, and demonstrate a potent ability to suppress proliferation and IFN-γ production in T cells from normal and tumor-bearing donors. Furthermore, these cells expressed hallmark suppressive factors of human MDSC including ARG1, iNOS2, TGF-β and IL-10. In summary our data demonstrate that MDSCs accumulate in the blood of dogs with advanced cancer and can be measured using this three-marker immunophenotype, thereby enabling prospective studies that can monitor MDSC burden.     

Circulating and Tumor-Infiltrating Myeloid-Derived Suppressor Cells in Patients with Colorectal Carcinoma

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of myeloid cells that suppress T cell immunity in tumor-bearing hosts. In patients with colon cancer, MDSCs have recently been described as Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ cells correlating with cancer stage, metastasis and chemotherapy response. To learn in more detail the dynamic change and clinical relevance of circulating and tumor-infiltrating Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSC in colorectal cancer, we harvested the blood from 64 patients with varying stage of colorectal cancer and tumor and matched paraneoplastic tissues from 5 patients with advanced colorectal cancer, subjected them to multicolor flow cytometric analysis of percentage, absolute number and phenotype of MDSC and finally characterized their immunosuppressive functions. Our results demonstrate that peripheral blood from colorectal cancer patients contains markedly increased percentage and absolute number of Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSCs compared with healthy individuals, and this increase is closely correlated with clinical cancer stage and tumor metastasis but not primary tumor size and serum concentrations of cancer biomarker. A similar increase of MDSCs was also observed in the tumor tissues. Phenotyping MDSCs shows that they express high CD13 and CD39, low CD115, CD117, CD124 and PD-L1, and devoid of CD14, CD15 and CD66b, reminiscent of precursor myeloid cells. MDSCs from cancer patients but not healthy donors have the immunosuppressive activity and were able to inhibit in vitro autologous T-cell proliferation. Collectively, this study substantiates the presence of increased immunosuppressive circulating and tumor-resident Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSCs in patients with colorectal cancers correlating with cancer stage and metastasis, and suggests that pharmacologic blockade of MDSCs should be considered in future clinical trials.     

Metabolic reprogramming of myeloid-derived suppressor cells in the context of organ transplantation

Myeloid-derived suppressor cells (MDSCs) are naturally occurring leukocytes that develop from immature myeloid cells under inflammatory conditions that were discovered initially in the context of tumor immunity. Because of their robust immune inhibitory activities, there has been growing interest in MDSC-based cellular therapies for transplant tolerance induction. Indeed, various pre-clinical studies have introduced in vivo expansion or adoptive transfer of MDSC as a promising therapeutic strategy leading to a profound extension of allograft survival due to suppression of alloreactive T cells. However, several limitations of cellular therapies using MDSCs remain to be addressed, including their heterogeneous nature and limited expansion capacity. Metabolic reprogramming plays a crucial role for differentiation, proliferation and effector function of immune cells. Notably, recent reports have focused on a distinct metabolic phenotype underlying the differentiation of MDSCs in an inflammatory microenvironment representing a regulatory target. A better understanding of the metabolic reprogramming of MDSCs may thus provide novel insights for MDSC-based treatment approaches in transplantation. In this review, we will summarize recent, interdisciplinary findings on MDSCs metabolic reprogramming, dissect the underlying molecular mechanisms and discuss the relevance for potential treatment approaches in solid-organ transplantation.     

Deregulation of Apoptotic Factors Bcl-xL and Bax Confers Apoptotic Resistance to Myeloid-derived Suppressor Cells and Contributes to Their Persistence in Cancer

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that accumulate in response to tumor progression. Compelling data from mouse models and human cancer patients showed that tumor-induced inflammatory mediators induce MDSC differentiation. However, the mechanisms underlying MDSC persistence is largely unknown. Here, we demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis as compared with myeloid cells with the same phenotypes from tumor-free mice. Consistent with the decreased apoptosis, cell surface Fas receptor decreased significantly in tumor-induced MDSCs. Screening for changes of key apoptosis mediators downstream the Fas receptor revealed that expression levels of IRF8 and Bax are diminished, whereas expression of Bcl-xL is increased in tumor-induced MDSCs. We further determined that IRF8 binds directly to Bax and Bcl-x promoter in primary myeloid cells in vivo, and IRF8-deficient MDSC-like cells also exhibit increased Bcl-xL and decreased Bax expression. Analysis of CD69 and CD25 levels revealed that cytotoxic T lymphocytes (CTLs) are partially activated in tumor-bearing hosts. Strikingly, FasL but not perforin and granzymes were selectively activated in CTLs in the tumor-bearing host. ABT-737 significantly increased the sensitivity of MDSCs to Fas-mediated apoptosis in vitro. More importantly, ABT-737 therapy increased MDSC spontaneous apoptosis and decreased MDSC accumulation in tumor-bearing mice. Our data thus determined that MDSCs use down-regulation of IRF8 to alter Bax and Bcl-xL expression to deregulate the Fas-mediated apoptosis pathway to evade elimination by host CTLs. Therefore, targeting Bcl-xL is potentially effective in suppression of MDSC persistence in cancer therapy.     

Establishment of lal-/- Myeloid Lineage Cell Line That Resembles Myeloid-Derived Suppressive Cells

Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4⁺ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions.     

Myeloid-derived suppressor cell cytokine secretion as prognostic factor in myelodysplastic syndromes

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that play a critical immunosuppressive role in the tumour micro-environment. Although biological research on MDSCs has made progress, the relationship between the secretion of cytokines by MDSCs and poor prognosis is not clear, and there are no criteria to measure the functional status of MDSCs. Here, we detected the mRNA expression of IL-10, IL-12, TGF-β and TNF-α in MDSCs and the levels of these cytokines in MDSC culture supernatants of patients with myelodysplastic syndromes, and quantified the functional status of MDSCs by IL-10/IL-12 ratio and TGF-β/TNF-α ratio. We found that the ratio of IL-10/IL-12 and TGF-β/TNF-α was significantly higher in higher-risk MDS than in lower-risk MDS and normal control groups. The TGF-β/TNF-α ratio in MDSCs was positively correlated with the percentage of blast cells and was negatively correlated with the percentage of CD3⁺CD8⁺ T lymphocytes. Meanwhile, the TGF-β/TNF-α ratio was higher in patients with a lower absolute neutrophil count. It suggested that MDSCs in higher-risk MDS have a stronger immunosuppressive effect and might be related to poor prognosis. Quantifying the functional status of MDSCs with IL-10/IL-12 and TGF-β/TNF-α ratio might help to evaluate the balance of cellular immunity of MDSCs in MDS.     

Isolation, growth and identification of colony-forming cells with erythroid, myeloid, dendritic cell and NK-cell potential from human fetal liver

The study of hematopoietic stem cells (HSCs) and the process by which they differentiate into committed progenitors has been hampered by the lack of in vitro clonal assays that can support erythroid, myeloid and lymphoid differentiation. We describe a method for the isolation from human fetal liver of highly purified candidate HSCs and progenitors based on the phenotypes CD38⁻CD34⁺⁺ and CD38⁺CD34⁺⁺, respectively. We also describe a method for the growth of colony-forming cells (CFCs) from these cell populations, under defined culture conditions, that supports the differentiation of erythroid, CD14/CD15⁺ myeloid, CD1a⁺ dendritic cell and CD56⁺ NK cell lineages. Flow cytometric analyses of individual colonies demonstrate that CFCs with erythroid, myeloid and lymphoid potential are distributed among both the CD38⁻ and CD38⁺ populations of CD34⁺⁺ progenitors. Published: June 11, 2002.     

Systemic Monocytic-MDSCs Are Generated from Monocytes and Correlate with Disease Progression in Breast Cancer Patients

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-DR^low/-CD86^low/-CD80^low/-CD163^low/-) 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.     

A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues

Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b^- DC, and CD11b⁺ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.     

Circulating myeloid-derived suppressor cells: An independent prognostic factor in patients with breast cancer

Evading immune destruction is a hallmark of cancer. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid immune cells, are thought to foster the establishment of an immunosuppressive tumor microenvironment, but it remains unclear how. This study aims to determine the levels of circulating MDSCs and their subpopulations and test their immunosuppressive functions in patients with breast cancer (BC). We analyzed the fractions of MDSCs in freshly isolated peripheral blood mononuclear cells of patients with BC and healthy donors using flow cytometry. Circulating MDSCs were further phenotyped using fluorescently labeled antihuman monoclonal antibodies. Coculture experiments revealed the effects of MDSCs on CD3⁺ T cell response. Moreover, we correlated circulating MDSC levels with clinicopathological features of patients with BC. We show that the fraction of HLA-DR ^– CD33 ⁺ MDSCs in peripheral blood is about 10-fold higher in patients with BC than in healthy control individuals. The levels of all MDSC subpopulations, including monocytic and granulocytic MDSCs, are significantly elevated. Coculture experiments of purified HLA-DR ^– CD33 ⁺ MDSCs and CD3 ⁺ T cells demonstrate that T cell proliferation is more effectively inhibited by BC patient-derived MDSCs than by healthy control MDSCs. Moreover, increased circulating MDSC levels robustly associate with advanced BC stage and positive lymph node status. By being more abundant and more effective T cell suppressors, BC patient-derived circulating MDSCs exert a dual immunosuppressive effect. Our findings pave the way to develop novel diagnostic and immunotherapeutic strategies, aimed at detecting and inhibiting MDSCs in patients with BC.     

CD8+ T cells exhaustion induced by myeloid‐derived suppressor cells in myelodysplastic syndromes patients might be through TIM3/Gal‐9 pathway

CD8⁺ T cells play a central role in antitumour immunity, which often exhibit ‘exhaustion’ in the setting of malignancy and chronic viral infection due to T cell immunoglobulin and mucin domain 3 (TIM3) and myeloid‐derived suppressor cells (MDSCs). Our team previously found that overactive MDSCs and exhausted TIM3⁺CD8⁺ T cells were observed in myelodysplastic syndromes (MDS) patients. However, it is not obvious whether MDSCs suppress CD8⁺ T cells through TIM3/Gal‐9 pathway. Here, Gal‐9, as the ligand of TIM3, was overexpressed in MDSCs. The levels of Gal‐9 in bone marrow supernatants, serum and culture supernatants of MDSCs from MDS patients were elevated. CD8⁺ T cells from MDS or normal controls produced less perforin and granzyme B and exhibited increased early apoptosis after co‐culture with MDSCs from MDS. Meanwhile, the cytokines produced by CD8⁺ T cells could be partially restored by TIM3/Gal‐9 pathway inhibitors. Furthermore, CD8⁺ T cells produced less perforin and granzyme B after co‐culture with excess exogenous Gal‐9, and the function of CD8⁺ T cells was similarly restored by TIM3/Gal‐9 pathway inhibitors. Expression of Notch1, EOMES (associated with perforin and granzyme B secretion), p‐mTOR and p‐AKT (associated with cell proliferation) was decreased in CD8⁺ T cells from MDS after co‐culture with excess exogenous Gal‐9. These suggested that MDSCs might be the donor of Gal‐9, and TIM3/Gal‐9 pathway might be involved in CD8⁺ T cells exhaustion in MDS, and that TIM3/Gal‐9 pathway inhibitor might be the promising candidate for target therapy of MDS in the future.     

CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56^bright, NK cells. Most NK cells in healthy human donors are CD45RA⁺CD45RO^-. The CD45RA^-RO⁺ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RA^dimRO^- (CD45RA^dim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RA^dim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RA^dim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.     

Age-related changes in the distribution and frequency of myeloid and T cell populations in the small intestine of calves

Mucosal dendritic cells (DCs) play a key role in discriminating between dietary antigens, commensal microflora and pathogens but little is known regarding age-related changes in mucosal DC populations. We analyzed lymphoid and myeloid populations within the epithelium and lamina propria (LP) of the ileum and jejunum of weaned calves (6 months old) and compared their frequency and distribution with newborn calves (3–5 weeks old). CD4, CD8 and γδ TcR T cells and CD11c^HiMHC Class II⁺ myeloid cell frequency were significantly different when comparing ileum and jejunum of weaned calves. In particular, the number of CD8 and γδ TcR T cells, and CD11c^HiCD14⁺ macrophages was significantly greater in the ileum but CD11c⁺ and CD11b⁺ myeloid cell distribution was similar throughout the mucosal epithelium of the small intestine. Furthermore, significant age-related changes were apparent when comparing the frequency and abundance of mucosal leukocyte subpopulations in newborn and weaned calves. Total mucosal leukocytes (CD45⁺) increased significantly with age in both ileum and jejunum and much of this increase was attributed to mucosal T cells (CD3⁺). In particular, CD4 T cells and NK cells increased significantly in the jejunum and CD8, and γδ TcR T cells increased significantly with age throughout the small intestine. In contrast, CD11c^HiMHC Class II⁺ myeloid cells remained numerically unchanged with age but DCs (CD13⁺, CD26⁺, CD205⁺) were enriched and macrophages (CD14⁺, CD172a⁺) were depleted in older animals. Therefore, regional differences between ileal and jejunal mucosal leukocytes changed with age and there was also a marked age-dependent change in the composition of mucosal myeloid cells. These observations have significant implications for host responses to both pathogens and commensal microflora.     

Myeloid-specific expression of human lysosomal acid lipase corrects malformation and malfunction of myeloid-derived suppressor cells in lal-/- mice

Lysosomal acid lipase (LAL) cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. LAL deficiency causes expansion of CD11b(+)Gr-1(+) immature myeloid cells, loss of T cells, and impairment of T cell function. To test how myeloid cell LAL controls myelopoiesis and lymphopoiesis, a myeloid-specific doxycycline-inducible transgenic system was used to reintroduce human lysosomal acid lipase (hLAL) expression into LAL gene knockout (lal(-/-)) mice. Expression of hLAL in myeloid cells of lal(-/-) mice reversed abnormal myelopoiesis in the bone marrow starting at the granulocyte-monocyte progenitor stage and reduced systemic expansion of myeloid-derived suppressor cells (MDSCs). Myeloid hLAL expression inhibited reactive oxygen species production and arginase expression in CD11b(+)Gr-1(+) cells of lal(-/-) mice. Structural organization of the thymus and spleen was partially restored in association with reduced infiltration of CD11b(+)Gr-1(+) cells in these mice. In the thymus, reconstitution of myeloid cell LAL restored development of thymocytes at the double-negative DN3 stage. Myeloid cell LAL expression improved the proliferation and function of peripheral T cells. In vitro coculture experiments showed that myeloid hLAL expression in lal(-/-) mice reversed CD11b(+)Gr-1(+) myeloid cell suppression of CD4(+) T cell proliferation, T cell signaling activation, and lymphokine secretion. Blocking stat3 and NF-κB p65 signaling by small-molecule inhibitors in MDSCs achieved a similar effect. Injection of anti-Gr-1 Ab into lal(-/-) mice to deplete MDSCs restored T cell proliferation. These studies demonstrate that LAL in myeloid cells plays a critical role in maintaining normal hematopoietic cell development and balancing immunosuppression and inflammation.     

Characterization of Liver Monocytic Myeloid-Derived Suppressor Cells and Their Role in a Murine Model of Non-Alcoholic Fatty Liver Disease

Myeloid-derived suppressor cells (MDSCs) are potent suppressors of T cell immunity in tumors and inflammatory diseases. They are identified by surface expression of CD11b⁺Gr1⁺ in mice, and CD11b⁺Gr1⁺ cells accumulate in the livers of obese mice. However, many myeloid cells share these CD11b⁺Gr1⁺ markers. Accordingly, the aim of this study was to identify the authentic phenotype of MDSCs and investigate their functions in non-alcoholic fatty liver disease (NAFLD). C57BL/6J mice were divided into 2 diet groups: a normal control group and high-fat group to induce NAFLD. We demonstrated that monocytic CD11b⁺Gr1^dim cells could be further divided into 2 populations based on side scatter (SSC) during flow cytometry. We found that SSC^lowCD11b⁺Gr1^dim cells accumulated in the livers of NAFLD mice over time, and that these cells were recruited by the chemokine CCL2 and its receptor CCR2 and might expand in the liver via macrophage colony-stimulating factor stimulation. Furthermore, SSC^lowCD11b⁺Gr1^dim cells had a strong suppressive ability on T cells; this effect was not observed for SSC^highCD11b⁺Gr1^dim cells, and was dependent on nitric oxide production by inducible nitric oxide synthase. Our findings demonstrate that SSC^lowCD11b⁺Gr1^dim cells represent authentic MDSCs in NAFLD livers, and might serve an important negative feedback function in liver inflammation.