Development Of Myeloid-derived Suppressor Cells Research Articles

LncRNA HOTAIRM1 promotes MDSC expansion and suppressive functions through the HOXA1-miR124 axis during HCV infection

HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1) is a long non-coding RNA (lncRNA) that plays a pivotal role in regulating myeloid cell development via targeting HOXA1 gene expression. We and others have previously shown that myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature myeloid cells, expand during chronic viral (HCV, HIV) infections. However, the role of HOTAIRM1 in the development and suppression of MDSCs during viral infection remains unknown. In this study, we demonstrate that the expressions of HOTAIRM1 and its target HOXA1 are substantially upregulated to promote the expressions of immunosuppressive molecules, including arginase 1, inducible nitric oxide synthase, signal transducer and activator of transcription 3, and reactive oxygen species, in CD33+ myeloid cells derived from hepatitis C virus (HCV)-infected patients. We show that HCV-associated exosomes (HCV-Exo) can modulate HOTAIRM1, HOXA1, and miR124 expressions to regulate MDSC development. Importantly, overexpression of HOTAIRM1 or HOXA1 in healthy CD33+ myeloid cells promoted the MDSC differentiation and suppressive functions; conversely, silencing of HOTAIRM1 or HOXA1 expression in MDSCs from HCV patients significantly reduced the MDSC frequency and their suppressive functions. In essence, these results indicate that the HOTAIRM1-HOXA1-miR124 axis enhances the differentiation and suppressive functions of MDSCs and may be a potential target for immunomodulation in conjunction with antiviral therapy during chronic viral infection.

Long noncoding RNA HOTAIRM1 promotes myeloid-derived suppressor cell expansion and suppressive functions through up-regulating HOXA1 expression during latent HIV infection.

Myeloid-derived suppressor cells (MDSCs) contribute to HIV progression by impairing antiviral immunity; however, the mechanisms responsible for MDSC development during HIV infection are incompletely understood. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) is a long noncoding RNA (lncRNA) that plays a pivotal role in regulating myeloid cell development via targeting HOXA1. The role of HOTAIRM1--HOXA1 in the differentiation and functions of MDSCs during HIV infection remains unclear. In this study, we measured MDSC induction and suppressive functions by flow cytometry, RT-PCR, and co-culture experiments using CD33 myeloid cells derived from people living with HIV (PLHIV) on antiretroviral therapy (ART). We also manipulated the HOTAIRM1--HOXA1 axis in myeloid cells using knockdown and overexpression approaches. We demonstrate that HOTAIRM1 and HOXA1 expressions are reciprocally upregulated and are responsible for increased levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS), in CD33 myeloid cells derived from PLHIV on ART. We found that overexpression of HOTAIRM1 or HOXA1 in CD33 cells isolated from healthy individuals promoted the differentiation and suppressive functions of MDSCs, whereas silencing of HOTAIRM1 or HOXA1 expression in MDSCs derived from PLHIV significantly inhibited the frequency of MDSCs and expressions of the immunosuppressive molecules and reduced their immunosuppressive effects on T cells. These results indicate that the HOTAIRM1--HOXA1 axis enhances differentiation and suppressive functions of MDSCs and could be a potential therapeutic target for immunomodulation during latent HIV infection.

Connections between Metabolism and Epigenetic Modification in MDSCs.

Myeloid-derived suppressor cells (MDSCs) are major immunosuppressive cells in the tumor microenvironment (TME). During the differentiation and development of MDSCs from myeloid progenitor cells, their functions are also affected by a series of regulatory factors in the TME, such as metabolic reprogramming, epigenetic modification, and cell signaling pathways. Additionally, there is a crosstalk between these regulatory factors. This review mainly introduces the metabolism (especially glucose metabolism) and significant epigenetic modification of MDSCs in the TME, and briefly introduces the connections between metabolism and epigenetic modification in MDSCs, in order to determine the further impact on the immunosuppressive effect of MDSCs, so as to serve as a more effective target for tumor therapy.

MicroRNAs and lncRNAs-A New Layer of Myeloid-Derived Suppressor Cells Regulation.

Myeloid-derived suppressor cells (MDSCs) constitute an important component in regulating immune responses in several abnormal physiological conditions such as cancer. Recently, novel regulatory tumor MDSC biology modulating mechanisms, including differentiation, expansion and function, were defined. There is growing evidence that miRNAs and long non-coding RNAs (lncRNA) are involved in modulating transcriptional factors to become complex regulatory networks that regulate the MDSCs in the tumor microenvironment. It is possible that aberrant expression of miRNAs and lncRNA contributes to MDSC biological characteristics under pathophysiological conditions. This review provides an overview on miRNAs and lncRNAs epiregulation of MDSCs development and immunosuppressive functions in cancer.

Sympathetic nervous tone limits the development of myeloid-derived suppressor cells.

Sympathetic nerves that innervate lymphoid organs regulate immune development and function by releasing norepinephrine that is sensed by immune cells via their expression of adrenergic receptors. Here, we demonstrate that ablation of sympathetic nervous system (SNS) signaling suppresses tumor immunity, and we dissect the mechanism of such immune suppression. We report that disruption of the SNS in mice removes a critical α-adrenergic signal required for maturation of myeloid cells in normal and tumor-bearing mice. In tumor-bearing mice, disruption of the α-adrenergic signal leads to the accumulation of immature myeloid-derived suppressor cells (MDSCs) that suppress tumor immunity and promote tumor growth. Furthermore, we show that these SNS-responsive MDSCs drive expansion of regulatory T cells via secretion of the alarmin heterodimer S100A8/A9, thereby compounding their immunosuppressive activity. Our results describe a regulatory framework in which sympathetic tone controls the development of innate and adaptive immune cells and influences their activity in health and disease.

Umbilical cord blood-derived mesenchymal stromal cells promote myeloid-derived suppressor cell proliferation by secreting HLA-G to reduce acute graft-versus-host disease after hematopoietic stem cell transplantation

Background aimsMesenchymal stem cells (MSCs) use multiple mechanisms to constrain both innate and adaptive immune responses to prevent graft-versus-host disease (GVHD). Myeloid-derived suppressor cells (MDSCs), as a heterogeneous population of early myeloid progenitor cells originating from bone marrow, are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses, indicating their potential for GVHD therapy. There is accumulating evidence that MSCs and MDSCs do not act independently, but rather establish crosstalk. However, the role of MSCs in MDSC expansion and activation in GVHD remains unexplored. MethodsIn vitro experiments included 2 groups: peripheral blood mononuclear cells (PBMCs) after mobilization and human umbilical cord blood-derived MSCs (UCB-MSCs) co-cultured with PBMCs. The number and functional difference of MDSCs in PBMCs were determined by flow cytometry. The culture supernatants of co-cultured cells were analyzed to identify cytokines involved in MDSC proliferation. The relationship between MSCs and MDSCs was clarified in GVHD and graft-versus-leukemia (GVL) animal models. ResultsIn vitro experiments confirmed that UCB-MSCs secreted HLA-G protein to promote and maintain the proliferation of MDSCs in peripheral blood after granulocyte colony-stimulating factor mobilization, and UCB-MSCs mediated the function of MDSCs to inhibit the proliferation of T cells and promote the proliferation of regulatory T cells. UCB-MSCs overexpressing HLA-G induced MDSC production in recipient mice, improved the ability of MDSCs to suppress T cells and further reduced acute GVHD (aGVHD) symptoms and survival time without influencing GVL effects. ConclusionsUCB-MSCs expanded MDSCs via HLA-G/Ig-like transcript 4, reducing the severity of aGVHD without affecting GVL. The immunosuppressive potential of MSCs for the treatment of aGVHD significantly affects the development of MDSCs, thereby consolidating the position of MSCs in the prevention and treatment of aGVHD.

A novel methodology of the myeloid-derived suppressor cells (MDSCs) generation with splenic stroma feeder cells

Myeloid-derived suppressor cells (MDSCs) are a significant obstacle for immunotherapy of cancer. It is of great clinical relevance to study the mechanism of MDSCs accumulation in mouse spleens and establish a stable method to obtain high-purity MDSCs in vitro for further research. Here, we established a new method for amplifying a large number of highly pure MDSCs in vitro. To mimic the microenvironment of MDSCs development in vivo, mouse splenic stroma feeder cells and serum-free medium containing granulocyte-macrophage colony stimulating factor (GM-CSF) were used to induce myeloid precursors in mouse bone marrow cells, which differentiate into MDSCs. Development and immunological functions of the cells were monitored both in vivo and in vitro. A total of 4 × 108 MDSCs could be obtained from the bone marrow from one mouse, the ratio of CD11b+Gr-1+ MDSCs could reach 93.8% ± 3.3% after nine days of culture in vitro. Cultured MDSCs maintained a similar immunophenotype with MDSCs found in tumor-bearing mice. Colony forming assay in vitro and in vivo demonstrated that these were myeloid precursor cells. These cells generated high levels of reactive oxygen species and arginase 1 to prevent proliferation of CD8+ T cells in vitro. These also increased regulatory T (Treg) cells in blood while promoting the growth of lymphoma in vivo. In addition, cultured MDSCs effectively inhibited acute graft-versus-host disease (aGVHD). Our findings suggest that mouse splenic stroma plays an important role in the generation of MDSCs and represent a preliminary mechanism for the accumulation of MDSCs in spleens, and thereby lay the foundation for basic research and the clinical application of MDSCs.

Systemic but not MDSC-specific IRF4 deficiency promotes an immunosuppressed tumor microenvironment in a murine pancreatic cancer model

Pancreatic ductal adenocarcinoma is characterized by a strong immunosuppressive network with a dense infiltration of myeloid cells including myeloid-derived suppressor cells (MDSC). Two distinct populations of MDSC have been defined: polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC). Several factors influence the development and function of MDSC including the transcription factor interferon regulatory factor 4 (IRF4). Here, we show that IRF4 deficiency accelerates tumor growth and reduces survival, accompanied with a dense tumor infiltration with PMN-MDSC and reduced numbers of CD8+ T cells. As IRF4 has been described to modulate myeloid cell development and function, particularly of PMN-MDSC, we analyzed its role using MDSC-specific IRF4 knockout mice with the Ly6G or LysM knock-in allele expressing Cre recombinase and Irf4flox. In GM-CSF-driven bone marrow cultures, IRF4 deficiency increased the frequency of MDSC-like cells with a strong T cell suppressive capacity. Myeloid (LysM)-specific depletion of IRF4 led to increased tumor weight and a moderate splenic M-MDSC expansion in tumor-bearing mice. PMN cell (Ly6G)-specific depletion of IRF4, however, did not influence tumor progression or MDSC accumulation in vivo in accordance with our finding that IRF4 is not expressed in PMN-MDSC. This study demonstrates a critical role of IRF4 in the generation of an immunosuppressive tumor microenvironment in pancreatic cancer, which is independent of IRF4 expression in PMN-MDSC.

Carboplatin chemoresistance is associated with CD11b+/Ly6C+ myeloid release and upregulation of TIGIT and LAG3/CD160 exhausted T cells

Immunosuppressive chemoresistance is a major barrier in lung cancer treatment. Tumor immunosuppressive environments mediated by myeloid-derived suppressor cells (MDSCs) play a key role in chemotherapy induced MDSC development and differentiation but their mechanistic role has not been elucidated. Here, we define a role for carboplatin based chemotherapy in potentiating an MDSC-dependent pathway that triggers the chemoresistance mechanism. Findings reveal MDSC differentiation and activation of IL-13/IL-33-mediated pathway through VCAM/RANTES following carboplatin treatment. Furthemore, secretion of T regulatory IL-10-producing CD4+Foxp3+ cells was increased followed by expression of co-inhibitory receptor TIGIT on T cells, leading to a dysfunctional T cell phenotype. These cells were characterized by an immunosuppressive phenotype with impaired activation, proliferation and cytokine production. Lung cancer tissues expressed CD155, which bound TIGIT receptors and inactivated CD8 T cells. This TIGIT expression on tumor-infiltrating T cells was found to be associated with tumor progression and was linked to functional exhaustion of T cells. In addition, the presence of plasmacytoid dendritic cells (pDCs) exposed to tumor-derived factors further enhanced tumor progression through IL-10 production and up-regulation of the inducible co-stimulatory ligand (ICOS-L). Deciphering these deranged immune mechanisms and how they are impacted by chemotherapy induction is essential for incorporation of novel immune-based strategies in order to restore immunity and inhibit the immunosuppressive phenotype of metastatic lung cancer.

Abstract A18: Angiotensin receptor blockers normalize the pancreatic ductal adenocarcinoma stroma by reprogramming carcinoma-associated fibroblasts

Abstract The resistance to therapy in pancreatic ductal adenocarcinoma (PDAC) is due in part to carcinoma-associated fibroblasts (CAFs) that produce a highly desmoplastic and proinflammatory tumor microenvironment (TME). Our laboratory has shown that the angiotensin receptor blocker (ARB) losartan reduces the CAF-density and desmoplasia, and improves vascular perfusion, and the uptake and efficacy of drugs in animal models of PDAC (Diop-Frimpong et al., PNAS 2011; Chauhan et al., Nature Comm 2013). These preclinical findings led to a phase II trial at Massachusetts General Hospital in locally advanced PDAC patients, which showed that losartan combined with neoadjuvant-FOLFIRINOX chemotherapy followed by chemoradiation more than doubled the resection rate (Murphy et al., JAMA Oncology 2019). Moreover, in a retrospective analysis, we found that the chronic use of angiotensin system inhibitors was independently associated with longer overall survival in nonmetastatic PDAC patients (Liu et al., Clin Cancer Res 2017). However, how ARBs affect CAFs and desmoplasia in human PDAC remains unknown. Here we performed immunohistochemistry in formalin-fixed, paraffin-embedded sections to examine the effect of chronic use of ARB on CAF phenotypes and the extracellular matrix (ECM) in resected PDAC samples from untreated patients and patients treated with ARBs to control their hypertension. We found that the chronic use of ARBs did not reduce the density of CAFs expressing the fibroblast activation protein (FAP), α-smooth muscle actin (α-SMA), or platelet-derived growth factor receptor-beta alone. However, ARBs increased the fraction of CAFs expressing both the angiotensin receptor 1 (AT1) and α-SMA and decreased the fraction of CAFs expressing both AT1 and FAP. ARBs also reduced the intratumoral levels of hyaluronan, suggesting that ARBs reduce desmoplasia in human PDAC. To reveal the underlying mechanisms, we performed a transcriptomic analysis of the bulk tumor as well as α-SMA+ cells isolated from mice bearing orthotopic PDAC tumors and treated with losartan. Gene expression profiling revealed that in both the tumor bulk and α-SMA+ cells, losartan “normalized” the ECM/cytoskeleton interaction, glycosaminoglycan metabolism and platelet-derived growth factor gene sets, and significantly reduced gene transcripts ofS100A8 and S100A9 . Gene products of S100A8 and S100A9 are S100 proteins that stimulate the development of myeloid-derived suppressor cells. In α-SMA+ cells—but not in tumor bulk—losartan reduced cell cycle and muscle contraction gene sets. These results suggest that ARBs can reprogram α-SMA+ cells and normalize the TME in PDAC. Citation Format: Hao Liu, William W. Ho, Kamila Naxerova, Jelena Grahovac, Hadi Nia, Ivy Chen, Jessica M. Posada, Daniel H. Schanne, Matthias Pinter, Jonathan Crain, Xialong Qi, Jeffrey W. Clark, Theodore S. Hong, David P. Ryan, Peigen Huang, Keith D. Lillemoe, Carlos Fernández-Del Castillo, Cristina R Ferrone, Michael Downes, Ronald M. Evans, Vikram Deshpande, Yves Boucher, Rakesh K. Jain. Angiotensin receptor blockers normalize the pancreatic ductal adenocarcinoma stroma by reprogramming carcinoma-associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A18.

IMMU-36. THE ROLE OF PD-L1 IN GLIOBLASTOMA-DERIVED EXTRACELLULAR VESICLES IN THE INDUCTION OF IMMUNOSUPPRESSIVE MONOCYTES

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor, and novel therapeutic strategies that make a substantial impact on outcomes are sorely needed. Immunotherapies have shown great promise in the treatment of a number of cancers in recent year, and a concerted effort is being made to apply these treatment paradigms to GBM. However, many GBM patients exhibit profound immunosuppression, likely limiting the efficacy of such approaches. The mechanisms of this immunosuppression are poorly understood, but tumor-derived extracellular vesicles (EVs), may play a role. We demonstrate that GBM-derived EVs induce the development of myeloid-derived suppressor cells (MDSCs) and non-classical monocytes (NCMs). We further demonstrate that these EV-induced monocytic cells are immunosuppressive, resulting in impaired T cell proliferation upon co-culture. We found that the immunosuppressive effects of tumor-derived EVs appear to be driven by the induction of these immunosuppressive monocytes, as EV treatment of T cells did not significantly impact T cell proliferation. Further, we sought to characterize the important of programmed death ligand 1 (PD-L1) in the induction of these immunosuppressive monocyte populations. We found PD-L1 to be expressed in the EVs from GBM cell lines, and that modulation in PD-L1 expression via either constitutive overexpression or shRNA-mediated knockdown resulted in concordant changes in expression in tumor-derived EVs. We demonstrate that PD-L1 is important for the induction of NCM but not for MDSCs. Taken together, these findings point to a significant role for tumor-derived EVs in the induction of immunosuppressive monocytes in GBM, and that these cells may be a driving force of systemic immunosuppression. PD-L1 is one factor expressed in EVs that has immunomodulatory properties, but additional EV cargo likely plays a major role in the induction of immunosuppressive cells.

Hepatic Stellate Cells Enhance Liver Cancer Progression by Inducing Myeloid-Derived Suppressor Cells through Interleukin-6 Signaling.

The tumor microenvironment, which consists of fibroblasts, smooth muscle cells, endothelial cells, immune cells, epithelial cells, and extracellular matrices, plays a crucial role in tumor progression. Hepatic stellate cells (HSCs), a class of unique liver stromal cells, participate in immunomodulatory activities by inducing the apoptosis of effector T-cells, generation of regulatory T-cells, and development of myeloid-derived suppressor cells (MDSCs) to achieve long-term survival of islet allografts. This study provides in vitro and in vivo evidences that HSCs induce the generation of MDSCs to promote hepatocellular carcinoma (HCC) progression through interleukin (IL)-6 secretion. HSC-induced MDSCs highly expressed inducible nitric oxide synthase (iNOS) and arginase 1 mRNA and presented potent inhibitory T-cell immune responses in the tumor environment. Wild-type HSC-induced MDSCs expressed lower levels of CD40, CD86, and MHC II, and a higher level of B7-H1 surface molecules, as well as increased the production of iNOS and arginase I compared with MDSCs induced by IL-6-deficient HSCs in vitro. A murine-transplanted model of the liver tumor showed that HCCs cotransplanted with HSCs could significantly enhance the tumor area and detect more MDSCs compared with HCCs alone or HCCs cotransplanted with HSCs lacking IL-6. In conclusion, the results indicated that MDSCs are induced mainly by HSCs through IL-6 signaling and produce inhibitory enzymes to reduce T-cell immunity and then promote HCC progression within the tumor microenvironment. Therapies targeting the pathway involved in MDSC production or its immune-modulating pathways can serve as an alternative immunotherapy for HCC.

SUMO-Specific Protease 1 Is Critical for Myeloid-Derived Suppressor Cell Development and Function.

Myeloid-derived suppressor cells (MDSC) can suppress immunity and promote tumorigenesis, and their abundance is associated with poor prognosis. In this study, we show that SUMO1/sentrin-specific peptidase 1 (SENP1) regulates the development and function of MDSC. SENP1 deficiency in myeloid cells promoted MDSC expansion in bone marrow, spleen, and other organs. Senp1-/- MDSC showed stronger immunosuppressive activity than Senp1+/+ MDSC; we observed no defects in the differentiation of myeloid precursor cell in Senp1-/- mice. Mechanistically, SENP1-mediated regulation of MDSC was dependent on STAT3 signaling. We identified CD45 as a specific STAT3 phosphatase in MDSC. CD45 was SUMOylated in MDSC and SENP1 could deconjugate SUMOylated CD45. In Senp1-/- MDSC, CD45 was highly SUMOylated, which reduced its phosphatase activity toward STAT3, leading to STAT3-mediated MDSC development and function. These results reveal a suppressive function of SENP1 in modulating MDSC expansion and function via CD45-STAT3 signaling axis. SIGNIFICANCE: These findings show that increased SUMOylation of CD45 via loss of SENP1 suppresses CD45-mediated dephosphorylation of STAT3, which promotes MDSC development and function, leading to tumorigenesis.

Interleukin‑12 exacerbates Sj�gren's syndrome through induction of myeloid‑derived suppressor cells

Interleukin (IL)-12 modulates the generation and function of various immune cells and plays a vital role in the pathogenesis of Sjögren's syndrome (SS). Myeloid-derived suppressor cells (MDSCs) are involved in autoimmune diseases by regulating various immune responses. However, it has not been confirmed whether inflammatory IL-12 participates in the progression of SS via regulating MSDCs. In the present study, the plasma levels of IL-12 were detected by ELISA in SS-like non-obese diabetic (NOD) mice. The mice were treated by intraperitoneal injection of IL-12 and anti-IL-12 antibody, respectively, and then the salivary flow rate was detected. The pathology of submandibular glands was evaluated in tissue sections stained with hematoxylin and eosin. The proportion of MDSCs was assessed by flow cytometry. The results showed that plasma IL-12 was significantly increased in the SS-like NOD mice comparing with that noted in the control mice. The exogenous IL-12 exacerbated SS-like symptoms of NOD mice and promoted the generation of both bone marrow (BM) and splenic MDSCs in the SS-like NOD mice. Of note, anti-IL-12 alleviated SS-like symptoms of NOD mice and inhibited the generation of BM and splenic MDSCs. Moreover, the generation of MDSCs was crippled in the IL-12-deficient C57BL/6 (Il-12−/− B6) mice. Our findings suggest that aggravation of SS-like symptoms by IL-12 in NOD mice may be attributed to its promotion of MDSC development.

The Pseudogene Olfr29-ps1 Promotes the Suppressive Function and Differentiation of Monocytic MDSCs.

Long noncoding RNA (lncRNA) plays a critical role in many biological processes, such as cell differentiation and development. However, few studies about lncRNAs regulating the differentiation and development of myeloid-derived suppressor cells (MDSCs) exist. In this study, we identified a lncRNA pseudogene, Olfr29-ps1, which was expressed in MDSCs and upregulated by the proinflammatory cytokine IL6. The Olfr29-ps1 in vertebrates is conserved, and the similarity between the Olfr29-ps1 and human OR1F2P sequence is 43%. This lncRNA promoted the immunosuppressive function and differentiation of monocytic (Mo-)MDSCs in vitro and in vivo It directly sponged miR-214-3p to downregulate miR-214-3p, which may target MyD88 to modulate the differentiation and development of MDSCs. The functions of Olfr29-ps1 were dependent on IL6-mediated N 6-methyladenosine (m6A) modification, which not only enhanced Olfr29-ps1, but also promoted the interaction of Olfr29-ps1 with miR-214-3p Thus, our results demonstrated that the pseudogene Olfr29-ps1 may regulate the differentiation and function of MDSCs through a m6A-modified Olfr29-ps1/miR-214-3p/MyD88 regulatory network, revealing a mechanism for the regulation of myeloid cells and also providing potential targets for antitumor immunotherapy.

TRAF3 is a critical regulator of the expansion of myeloid-derived suppressor cells in mice

Abstract TRAF3 is an adaptor protein and E3 ligase that regulates the signal transduction pathways of the TNFR superfamily, pattern recognition receptors, and cytokine receptors. We previously showed that specific deletion of TRAF3 in myeloid cells leads to spontaneous inflammation and tumor development in aging M-TRAF3−/− mice, which is consistently associated with a striking expansion of CD11b+Gr1+ myeloid cells. Here we report that these CD11b+Gr1+ cells were myeloid-derived suppressor cells (MDSCs), as they exhibited a strong suppressive activity on CD8 T cell proliferation and also displayed a nuclear morphology of immature myeloid cells. Using a chronic inflammation model induced by repeated injections of heat-killed Bacillus Calmette-Guérin (BCG), we showed that young adult M-TRAF3−/− mice also exhibited hyperexpansion of MDSCs after BCG injections. Our in vivo BrdU labeling experiments revealed that MDSC hyperexpansion in M-TRAF3−/− mice was mainly due to the increased proliferation of MDSCs or progenitor cells in the spleen but not in the bone marrow (BM). Interestingly, TRAF3 does not appear to be an intrinsic regulator of the development, survival or proliferation of MDSCs, as suggested by the results of cultured BM progenitor cells and purified splenic MDSCs, in the absence or presence of CSFs or agonists of TLRs or CD40. Furthermore, we identified environmental factors that induced the aberrant expansion of MDSCs in M-TRAF3−/− mice using cytokine protein array analyses, which were further verified by macrophage cultures and in vivo neutralization studies. Taken together, our findings identify TRAF3 as a new regulator of MDSC expansion, and therefore also a critical regulator of chronic inflammation and immunosuppression.

Radiotherapy Both Promotes and Inhibits Myeloid-Derived Suppressor Cell Function: Novel Strategies for Preventing the Tumor-Protective Effects of Radiotherapy.

Cancer immunotherapies aimed at neutralizing the programmed death-1 (PD-1) immune suppressive pathway have yielded significant therapeutic efficacy in a subset of cancer patients. However, only a subset of patients responds to antibody therapy with either anti-PD-1 or anti-PD-L1 antibodies. These patients appear to have so-called “hot” tumors containing tumor-reactive T cells. Therefore, checkpoint blockade therapy may be effective in a larger percentage of cancer patients if combined with therapeutics that also activate tumor-reactive T cells. Radiotherapy (RT) is a prime candidate for combination therapy because it facilitates activation of both local antitumor immunity and antitumor immunity at non-radiated, distant sites (abscopal response). However, RT also promotes tumor cell expression of PD-L1 and facilitates the development of myeloid-derived suppressor cells (MDSC), a population of immune suppressive cells that also suppress through PD-L1. This article will review how RT induces MDSC, and then describe two novel therapeutics that are designed to simultaneously activate tumor-reactive T cells and neutralize PD-1-mediated immune suppression. One therapeutic, a CD3xPD-L1 bispecific T cell engager (BiTE), activates and targets cytotoxic T and NKT cells to kill PD-L1+ tumor cells, despite the presence of MDSC. The BiTE significantly extends the survival time of humanized NSG mice reconstituted with human PBMC and carrying established metastatic human melanoma tumors. The second therapeutic is a soluble form of the costimulatory molecule CD80 (sCD80). In addition to costimulating through CD28, sCD80 inhibits PD-1 suppression by binding to PD-L1 and sterically blocking PD-L1/PD-1 signaling. sCD80 increases tumor-infiltrating T cells and significantly extends survival time of mice carrying established, syngeneic tumors. sCD80 does not suppress T cell function via CTLA-4. These studies suggest that the CD3xPD-L1 BiTE and sCD80 may be efficacious therapeutics either as monotherapies or in combination with other therapies such as radiation therapy for the treatment of cancer.

Tolerogenic Role of Myeloid Suppressor Cells in Organ Transplantation

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells of myeloid origin with a specific immune inhibitory function that negatively regulates the adaptive immune response. Since MDSC participate in the promotion of tolerance in the context of organ transplantation, therapeutic strategies that regulate the induction and development of MDSC have been the center of scientist attention. Here we review literature regarding induction of MDSC with demonstrated suppressive function among different types of allografts and their mechanism of action. While manipulation of MDSC represents a potential therapeutic approach for the promotion of donor specific tolerance in solid organ transplantation, further characterization of their specific phenotype, which distinguishes MDSC from non-suppressive myeloid cells, and detailed evaluation of the inhibitory mechanism that determines their suppressive function, is necessary for the realistic application of MDSC as biomarkers in health and disease and their potential use as immune cell therapy in organ transplantation.

Development of myeloid-derived suppressor cells (MDSC) targeted therapy for the treatment of cisplatin-resistant bladder cancer.

415 Background: The emergence of immune checkpoint inhibitors (ICI) has brought hope for cure and survival for those suffering from various cancers, including bladder cancer. However, the response rate of ICI monotherapy is modest, and recent reports indicate that myeloid-derived suppressor cells (MDSC) might play a role in the resistant mechanism of ICI. In this study, we assess the effect of chemokine signal on the proliferation of bladder cancer and investigate whether MDSC could be a new target for the treatment of cisplatin-resistant bladder cancer. Methods: We established a cisplatin resistant strain (MB49R) of mice bladder cancer cell line MB49, and examined the alteration of the expression levels of inflammatory chemokines by chemokine array. Next, we isolated MDSCs from spleen and tumor in tumor-bearing mice to examine gene expression levels of chemokine receptors (CXCR2 and CCR2) and immunosuppression genes (Arg-1 and iNOS). Furthermore, we assessed the efficacy of CDDP, α-PD-L1 and chemokine antagonists against the proliferation of tumors in MB49 and MB49R xenograft models. Results: Expression levels of CCL2 and CXCL1/2, which are involved in the migration of MDSC, were significantly increased in the culture supernatant of MB49R compared to those in MB49 cell lines. This result was confirmed by real-time RT-qPCR of tumor extract, and this increase was also observed in human bladder cancer cell lines (T24 and T24R). CXCR2 and CCR2 were highly expressed in PMN-MDSC and M-MDSC, respectively, which were isolated from spleen or tumors in tumor-bearing mice, and gene expression levels of Arg-1 and iNOS were dramatically increased in M-MDSCs from the tumor tissues compared to those from spleen. Also, analysis by flow cytometry revealed that PMN-MDSC dramatically decreased in MB49R compared to parental MB49 tumors, while the proportion of M-MDSC was not changed in MB49R, which indicates that M-MDSC could be a target for the treatment of CDDP resistant bladder cancer. Conclusions: The results in the present study might indicate that the combination treatment with ICI and MDSC-targeting therapy could be an option for the treatment of cisplatin-resistant bladder cancer.

Myeloid-derived suppressor cells are bound and inhibited by anti-thymocyte globulin.

Myeloid-derived suppressor cells (MDSCs) inhibit T cell responses and are relevant to cancer, autoimmunity and transplant biology. Anti-thymocyte globulin (ATG) is a commonly used T cell depletion agent, yet the effect of ATG on MDSCs has not been investigated. MDSCs were generated in Lewis Lung Carcinoma 1 tumor-bearing mice. MDSC development and function were assessed in vivo and in vitro with and without ATG administration. T cell suppression assays, RT-PCR, flow cytometry and arginase activity assays were used to assess MDSC phenotype and function. MDSCs increased dramatically in tumor-bearing mice and the majority of splenic MDSCs were of the polymorphonuclear subset. MDSCs potently suppressed T cell proliferation. ATG-treated mice developed 50% fewer MDSCs and these MDSCs were significantly less suppressive of T cell proliferation. In vitro, ATG directly bound 99.6% of MDSCs. CCR7, L-selectin and LFA-1 were expressed by both T cells and MDSCs, and binding of LFA-1 was inhibited by ATG pre-treatment. Arg-1 and PD-L1 transcript expression were reduced 30–40% and arginase activity decreased in ATG-pretreated MDSCs. MDSCs were bound and functionally inhibited by ATG. T cells and MDSCs expressed common Ags which were also targets of ATG. ATG may be helpful in tumor models seeking to suppress MDSCs. Alternatively, ATG may inadvertently inhibit important T cell regulatory events in autoimmunity and transplantation.