Mitigating the prevalence and function of myeloid-derived suppressor cells by redirecting myeloid differentiation using a novel immune modulator.

Immune suppression is common in neoplasia and a major driver is tumor-induced myeloid dysfunction. Yet, overcoming such myeloid cell defects remains an untapped strategy to reverse suppression and improve host defense. Exposure of bone marrow progenitors to heightened levels of myeloid growth factors in cancer or following certain systemic treatments promote abnormal myelopoiesis characterized by the production of myeloid-derived suppressor cells (MDSCs) and a deficiency in antigen-presenting cell function. We previously showed that a novel immune modulator, termed 'very small size particle' (VSSP), attenuates MDSC function in tumor-bearing mice, which was accompanied by an increase in dendritic cells (DCs) suggesting that VSSP exhibits myeloid differentiating properties. Therefore, here, we addressed two unresolved aspects of the mechanism of action of this unique immunomodulatory agent: (1) does VSSP alter myelopoiesis in the bone marrow to redirect MDSC differentiation toward a monocyte/macrophage or DC fate? and (2) does VSSP mitigate the frequency and suppressive function of human tumor-induced MDSCs? To address the first question, we first used a murine model of granulocyte-colony stimulating factor-driven emergency myelopoiesis following chemotherapy-induced myeloablation, which skews myeloid output toward MDSCs, especially the polymorphonuclear (PMN)-MDSC subset. Following VSSP treatment, progenitors and their myeloid progeny were analyzed by immunophenotyping and MDSC function was evaluated by suppression assays. To strengthen rigor, we validated our findings in tumor-bearing mouse models. To address the second question, we conducted a clinical trial in patients with metastatic renal cell carcinoma, wherein 15 patients were treated with VSSP. Endpoints in this study included safety and impact on PMN-MDSC frequency and function. We demonstrated that VSSP diminished PMN-MDSCs by shunting granulocyte-monocyte progenitor differentiation toward monocytes/macrophages and DCs with heightened expression of the myeloid-dependent transcription factors interferon regulatory factor-8 and PU.1. This skewing was at the expense of expansion of granulocytic progenitors and rendered the remaining MDSCs less suppressive. Importantly, these effects were also demonstrated in a clinical setting wherein VSSP monotherapy significantly reduced circulating PMN-MDSCs, and their suppressive function. Altogether, these data revealed VSSP as a novel regulator of myeloid biology that mitigates MDSCs in cancer patients and reinstates a more normal myeloid phenotype that potentially favors immune activation over immune suppression.

Oliver L ，Alvarez R ，Diaz R ，Valdés A ，Colligan SH ，Nemeth MJ ，Twum DYF ，Fernández A ，Fernández-Medina O ，Carlson LM ，Yu H ，Eng KH ，Hensen ML ，Rábade-Chediak ML ，Fernández LE ，Lee KP ，Perez L ，Muhitch JB ，Mesa C ，Abrams SI ... -  《Journal for ImmunoTherapy of Cancer》

The Granulocyte Progenitor Stage Is a Key Target of IRF8-Mediated Regulation of Myeloid-Derived Suppressor Cell Production.

Alterations in myelopoiesis are common across various tumor types, resulting in immature populations termed myeloid-derived suppressor cells (MDSCs). MDSC burden correlates with poorer clinical outcomes, credited to their ability to suppress antitumor immunity. MDSCs consist of two major subsets, monocytic and polymorphonuclear (PMN). Intriguingly, the latter subset predominates in many patients and tumor models, although the mechanisms favoring PMN-MDSC responses remain poorly understood. Ordinarily, lineage-restricted transcription factors regulate myelopoiesis that collectively dictate cell fate. One integral player is IFN regulatory factor (IRF)-8, which promotes monocyte/dendritic cell differentiation while limiting granulocyte development. We recently showed that IRF8 inversely controls MDSC burden in tumor models, particularly the PMN-MDSC subset. However, where IRF8 acts in the pathway of myeloid differentiation to influence PMN-MDSC production has remained unknown. In this study, we showed that: 1) tumor growth was associated with a selective expansion of newly defined IRF8lo granulocyte progenitors (GPs); 2) tumor-derived GPs had an increased ability to form PMN-MDSCs; 3) tumor-derived GPs shared gene expression patterns with IRF8-/- GPs, suggesting that IRF8 loss underlies GP expansion; and 4) enforced IRF8 overexpression in vivo selectively constrained tumor-induced GP expansion. These findings support the hypothesis that PMN-MDSCs result from selective expansion of IRF8lo GPs, and that strategies targeting IRF8 expression may limit their load to improve immunotherapy efficacy.

Netherby CS ，Messmer MN ，Burkard-Mandel L ，Colligan S ，Miller A ，Cortes Gomez E ，Wang J ，Nemeth MJ ，Abrams SI ... -  《-》

Decidua-derived granulocyte macrophage colony-stimulating factor induces polymorphonuclear myeloid-derived suppressor cells from circulating CD15+ neutrophils.

Do decidua-derived factors stimulate the conversion of circulating neutrophils to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in early human pregnancy? Circulating neutrophils can acquire PMN-MDSC-like phenotypes and function via phosphorylated signal transducer and activator of transcription 5/programmed death ligand 2 (pSTAT5/PD-L2) signalling after stimulation with decidua-derived granulocyte macrophage colony-stimulating factor (GM-CSF). PMN-MDSCs are an important immunoregulatory cell type in early pregnancy. Neutrophils are of high heterogeneity and plasticity and can polarize to immunosuppressive PMN-MDSCs upon stimulation. For analysis of myeloid-derived suppressor cell (MDSC) subset proportions, 12 endometrium tissues and 12 peripheral blood samples were collected from non-pregnant women, and 40 decidua tissues and 16 peripheral blood samples were obtained from women with normal early pregnancy undergoing elective surgical pregnancy termination for nonmedical reasons with gestation age of 6-10 weeks. Twenty-nine decidua tissues were collected for isolation of CD15+ PMN-MDSCs. Twenty endometrium tissues and 30 decidua tissues were collected for cytokine analysis, immunohistochemistry or neutrophil stimulation. Peripheral blood samples were obtained from 36 healthy donors for isolation of CD3+ T cells and CD15+ neutrophils. The proportion of MDSC subsets in the decidua and peripheral blood of normal early pregnancy, endometrium and peripheral blood of non-pregnant women was analysed by flow cytometry. The phenotypes and function of decidual PMN-MDSCs and circulating neutrophils were compared by flow cytometry. Circulating neutrophils were stimulated with decidual explant supernatant (DES) and the phenotypes were measured by flow cytometry and immunofluorescence. The suppressive capacity of decidual PMN-MDSCs and DES-conditioned neutrophils was analysed by flow cytometry with or without anti-programmed cell death-1 (PD-1) antibody. Cytokines from DES and endometrial explant supernatant (EES) were detected by a Luminex assay. GM-CSF expression was determined by ELISA and immunohistochemistry. Neutrophils were stimulated with DES, EES, DES with anti-GM-CSF antibody or EES with GM-CSF. CD11b, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), PD-L2 and pSTAT5 expression were measured by flow cytometry. The frequency of PMN-MDSCs was significantly increased in the decidua of early pregnancy compared with peripheral blood of non-pregnant women, the endometrium of non-pregnant women or peripheral blood during early pregnancy. Decidual PMN-MDSCs suppressed T-cell proliferation and cytokine production. Phenotypes of decidual PMN-MDSCs were similar to mature activated neutrophils. DES-induced CD11b, LOX-1, PD-L2 expression and STAT5 phosphorylation in neutrophils. The PD-L2 expression in neutrophils was dependent on STAT5 phosphorylation. Both decidual PMN-MDSCs and DES-conditioned neutrophils suppressed T-cell proliferation via PD-1 signalling. GM-CSF was up-regulated in the decidua and induced CD11b, LOX-1 and PD-L2 expression on neutrophils. DES significantly induced CD11b, LOX-1, PD-L2 expression and STAT5 phosphorylation. Anti-GM-CSF antibody remarkably blocked such stimulation in neutrophils. EES did not induce CD11b, LOX-1, PD-L2 expression or STAT5 phosphorylation, while GM-CSF treatment sufficiently stimulated CD11b, LOX-1, PD-L2 expression and STAT5 phosphorylation in neutrophils. N/A. The study was based on in vitro experiments and we were not able to evaluate neutrophils differentiation to PMN-MDSCs in other sites before entering the maternal-foetal interface due to the limited availability of human samples. This needs to be explored using murine models. This is the first study demonstrating that decidual PMN-MDSCs are a group of immunoregulatory cells with mature status, and that neutrophils can be induced to a PMN-MDSC-like phenotype with decidua-derived GM-CSF via pSTAT5/PD-L2 signalling. This study indicates that GM-CSF can facilitate immune tolerance of early pregnancy through regulating PMN-MDSCs and further provides a potential role of GM-CSF in prevention and treatment for pregnancy complications. This work was supported by the National Natural Science Foundation of China (81671481) and National Natural Science Foundation of China (81871179). All authors have no competing interests to declare.

Li C ，Chen C ，Kang X ，Zhang X ，Sun S ，Guo F ，Wang Q ，Kou X ，Bai W ，Zhao A ... -  《-》

Interferon regulatory factor 4 (IRF4) controls myeloid-derived suppressor cell (MDSC) differentiation and function.

Myeloid-derived suppressor cells (MDSCs) are immature cells that do not differentiate into mature myeloid cells. Two major populations of PMN-MDSCs (Ly6GhighLy6ClowGr1highCD11b+) and MO-MDSCs (Ly6G-Ly6ChighGr-1intCD11b+) have an immune suppressive function. Interferon regulatory factor 4 (IRF4) has a role in the negative regulation of TLR signaling and is associated with lymphoid cell development. However, the roles of IRF4 in myeloid cell differentiation are unclear. In this study, we found that IRF4 expression was remarkably suppressed during the development of MDSCs in the tumor microenvironment. Both the mRNA and protein levels of IRF4 in MDSCs were gradually reduced, depending on the development of tumors in the 4T1 model. siRNA-mediated knockdown of IRF4 in bone marrow cells promoted the differentiation of PMN-MDSCs. Similarly, IRF4 inhibition in bone marrow cells using simvastatin, which has been known to inhibit IRF4 expression, increased PMN-MDSC numbers. In contrast, IRF4 overexpression in bone marrow cells inhibited the total numbers of MDSCs, especially PMN-MDSCs. Notably, treatment with IL-4, an upstream regulator of IRF4, induced IRF4 expression in the bone marrow cells, and consequently, IL-4-induced IRF4 expression resulted in a decrease in PMN-MDSC numbers. Finally, we confirmed that IRF4 expression in MDSCs can modulate their activity to inhibit T cell proliferation through IL-10 production and ROS generation, and myeloid-specific deletion of IRF4 leads to the increase of MDSC differentiation. Our present findings indicate that IRF4 reduction induced by tumor formation can increase the number of MDSCs, and increases in the IRF4 expression in MDSCs may infringe on the immune-suppressive function of MDSCs.

Nam S ，Kang K ，Cha JS ，Kim JW ，Lee HG ，Kim Y ，Yang Y ，Lee MS ，Lim JS ... -  《-》

Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells.

Fernández A ，Oliver L ，Alvarez R ，Hernández A ，Raymond J ，Fernández LE ，Mesa C ... -  《-》