Polymorphonuclear Myeloid-derived Suppressor Cells Research Articles

PDCT-17 (LTBK-11). PNOC007: H3.3K27M SPECIFIC PEPTIDE VACCINE COMBINED WITH POLY-ICLC FOR THE TREATMENT OF NEWLY DIAGNOSED HLA-A2+ H3.3K27M MIDLINE GLIOMAS

Abstract OBJECTIVE To assess within a multi-center trial the safety of H3.3K27M specific peptide vaccine in combination with poly-ICLC in HLA-A02.01+ patients diagnosed with H3.3K27M+ diffuse midline gliomas. METHODS Subjects 3–21 years of age with CLIA confirmed H3.3K27 mutation status were eligible. Patients were enrolled after completion of focal radiation therapy. The trial enrolled two strata: Stratum A included newly diagnosed patients with diffuse intrinsic pontine glioma (DIPG) (n=19); Stratum B included non-DIPG midline glioma patients including spinal cord tumors as an exploratory strata (n=10). The H3.3K27M vaccine was administered in combination with poly-ICLC every 3 weeks for a total of 8 doses followed by every 6 weeks for a maximum of 96 weeks of therapy. Immuno-monitoring and imaging occurred every 3 months. Modified iRANO criteria are used for ongoing assessment of disease status. Patients’ peripheral blood mononuclear cell (PBMC) samples were evaluated by mass cytometry. RESULTS From November 2016 until March 2019, 19 eligible patients (median age 11, range 5–17 yrs; 53 % female) were enrolled in Stratum A and 10 eligible patients (median age 13, range 7–18 yrs; 60 % female) in Stratum B. Treatment was well tolerated with 7 grade 3 and 0 grade 4 treatment related toxicities. The most common related side effect included injection site reaction. Median number of vaccine administrations per patient was 6 (range 1–11). Overall survival at 12 months was 40% (95% CI 22–73%) for Stratum A and 39% (95% CI 16–93%) for Stratum B. A mass cytometry-based analysis of patient PBMCs (n = 16) revealed a trend towards an inverse correlation between polymorphonuclear myeloid-derived suppressor cells and the expansion of H3.3K27M-specific CD8+ T-cells (p-value = 0.154). CONCLUSION H3.3K27M specific vaccine in combination with poly-ICLC is well tolerated. Further evaluation will include a combination with checkpoint inhibitor nivolumab.

IMMU-31. DRIVER GENE MUTATIONS DICTATE THE COMPOSITION OF THE IMMUNE LANDSCAPE OF GLIOBLASTOMA AND CONFER SELECTIVE RESPONSE TO IMMUNOTHERAPY

Abstract The characterization of the immune landscape of glioblastoma multiforme (GBM) is rapidly emerging. Current immunotherapeutic efforts for GBM would benefit from a comprehensive and mechanistic understanding of the relationship between distinct driver gene mutations and the composition and function of the immune tumor microenvironment. The majority of GBM tumors overexpress EGFR and EGFRvIII and ~40% are mutated for PTEN. Using genetically engineered and accurate preclinical mouse models of GBM built on combinations of these driver genes, we demonstrate that the compositions of the immune landscape and the response to checkpoint blockade immunotherapy vary according to the genotype of the GBM. Mechanistically, we show that signaling networks downstream of EGFR and PTEN establish chemokine and cytokine profiles that parallel the levels of intra-tumoral polymorphonuclear myeloid-derived suppressor cells and regulatory T cells. Additionally, tumor-associated microglia/macrophages number, function and polarization are heavily influenced by specific driver-gene mutation combinations. Furthermore, we demonstrate that efficacy of checkpoint blockade therapy using anti PD-1 as a single agent or in combination with anti CTLA-4 is dependent on EGFR and PTEN status. Taken together, our findings demonstrate that important components of the tumor immune microenvironment are influenced by specific driver gene mutations. Our results suggest that stratifying patients based on tumor genotype or signaling events may be informative for selection of appropriate candidates for checkpoint blockade therapy and present an opportunity to pharmacologically modulate GBM signaling with targeted therapeutics to sensitize patients to immunotherapy.

Bacterial Killing Activity of Polymorphonuclear Myeloid-Derived Suppressor Cells Isolated From Tumor-Bearing Dogs.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are implicated in the progression and outcome of a variety of pathological states, from cancer to infection. Our previous work has identified three antimicrobial peptides differentially expressed by PMN-MDSCs compared to conventional neutrophils isolated from dogs, mice, and human patients with cancer. We therefore hypothesized that PMN-MDSCs in dogs with cancer possess antimicrobial activity. In the current work, we observed that exposure of PMN-MDSCs to Gram-negative bacteria (Escherichia coli) increased the expression of reactive oxygen species by the PMN-MDSCs, indicating that they are capable of initiating an anti-microbial response. Electron microscopy revealed that the PMN-MDSCs phagocytosed Gram-negative and Gram-positive (Staphylococcus aureus) bacterial species. Lysis of bacteria within some of the PMN-MDSCs suggested bactericidal activity, which was confirmed by the recovery of significantly lower numbers of bacteria of both species following exposure to PMN-MDSCs isolated from tumor-bearing dogs. Our data therefore indicate that PMN-MDSCs isolated from dogs with cancer, in common with PMNs, have phagocytic and bactericidal activity. This nexus of immunosuppressive and antimicrobial activity reveals a hitherto unrecognized function of MDSCs.

TAM Family Receptor Kinase Inhibition Reverses MDSC-Mediated Suppression and Augments Anti-PD-1 Therapy in Melanoma.

Myeloid cell receptor tyrosine kinases TYRO3, AXL, and MERTK and their ligands, GAS6 and PROTEIN S, physiologically suppress innate immune responses, including in the tumor microenvironment. Here, we showed that myeloid-derived suppressor cells (MDSC) dramatically upregulated TYRO3, AXL, and MERTK and their ligands [monocytic MDSCs (M-MDSC)>20-fold, polymorphonuclear MDSCs (PMN-MDSC)>15-fold] in tumor-bearing mice. MDSCs from tumor-bearing Mertk-/-, Axl-/- , and Tyro3-/- mice exhibited diminished suppressive enzymatic capabilities, displayed deficits in T-cell suppression, and migrated poorly to tumor-draining lymph nodes. In coimplantation experiments using TYRO3-/-, AXL-/-, and MERTK-/- MDSCs, we showed the absence of these RTKs reversed the protumorigenic properties of MDSCs in vivo Consistent with these findings, in vivo pharmacologic TYRO3, AXL, and MERTK inhibition diminished MDSC suppressive capability, slowed tumor growth, increased CD8+ T-cell infiltration, and augmented anti-PD-1 checkpoint inhibitor immunotherapy. Mechanistically, MERTK regulated MDSC suppression and differentiation in part through regulation of STAT3 serine phosphorylation and nuclear localization. Analysis of metastatic melanoma patients demonstrated an enrichment of circulating MERTK+ and TYRO3+ M-MDSCs, PMN-MDSCs, and early-stage MDSCs (e-MDSC) relative to these MDSC populations in healthy controls. These studies demonstrated that TYRO3, AXL, and MERTK control MDSC functionality and serve as promising pharmacologic targets for regulating MDSC-mediated immune suppression in cancer patients.

Prim-O-glucosylcimifugin enhances the antitumour effect of PD-1 inhibition by targeting myeloid-derived suppressor cells

BackgroundMyeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that play an important role in immune evasion, PD-1/PD-L1 inhibitor tolerance and tumour progression. Therefore, MDSCs are potential targets for cancer immunotherapy. In this study, we screened an effective polymorphonuclear MDSC (PMN-MDSC) inhibitor from the Traditional Chinese Medicine Library and evaluated its synergistic antitumour effects with PD-1 inhibitor.MethodsIn the present study, we found that PMN-MDSCs accumulate heavily in the spleen and bone marrow of melanoma (B16-F10) tumour-bearing mice. Then, we determined the top 10 key proteins in the upregulated KEGG pathways of PMN-MDSCs in tumour-bearing mice through proteomics and Cytoscape analysis. The key proteins were then used as targets for the screening of PMN-MDSC inhibitors from the traditional Chinese Medicine Library (20000 compounds) through molecular docking and weight calculation of the docking score. Finally, the inhibitory effect of the inhibitor was verified through proteomics and metabolomics analysis in vitro and melanoma (B16-F10) and triple-negative breast cancer (4 T1) mouse tumour models in vivo.ResultsTraditional Chinese medicine saposhnikovia root extract Prim-O-glucosylcimifugin (POG) could bind well to the target proteins and inhibit the proliferation, metabolism and immunosuppressive ability of PMN-MDSCs by inhibiting arginine metabolism and the tricarboxylic acid cycle (TCA cycle). POG could also increase CD8 T-lymphocyte infiltration in the tumours and enhance the antitumour effect of PD-1 inhibitor in B16-F10 and 4 T1 mouse tumour models.ConclusionsPOG was successfully screened from the traditional Chinese Medicine library as a PMN-MDSC inhibitor. POG exhibited a good synergistic antitumour effect with PD-1 inhibitor. This study provided a potential option for enhancing the efficacy of PD-1 inhibitors in clinical applications.

Deletion of the RNA regulator HuR in tumor-associated microglia and macrophages stimulates anti-tumor immunity and attenuates glioma growth.

Glioblastoma is a malignant brain tumor that portends a poor prognosis. Its resilience, in part, is related to a remarkable capacity for manipulating the microenvironment to promote its growth and survival. Microglia/macrophages are prime targets, being drawn into the tumor and stimulated to produce factors that support tumor growth and evasion from the immune system. Here we show that the RNA regulator, HuR, plays a key role in the tumor-promoting response of microglia/macrophages. Knockout (KO) of HuR led to reduced tumor growth and proliferation associated with prolonged survival in a murine model of glioblastoma. Analysis of tumor composition by flow cytometry showed that tumor-associated macrophages (TAMs) were decreased, more polarized toward an M1-like phenotype, and had reduced PD-L1 expression. There was an overall increase in infiltrating CD4+ cells, including Th1 and cytotoxic effector cells, and a concomitant reduction in tumor-associated polymorphonuclear myeloid-derived suppressor cells. Molecular and cellular analyses of HuR KO TAMs and cultured microglia showed changes in migration, chemoattraction, and chemokine/cytokine profiles that provide potential mechanisms for the altered tumor microenvironment and reduced tumor growth in HuR KO mice. In summary, HuR is a key modulator of pro-glioma responses by microglia/macrophages through the molecular regulation of chemokines, cytokines, and other factors. Our findings underscore the relevance of HuR as a therapeutic target in glioblastoma.

Myeloid Derived Suppressor Cells Expansion Persists After Early ART and May Affect CD4 T Cell Recovery

Myeloid-derived suppressor cells (MDSC) are expanded during HIV-1 infection and correlated with disease progression. MDSC expand in the early phase of primary infection depending on TRAIL level. In this study we evaluated the effect of ART on the frequency of MDSC in patients with primary HIV infection (PHI), and their impact on CD4 T cell reconstitution. MDSC frequency was evaluated by flow-cytometry in 60 PHI patients at 12, 24 and 48 weeks after ART initiation. Cytokine plasma levels were evaluated by Luminex technology at the same time points. The capacity of MDSC to modulate hematopoietic early progenitor cells' expansion was evaluated using the OP9/Dl1 in vitro system. As previously described, polymorphonuclear-MDSC (PMN-MDSC) frequency was higher in PHI compared to healthy donors. Interestingly, 48 weeks of successful ART failed to normalize the PMN-MDSC frequency. Moreover, PMN-MDSC frequency was not correlated with residual viral load, suggesting that the persistence of PMN-MDSC was not due to residual viral replication. Interestingly, patients with low PMN-MDSC frequency (<6%) at T0 had a higher HIV DNA at the same time point than individuals with high PMN-MDSC frequency (>6%). We also found an inverse correlation between PMN-MDSC frequency and CD4-T cell count at 48 weeks post-ART, which was confirmed by multivariate analysis adjusting for age and CD4 T cell number at baseline. These data suggest that the persistence of PMN-MDSC may impact CD4 T cell recovery. Indeed, in vitro PMN-MDSC impaired the expansion of CD34+CD38- hematopoietic early progenitors. Further, a balance between TRAIL and GM-CSF may be necessary to maintain a low MDSC level. In conclusion, early ART initiation was not able to normalize PMN-MDSC frequency that might impact the CD4 T cell recovery. These data open new questions regarding the clinical impact of MDSC persistence in HIV+ patients, in particular on non-AIDS related diseases.

Optimization of the process for purifying icariin from Herba Epimedii by macroporous resin and the regulatory role of icariin in the tumor immune microenvironment

Pancreatic cancer is a digestive tract malignancy that poses a serious threat to human health. Compounds derived from traditional Chinese medicines have been an important source of anticancer drugs and adjuvant agents to regulate the tumor immune microenvironment in patients with pancreatic cancer. In this study, icariin was purified from Herba Epimedii using macropores, and its bioactivity against pancreatic cancer was also investigated. We found that icariin has direct inhibitory and immunomodulatory effects on tumor cells. In vitro experiments showed that icariin can inhibit the migration and proliferation of Panc02 pancreatic cancer cells and induce apoptosis. Our in vivo experiments show that icariin inhibits the development of mouse pancreatic cancer by inhibiting tumor-infiltrating M2 macrophages and polymorphonuclear myeloid-derived suppressor cells (MDSCs) (PMN-MDSCs). In addition, icariin inhibits the polarization of RAW 264.7 cells into M2 macrophages by inhibiting the expression of ARG1 and MRC1 and downregulating the IL4-STAT6 signaling pathway. In conclusion, the inhibitory effect of icariin on pancreatic cancer can not only directly affect tumor cells but also inhibit tumor development by regulating the tumor immune microenvironment.

Chemokine signaling axis between endothelial and myeloid cells regulates development of pulmonary hypertension associated with pulmonary fibrosis and hypoxia.

Pulmonary hypertension complicates the care of many patients with chronic lung diseases (defined as Group 3 pulmonary hypertension), yet the mechanisms that mediate the development of pulmonary vascular disease are not clearly defined. Despite being the most prevalent form of pulmonary hypertension, to date there is no approved treatment for patients with disease. Myeloid-derived suppressor cells (MDSCs) and endothelial cells in the lung express the chemokine receptor CXCR2, implicated in the evolution of both neoplastic and pulmonary vascular remodeling. However, precise cellular contribution to lung disease is unknown. Therefore, we used mice with tissue-specific deletion of CXCR2 to investigate the role of this receptor in Group 3 pulmonary hypertension. Deletion of CXCR2 in myeloid cells attenuated the recruitment of polymorphonuclear MDSCs to the lungs, inhibited vascular remodeling, and protected against pulmonary hypertension. Conversely, loss of CXCR2 in endothelial cells resulted in worsened vascular remodeling, associated with increased MDSC migratory capacity attributable to increased ligand availability, consistent with analyzed patient sample data. Taken together, these data suggest that CXCR2 regulates MDSC activation, informing potential therapeutic application of MDSC-targeted treatments.

Lnc-C/EBPβ Modulates Differentiation of MDSCs Through Downregulating IL4i1 With C/EBPβ LIP and WDR5.

Myeloid-derived suppressor cells (MDSCs), which play an important role in tumor and inflammatory diseases, are divided into two subsets CD11b+Ly6ChiLy6G− monocytic MDSC (Mo-MDSC) and CD11b+Ly6Clow/negLy6G+ polymorphonuclear MDSC (PMN-MDSC) with different immunosuppressive function. However, it is poorly understood the mechanism(s) to control differentiation of Mo-MDSCs and PMN-MDSCs. Here, we found that lnc-C/EBPβ may promote PMN-MDSC but impede differentiation of Mo-MDSCs in vitro and in vivo. We demonstrated that lnc-C/EBPβ mediated differentiation of MDSCs was through downregulating multiple transcripts such as IL4il. Lnc-C/EBPβ not only bound to C/EBPβ isoform LIP to inhibit the activation of C/EBPβ but also interacted with WDR5 to interrupt the enrichment of H3K4me3 mark on the promoter region of IL4i1. Data also imply that conserved homo lnc-C/EBPβ has a similar function with mouse lnc-C/EBPβ. Since MDSC subsets exert different suppressive function, lnc-C/EBPβ may be acted as a potential therapeutic target for inflammatory and tumor-associated diseases.

Abstract 1530: Polymorphonuclear myeloid derived suppressor cells secrete IL6 to promote colon tumorigenesis in an inflammatory vicious cycle

Abstract It is widely accepted now that chronic inflammation can create a favorable environment for neoplastic transformation and tumor progression. Tumor Microenvironment consists of various innate and adaptive immune cells, which secrete numerous inflammatory mediating and immunoregulatory cytokines. Among all immune cells infiltrated to the tumor site, myeloid cells have been considered as major players in the tumor associated inflammation. Recently, a subpopulation named myeloid-derived suppressor cells (MDSCs) have gain a great attention as a potential link between inflammation and tumor progression. MDSCs are heterogeneous population of immature myeloid cells that is subdivided into monocytic (M-MDSC) and polymorphonuclear (PMN-MDSC) subsets depending on different markers expressed differentially on their surface. Both PMN-MDSC and M-MDSC have been shown to be immune-suppressive and to exert an immunoregulatory role in the tumor microenvironment. However, a clear distinction in their functional and molecular properties has remained a matter of debate. Here we report that PMN-MDSC but not M-MDSC is the major secreting immune subset of the proinflammatory and tumor-promoting cytokine Interleukin 6 (IL6) in colon cancer. IL6 has been shown in literature to function as a tumor promoter through enhancing tumor growth, invasion and chemoresistance. We observed that IL6 upregulates DNMT1 and DNMT3b in colon carcinoma cells to boost their growth and survival in a STAT3 dependent mechanism. We also observed that tumor-expressed IL6 acts as a key modulator of the immune profile in colon tumor microenvironment. We generated an IL6-overexpressing colon carcinoma cell lines. We observed that IL6-overexpressing tumor triggers a significant increment in PMN-MDSCs but not M-MDSCs infiltration and accumulation in mouse colon carcinoma xenograft model. This remarkable PMN-MDSC tumor enrichment is associated with less effector T lymphocytes infiltration and more progressive and proliferative tumor. Moreover, DSS-induced colitis murine model is characterized with higher infiltration of PMN-MDSC but not M-MDSC, which secrete significant amount of IL6. Therefore, inflammation-derived IL6 induces neoplastic transformation of colonic epithelial cells through upregulation of DNMT1 and DNMT3b. Collectively; our data indicate that PMN-MDSC is the primary IL6 producer among all tumor-infiltrated leukocytes. PMN-MDSC-derived IL6 has a dual tumor favoring function through nurturing colon tumor cells in DNA-methylation dependent mechanism, and raising an immunosuppressive context enriched with more PMN-MDSCs, which secrete more IL6 in an endless vicious cycle. Thus, targeting this circuit, may have a promising implication in colon cancer immunotherapy. Citation Format: Mohammed L. Ibrahim, John D. Klement, Priscilla S. Redd, Chunwan Lu, Kebin Liu. Polymorphonuclear myeloid derived suppressor cells secrete IL6 to promote colon tumorigenesis in an inflammatory vicious cycle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1530.

Abstract 1510: Androgen regulated IL-8 expression in prostate cancer: Insights into tumor cell mediated immunosuppression

Abstract Androgen deprivation therapy (ADT) results in castration-resistant prostate cancer (CRPC) in a significant fraction of patients. We have previously reported that the protein levels of interleukin-8 (IL-8) were inversely correlated with disease progression in men with biochemical recurrent prostate cancer treated with Lenalidomide. Recently, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were implicated as potential drivers of CRPC. Here we show that IL-8 expression is upregulated as a consequence of ADT and mediates the recruitment of PMN-MDSCs to the tumor microenvironment. We found that IL-8 expression is regulated by both an inflammatory stimulus (NF-kβ mediated) and loss of androgen receptor (AR) signaling following ADT. We confirmed direct binding of both the p65 subunit of NF-kβ and AR to the IL-8 promoter, and their respective effects on promoter activity. The suppressive activity of AR was further supported by a reduction in active transcription markers at the chromatin level surrounding the IL-8 promoter. Accordingly, intratumoral infiltration of PMN-MDSCs correlated with IL-8 expression, and was reduced in IL-8 knockouts. Taken together, these results suggest an innate inflammatory response, loss of AR suppressive activity, and subsequent chemokine upregulation as a potential mechanism that regulates the infiltration of PMN-MDSCs to the tumor microenvironment of CRPC after ADT. These findings open a window of opportunity for therapeutic interventions aiming to improve responses to checkpoint blockade in prostate cancer. Citation Format: Zoila A. Lopez Bujanda, Michael C. Haffner, Matthew G. Chaimowitz, Nivedita Chowdhury, Paula J. Hurley, Angela M. Christiano, Charles G. Drake. Androgen regulated IL-8 expression in prostate cancer: Insights into tumor cell mediated immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1510.

Abstract 147: Deletion of the RNA regulator HuR in microglia/macrophages promotes an anti-tumor microenvironment in glioblastoma

Abstract Glioblastoma is a malignant brain tumor that portends a poor prognosis. Its resilience, in part, is related to a remarkable capacity for manipulating the microenvironment to promote its growth and survival. Microglia/macrophages are a prime target, being drawn into the tumor and stimulated to produce factors that support tumor growth and immune system evasion. Here we show that the RNA regulator, HuR, plays a key role in the tumor-promoting response of microglia/macrophages. Knockout of HuR in microglia/macrophages led to reduced tumor growth and prolonged survival in a murine model of glioblastoma. Analysis of tumor composition by flow cytometry showed that tumor associated macrophages were decreased, more polarized toward an M1-like phenotype, and had attenuated PD-L1 expression. There was a concomitant reduction in tumor-associated polymorphonuclear myeloid-derived suppressor cells, but an increase in Treg cells. In vitro assays showed reduced migration of HuR-/- microglia toward secreted factors derived from glioblastoma cells, and a decrease in migration of glioblastoma cells toward secreted factors derived from HuR-/- microglia. The molecular response of HuR-/- microglia was altered including reduced CXCL1, 2, MMP2, PD-L1 and VEGF expression and an increase in CXCL10 and several MMPs. There was a mixed effect on cytokines and other factors associated with both proinflammatory and alternatively activated phenotypes. In summary, HuR is a key modulator of the tumor microenvironment in glioblastoma, promoting tumor progression through its molecular regulation of factors produced by microglia/macrophages. These findings underscore the relevance of HuR as a therapeutic target in glioblastoma. Citation Format: Jiping Wang, Jianmei Leavenworth, Anita Hjelmeland, Ben Borg, Peter H. King. Deletion of the RNA regulator HuR in microglia/macrophages promotes an anti-tumor microenvironment in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 147.

Identification of monocyte-like precursors of granulocytes in cancer as a mechanism for accumulation of PMN-MDSCs.

We have identified a precursor that differentiates into granulocytes in vitro and in vivo yet belongs to the monocytic lineage. We have termed these cells monocyte-like precursors of granulocytes (MLPGs). Under steady state conditions, MLPGs were absent in the spleen and barely detectable in the bone marrow (BM). In contrast, these cells significantly expanded in tumor-bearing mice and differentiated to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Selective depletion of monocytic cells had no effect on the number of granulocytes in naive mice but decreased the population of PMN-MDSCs in tumor-bearing mice by 50%. The expansion of MLPGs was found to be controlled by the down-regulation of Rb1, but not IRF8, which is known to regulate the expansion of PMN-MDSCs from classic granulocyte precursors. In cancer patients, putative MLPGs were found within the population of CXCR1+CD15-CD14+HLA-DR-/lo monocytic cells. These findings describe a mechanism of abnormal myelopoiesis in cancer and suggest potential new approaches for selective targeting of MDSCs.

Low-density granulocytes activate T cells and demonstrate a non-suppressive role in systemic lupus erythematosus

ObjectivesThe presence of proinflammatory low-density granulocytes (LDG) has been demonstrated in autoimmune and infectious diseases. Recently, regulatory neutrophilic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) were identified in systemic lupus erythematosus (SLE)....

PF658 THE NEUTROPHIL TO LYMPHOCYTE RATIO (NLR) IS A CHEAP SURROGATE OF EXPANSION OF MYELOID‐DERIVED SUPPRESSOR CELLS IN MYELOFIBROSIS BEFORE AND AFTER TREATMENT WITH RUXOLITINIB: A SINGLE‐CENTER EXPERIENCE

Background:In myelofibrosis (MF), majority clinical benefits of ruxolitinib are presumably derived from reduction of inflammatory cytokines even if the exact mechanism remains unclear. Polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSC) are important regulators of immune responses in cancer, directly implicated in promotion of tumor progression. NLR, the ratio between absolute neutrophils count (ANC) and absolute lymphocyte count (ALC), mirrors the inflammatory status and the myeloid associated immune suppression, predictor of progression free survival (PFS) and overall survival (OS).Aims:To investigate the amount of circulating MDSC in MF patients undergoing treatment with ruxolitinib and any potential association with clinical variables at baseline and during treatment.Methods:From September 2016 through January 2019, we prospectively evaluated 40 healthy volunteers and 52 consecutive MF patients before and during treatment with ruxolitinib and measured NLR, MDSC by flow cytometry defined as monocytic‐like CD45+CD11b+ CD15−, CD14+ HLA‐DR− (mo‐MDSC) and granulocytic‐like CD45+CD11b+ CD15+, Lox1+ CD14− HLA‐DR− (PMN‐MDSC). Immune‐suppressive cytokines IL‐6, IL‐8, TGF‐beta and aminoacid‐degrading enzymes Arginase‐1 (Arg‐1) and indoleamine‐deoxygenase1 (IDO‐1) were evaluated in peripheral sera as functional markers of MDSC expansion.Spleen diameters were evaluated by physical examination before treatment and after 12 and 24 weeks of ruxolitinib continuous treatment according to drug label in a real‐life setting. NLR was calculated using data obtained from the complete blood count at diagnosis and correlated with driver mutations and clinical response outcome.Results:While there were not significant differences in mo‐MDSC percentages among MF and healthy subjects, PMN‐MDSC were increased (p &lt; 0.001) and showed increased p‐STAT3 and p‐STAT5 (p &lt; 0.001), markers of immune‐suppression in myeloid regulatory cells. Accordingly, immune‐suppressive cytokines IL‐6, IL‐8, TGF‐beta and aminoacid‐degrading enzymes Arg‐1 and IDO‐1 were significantly increased (p &lt; 0.001).For 26 patients, matched samples before and after 12‐weeks treatment with ruxolitinib were available and showed a significant decrease of both PMN‐MDSC (p &lt; 0.0001), associated to reduction of p‐STAT5 up to values measurable in healthy subjects (p = 0.003), but not p‐STAT3.PMN‐MDSC percentage was positively associated to NLR (r‐square 0.52, p = 0.003). NLR was increased upon progression of disease, and positively associated to fibrosis grading as detected by immune‐histochemistry. Patients with lower bone marrow fibrosis had lower median NLR (grade 0: 4.1, G1: 4.8, G2: 5.2, G3: 8.1, p &lt; 0.001), while cases positive for JAK2V617F had increased NLR (p = 0.002). Receiving operator curve identified NLR = 6 as cut‐off to distinguish healthy and MF subjects with 97 % sensitivity and 89% specificity (AUC = 0.92, p &lt; 0.001), and it was applied for further evaluations.Patients with NLR&gt;6 before starting ruxolitinib treatment did not achieve spleen reduction higher than 50% in the first 12 weeks (p = 0.001). At 24 weeks, patients with lower NLR had more chances to reduce spleen size of 100% (p = 0.001). After a median follow‐up of 30.2 months, NLR at diagnosis was not predictor of neither TTP nor OS.Summary/Conclusion:In MF, PMN‐MDSC are increased and positively associated to NLR, mutational status and bone marrow fibrosis. NLR is an useful, simple and early predictor of response and spleen reduction in patients treated with ruxolitinib, despite it does not have any impact on TTP and OS.

Targeting monocyte-intrinsic enhancer reprogramming improves immunotherapy efficacy in hepatocellular carcinoma

ObjectiveHepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its...

Temporal and spatial profile of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in ischemic stroke in mice.

Although T cells play important roles in the pathophysiology of ischemic stroke, the dynamics of T cells remains unclear. In cancer, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) contribute to the maintenance of the tumor microenvironment by suppressing T cells. However, the presence of these cells has never been examined in ischemic brain. Therefore, we examined the temporal and spatial profiles of PMN-MDSCs, which are defined as the CD11b+Ly6ClowLy6G+ cells with higher expression levels of Nox2 and C/EBP Homologous Protein (CHOP) mRNA than normal neutrophil. Fluorescence-activated cell sorter (FACS) analysis showed that the count of CD11b+Ly6ClowLy6G+ cells was increased in the ischemic hemisphere and bone marrow at 72 hours, as well as in the spleen 24 hours after transient middle cerebral artery occlusion in mice. In contrast, the contralateral hemisphere, normal bone marrow, and normal spleen contained few CD11b+Ly6ClowLy6G+ cells. Real-time reverse transcription polymerase chain reaction revealed that CD11b+Ly6ClowLy6G+ cells sorted from brain and spleen 72 hours after ischemia had greater expression of Nox2 and CHOP mRNA than neutrophils in bone marrow, suggesting that these cells constitute PMN-MDSCs. Immunohistochemistry showed that CD11b+Ly6G+ cells were located in the ischemic core and border zone, indicating that PMN-MDSCs might be endemic to these regions. Although neutrophils are believed to invade infarct regions 48–72 hours after ischemia, the present study suggested that some of these cells are in fact PMN-MDSCs. Further studies on the function of PMN-MDSCs might unveil the unknown mechanisms of T cell activation and recruitment in ischemic stroke.

Dynamics of migration patterns of polymorphonuclear myeloid-derived suppressor cells during tumor progression

Abstract Although recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) to the tumor site is well documented, little is known about their migration patterns within bone marrow in tumor progression. We found that the activation of neutrophils migration in cancer is a two-phase process. The first phase was characterized by the accumulation of a previously uncharacterized population of neutrophils that lack immunosuppressive activity but display a potent ability to spontaneously migrate, whereas the later phase is associated with accumulation of neutrophils with typical features of PMN-MDSC with low migratory activity in the bone marrow of mice with various subcutaneous (ectopic) tumors and in mice with advanced orthotopic lung cancer. Mechanistically, PM-LCs displayed increased metabolic flux through oxidative phosphorylation and glycolysis and have more ATP than that of control neutrophils. However, PMN-MDSC were indistinguishable from neutrophils from tumor-free mice in their metabolic activity. In line with studies of mice, CD15+ neutrophils exhibited greater spontaneous migration than that of PMN-MDSCs from the same patient with cancer. These results elucidate the dynamic changes that neutrophils undergo in cancer.

Sympathetic nervous tone influences anti-tumor immunity by mediating accumulation of myeloid-derived suppressor cells and regulatory T-cells

Abstract Sympathetic nerves innervate primary and secondary lymphoid organs, providing a means by which nervous system signaling can affect cellular immunity. For example, signaling by sympathetic nerves that innervate the spleen and bone marrow can regulate the development of myeloid cells. In cancer, aberrant myeloid development has been shown to drive accumulation of myeloid-derived suppressor cells (MDSCs) that suppress the anti-tumor immune response and are associated with poor prognosis. Given the known regulatory effect of sympathetic nervous system (SNS) signaling on myelopoiesis and the consequences of aberrant myeloid development in cancer, we hypothesized that the SNS plays an important role in regulating the immune response to cancer by influencing accumulation of MDSCs. Here, we demonstrate that inhibition of tonic SNS signaling compromises the anti-tumor immune response and results in increased tumor growth. We show that chemical ablation of the SNS by peripheral administration of 6-hydroxydopamine disrupts normal myeloid development in the bone marrow and results in expansion of CD11b+Ly6G+ polymorphonuclear MDSC and CD11b+ Ly6Chi monocytic MDSC in tumor-bearing mice. We show that these MDSC are able to potently suppress T-cell proliferation and mediate the expansion of regulatory T-cells that further inhibit the anti-tumor immune response. These data demonstrate how SNS-mediated regulation of immune development and function plays an important role in instructing the anti-tumor immune response. These findings have the potential to improve our understanding of the interplay between the nervous and immune systems, and better inform treatment of cancer and other immune-mediated pathologies.