Changes Of Myeloid-derived Suppressor Cells Research Articles

Exploratory phase I study of HF1K16 for the treatment of patients with refractory/recurrent advanced glioma.

TPS32 Background: Glioblastoma (GBM) is the most common primary and aggressive malignant brain tumor in adults. Almost all patients with glioblastoma eventually have disease relapse and the median overall survival (OS) remains at only 30 weeks for those recurrent GBM patients. However, very few therapies had been approved over the past two decades, emphasizing the need for novel treatments. It is now wildly acknowledged that the immune microenvironment in brain offers adequate opportunities to implement immunotherapy for the treatment. Since most recurrent tumors had previously been exposed to the genotoxic stress of irradiation and/or chemotherapy, it is predicted to resulted in a higher mutational load and more immunogenic. However, The limited success of T cell immunomodulatory approach for advanced glioma has prompted a deeper understanding of the brain tumor and the immune microenvironment. GBM tumors are well-appreciated to be a immunosuppressive and a hallmark of GBM immunosuppression is the appearance of circulating myeloid-derived suppressor cells (MDSCs) at higher levels. Our preliminary data suggests that HF1K16, a liposome ATRA (all trans retinoic acid) suspension, is capable of relieving immune suppression induced by MDSCs. Further, we recently completed a phase I dose escalation study which showed that HF1K16 is safe, well-tolerated with preliminary efficacy in brain tumor. Thus, we hypothesized that HF1K16 may reverse GBM-associated immune suppression and leading to an increase in TILs and improved survival. Methods: This is an advanced glioma-specific expansion arm for adult patients with prior confirmed brain tumor and failed standard treatment. We plan to enroll 20~30 evaluable patients. Key eligibility criteria include candidates age ≥ 18 years, Karnofsky performance status ≥ 60 with and at least one measurable lesion. The primary endpoint is determination of overall response rate (ORR), duration of response (DOR), disease control rate (DCR) and progression-free survival (PFS) according to RANO criteria. Secondary endpoints include longitudinal assessment of the peripheral immune response by changes in MDSC and T cell subsets and numbers. HF1K16 infusions were administered in 21-day cycles (q.o.d days 1-14). Prior to and during treatment, peripheral blood mononuclear cells were collected and analyzed with flow cytometry to monitor the changes in myeloid cell phenotype and T cell composition. The recruitment of patients is anticipated completed in 2024. Clinical trial information: NCT05388487 .

Targeting myeloid-derived suppressor cells by inhibiting hypoxia-inducible factor 1α could improve tumor progression.

Myeloid-derived suppressor cells (MDSCs) are a subset of immature myeloid cells that inhibit anti-tumor immunity and contribute to poor cancer outcomes. In this study, the authors used multi-color flow cytometry to detect changes in MDSCs in patients with cancer and tumor-bearing mice. Then the authors studied changes in MDSCs ratio and mouse tumors after administration of hypoxia-inducible factor 1α (HIF-1α) inhibitor. The results showed that the ratio of MDSCs, specifically polymorphonuclear MDSCs (PMN-MDSCs), was higher in patients with cancer, and both PMN-MDSCs and monocytic MDSCs (M-MDSCs) ratio were higher in tumor-bearing mice. When provided with the HIF-1α inhibitor LW-6, the ratio of MDSCs decreased in tumor-bearing mice, particularly PMN-MDSCs, and the volume of liver metastases also decreased. The authors' findings suggest that reducing MDSCs by inhibiting hypoxia-inducible factor 1α may slow tumor progression.

501 Systemic Immunological Suppression in Glioblastoma Is Targeted by Concurrent VEGF and PD-1 Inhibition in a Dose-Dependent Manner: Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) From a Randomized Controlled Trial

INTRODUCTION: Anti-PD1 blockade has had poor efficacy in recurrent glioblastoma (rGBM) across three RCTs. VEGF, a proangiogenic factor that is upregulated in rGBM, contributes to tumor-associated immunosuppression and is widely targeted through bevacizumab. Preclinical data indicate a potential dose-dependent effect of anti-VEGF therapy on immunomodulation. Hence, a combination of anti-PD-1 and anti-VEGF agents is a promising approach for rGBM, for which an RCT was conducted. METHODS: Patients with rGBM were administered nivolumab (240mg IV q2weeks) plus either standard dose bevacizumab (10mg/kg) or low dose bevacizumab (3mg/kg) IV q2weeks, with 1:1 randomization. Stratification factors were age, performance status, extent of resection, and MGMT methylation status. Primary endpoint was overall survival (OS) at 12 months. CITE-Seq was performed on 16 patients across trial arms, both pre- and on-therapy, yielding total 32 immunological profiles. For control, CITE-seq was done on three patients receiving anti-VEGF therapy alone. RESULTS: 90 patients were enrolled with median age of 60.6 years and 45 patients per arm. Both arms had similar OS at 12 months (P = 0.14). Significantly higher OS was found in posthoc analysis for patients aged = 60 years with standard dose. Distinct immune cell populations were identified and differential changes in myeloid-derived suppressor cells (MDSCs) found across trial arms on-therapy. Network medicine approach identified VEGF as target to reduce MDSCs, with VEGF primarily expressed by responders’ MDSCs. Differential gene expression analysis identified increased pro-inflammatory signatures in standard dose arm. CONCLUSIONS: Standard dose bevacizumab was associated with increased inflammatory response and reduction of immunosuppressive MDSC. These mechanistic insights potentially explain survival benefit seen with concurrent VEGF and PD-1 inhibition in elderly rGBM patients.

Decreased circulating myeloid-derived suppressor cell count at the engraftment is one of the risk factors for multiple myeloma relapse after autologous hematopoietic stem cell transplantation

Recent studies demonstrated that myeloid-derived suppressor cells (MDSCs) are involved in the pathogenesis and progression of multiple myeloma (MM). Nevertheless, data on the quantitative and functional changes in MDSCs during standard MM treatment remain poorly understood. Here, we determined that monocytic MDSCs (M-MDSC; CD14+HLA-DRlow/–) and granulocytic MDSCs (PMN-MDSC; Lin–HLA-DR–CD33+CD66b+) in MM patients in remission following induction therapy (IT) were significantly increased, while early MDSCs (E-MDSCs; Lin–HLA-DR–CD33+CD66b–) were decreased compared to the donor group. In progression, MM patients had the most pronounced decrease in E-MDSCs and enhanced levels of PMN-MDSCs. IT was accompanied with a decrease in the expression of arginase-1 (Arg-1). In MM patients with relapse or resistance to IT, Arg-1+ cell frequency in M-MDSCs and E-MDSCs, as well as PD-L1+ M-MDSCs, was increased, which may facilitate tumor immunosuppression. G-CSF administration led to a significant increment in the MDSC subsets. At the engraftment, circulating M-MDSC and PMN-MDSCs were temporarily increased, with a gradual decline to the pre-transplant levels in 12 months. The percentage of E-MDSCs was decreased at the leukocyte recovery. Patients with a higher (>Me) M-MDSC count at the engraftment had a shorter post-transplant leukopenia duration (Me 11 vs. 13 days; pU = 0.0086). The advanced MM stage, depth of response, and lower relative count of circulating E-MDSCs at the engraftment were independent risk factors associated with a lower progression-free survival.The obtained data allow us to hypothesize that MDSCs may play a positive role at the stage of leukocyte recovery by ameliorating the long-term anti-tumor response in MM.

IMMU-34. SYSTEMIC IMMUNE SUPPRESSION IN GLIOBLASTOMA IS TARGETED BY CONCURRENT VEGF AND PD-1 INHIBITION IN A DOSE-DEPENDENT MANNER: TRANSCRIPTOMIC FINDINGS THROUGH CITE-SEQ FROM A RANDOMIZED CONTROLLED TRIAL

Abstract INTRODUCTION Anti-PD1 blockade has had poor efficacy in recurrent glioblastoma (rGBM) across three RCTs. VEGF, a proangiogenic factor that is upregulated in rGBM, contributes to tumor-associated immunosuppression and is widely targeted through bevacizumab. Preclinical data indicate a potential dose-dependent effect of anti-VEGF therapy on immunomodulation. Hence, a combination of anti-PD-1 and anti-VEGF agents is a promising approach for rGBM, for which an RCT was conducted. This work sought to utilize a multimodal approach capturing single-cell gene and protein expression by ‘cellular indexing of transcriptomes and epitopes by sequencing’ (CITE-Seq) to interrogate the systemic immunological changes in response to concurrent VEG and PD-1 inhibition. METHODS Patients with rGBM were administered nivolumab (240mg IV q2weeks) plus either standard-dose bevacizumab (10mg/kg) or low-dose bevacizumab (3mg/kg) IV q2weeks, with 1:1 randomization. Stratification factors were age, performance status, extent of resection, and MGMT methylation status. Primary endpoint was overall survival (OS) at 12 months. CITE-Seq was performed on 16 patients across trial arms, both pre- and on-therapy, yielding total 32 immunological profiles. For control, CITE-seq was done on three patients receiving anti-VEGF therapy alone. RESULTS 90 patients were enrolled with median age of 60.6 years and 45 patients per arm. Both arms had similar OS at 12 months (P=0.14). Significantly higher OS was found in posthoc analysis for patients aged ≥60 years with standard dose. Distinct immune cell populations were identified, and differential changes in myeloid-derived suppressor cells (MDSCs) found across trial arms on-therapy. Network medicine approach identified VEGF as target to reduce MDSCs, with VEGF primarily expressed by responders’ MDSCs. Differential gene expression analysis identified increased pro-inflammatory signatures in standard-dose arm. CONCLUSIONS Standard dose bevacizumab was associated with increased inflammatory response and reduction of immunosuppressive MDSC. These mechanistic insights potentially explain survival benefit seen with concurrent VEGF and PD-1 inhibition in elderly rGBM patients.

Increased levels of circulating granulocytic myeloid‑derived suppressor cells in lumbar disc herniation.

Myeloid-derived suppressor cells (MDSCs) expand when the body undergoes inflammatory diseases and chronic diseases. However, its role in intervertebral disc degeneration remains unclear. The present study aimed to characterize specific subsets of MDSCs as potential indicators of disease progression in patients with lumbar disc herniation (LDH). The Gene Expression Omnibus (GEO) database was used to analyze the changes in granulocyte MDSCs (G-MDSCs). Peripheral blood samples were collected from 40 patients with LDH and 15 healthy controls, and flow cytometry was used to characterize different subsets of MDSCs. All subjects underwent lumbar spine magnetic resonance imaging. Then, t-distributed stochastic neighborhood embedding and FlowSOM were used to analyze the data obtained by CytoFlex. The correlation between circulating MDSCs and the clinicopathological stage of LDH was then further analyzed. The GEO database predicted that G-MDSCs were highly expressed in patients with LDH. The frequency of circulating G-MDSCs increased with Pfirrmann stage III and IV, while the percentage of mononuclear MDSCs (M-MDSCs) only increased. Patient age and sex did not correlate with the frequency of circulating G-MDSCs and M-MDSCs. The results of the computer algorithm analysis were consistent with those of our manual gating. The present study showed that the occurrence of LDH led to changes in the MDSC subpopulation in the circulating peripheral blood of patients, and the frequency of circulating G-MDSCs in patients with clinical stage III and IV LDH increased with the degree of degeneration. The determination of G-MDSCs can be used as an auxiliary examination item for LDH.

Dynamic changes in myeloid-derived suppressor cells during the menstrual cycle: A pilot study.

Various studies have described the roles of myeloid-derived suppressor cells (MDSCs) in pathological conditions, but relatively few have described them under normal physiological conditions. Accumulation of MDSCs is important creating an anti-inflammation environment, which is essential for fertilized egg implantation. This study was designed to record the dynamic changes in MDSC-like cells composition during the menstrual period (MP) and ovulation period (OP) in healthy volunteers over the course of a single menstrual cycle to explore the association between MDSCs and the menstrual cycle under normal physiological conditions. The ratio of MDSC-like cells was higher in MP samples, whereas the activity of Arg-1 was higher during the OP window. There was a negative correlation between the ratio of MDSC-like cells and the percentage of lymphocytes and a positive correlation between MDSC-like cells and prostaglandin E2 (PGE2). Furthermore, regular changes in the ratio and function of MDSC-like cells in the peripheral blood were observed during menstruation, all of which corresponded to the cycle stage. During menstruation, MDSCs may promote endometrial repair, whereas they promote pregnancy during the OP. These findings may help to better understand the pathophysiology of pregnancy-related complications and lay a foundation for improving perinatal outcomes.

Immune Signature of Pembrolizumab Plus Gemcitabine, Vinorelbine, and Liposomal Doxorubicin As Second-Line Therapy for Relapsed or Refractory Classical Hodgkin Lymphoma

Introduction: In our phase II study (Moskowitz AJ et al, JCO 2021), pembrolizumab plus gemcitabine, vinorelbine, and liposomal doxorubicin (P-GVD) was found to be highly effective as salvage for patients (pts) with relapsed or refractory (R/R) classic Hodgkin lymphoma (cHL) proceeding to high dose therapy and autologous hematopoietic cell transplantation (AHCT). We present analyses assessing immunologic changes associated with P-GVD treatment as well as updated clinical outcome. Methods: Transplant eligible pts with R/R cHL following failure of 1-line of therapy were eligible. Treatment consisted of 2 to 4 cycles of P-GVD followed by AHCT. Peripheral blood samples were collected at baseline, after 2 cycles of P-GVD, before AHCT, 3 and 9 months after AHCT. Peripheral blood was analyzed for serum thymus and activation-regulated chemokine (TARC) and immune cell subsets. TARC was measured by enzyme-linked immunosorbent assay. Immune cell subsets, including T-cells and myeloid-derived suppressor cells (MDSC), were measured by multi-parametric flow cytometry (FC). Metabolic tumor volume (MTV) was evaluated at baseline. Changes in MDSC and TARC levels were assessed using Wilcoxon rank sum and Friedman tests, respectively. Correlations between MTV and either TARC or MDSC were assessed using Pearson's R. Results: Of 38 evaluable pts, the complete response (CR) rate after 2 or 4 cycles of P-GVD was 95%. Two pts declined AHCT and 36 proceeded to AHCT. After a median follow-up of 30 months (range: 2-43), only 1 pt experienced disease progression 23 months after AHCT. The estimated 30-month progression-free survival (PFS) is 96%. The median baseline MTV value was 37 cm3 (range 4- 845). 36/38 and 27/38 pts had sufficient correlative samples to perform analysis for TARC and MDSC analysis, respectively. The median baseline TARC level was 526 pg/mL (range: 12-4017). TARC levels decreased significantly after 2 cycles of P-GVD (p < 0.001) and increased post-AHCT. Circulating MDSCs were 0.05% of total mononuclear cells (range: 0.003-0.59%) and significantly increased after 2 cycles of P-GVD (p<0.001). Higher baseline TARC and circulating MDSC levels correlated with higher baseline MTV (r=0.45, 95% CI: 0.11-0.70, p<0.01 and r=0.46, 95%CI 0.03-0.76, p=0.03, respectively). High-dimensional analysis of FC data revealed several distinct immune clusters (Figure 1), of which non-proliferating PD-1+ CD4+ T-cells decreased most significantly following treatment (Figure 2). Additional markers (ICOS, CTLA-4, LAG-3, FOXP3) of immune exhaustion showed only a mild decrease over the treatment course. Conclusions: After a median of 30-months follow-up, second-line P-GVD continues to be associated with durable remissions after AHCT. These analyses provide insights into key immune changes associated with chemoimmunotherapy in HL. Baseline TARC correlated with baseline MTV and was significantly lower after P-GVD, illustrating its role as a marker of tumor bulk. The subsequent rise in serum TARC post AHCT suggests immune reconstitution of TARC-secreting cells. Higher circulating levels of MDSCs correlated with baseline MTV, which may reflect an association between immune suppression and tumor bulk. MDSC levels unexpectedly increased after P-GVD, indicating that a rise in MDSCs after treatment may not necessarily predict for poor outcomes. Ongoing single-cell RNA sequencing analysis of peripheral blood mononuclear cells (PBMC) may provide further understanding into immune dynamics associated with P-GVD treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tristetraprolin limits age-related expansion of myeloid-derived suppressor cells.

Aging results in enhanced myelopoiesis, which is associated with an increased prevalence of myeloid leukemias and the production of myeloid-derived suppressor cells (MDSCs). Tristetraprolin (TTP) is an RNA binding protein that regulates immune-related cytokines and chemokines by destabilizing target mRNAs. As TTP expression is known to decrease with age in myeloid cells, we used TTP-deficient (TTPKO) mice to model aged mice to study TTP regulation in age-related myelopoiesis. Both TTPKO and myeloid-specific TTPKO (cTTPKO) mice had significant increases in both MDSC subpopulations M-MDSCs (CD11b+Ly6ChiLy6G-) and PMN-MDSCs (CD11b+Ly6CloLy6G+), as well as macrophages (CD11b+F4/80+) in the spleen and mesenteric lymph nodes; however, no quantitative changes in MDSCs were observed in the bone marrow. In contrast, gain-of-function TTP knock-in (TTPKI) mice had no change in MDSCs compared with control mice. Within the bone marrow, total granulocyte-monocyte progenitors (GMPs) and monocyte progenitors (MPs), direct antecedents of M-MDSCs, were significantly increased in both cTTPKO and TTPKO mice, but granulocyte progenitors (GPs) were significantly increased only in TTPKO mice. Transcriptomic analysis of the bone marrow myeloid cell populations revealed that the expression of CC chemokine receptor 2 (CCR2), which plays a key role in monocyte mobilization to inflammatory sites, was dramatically increased in both cTTPKO and TTPKO mice. Concurrently, the concentration of CC chemokine ligand 2 (CCL2), a major ligand of CCR2, was high in the serum of cTTPKO and TTPKO mice, suggesting that TTP impacts the mobilization of M-MDSCs from the bone marrow to inflammatory sites during aging via regulation of the CCR2-CCL2 axis. Collectively, these studies demonstrate a previously unrecognized role for TTP in regulating age-associated myelopoiesis through the expansion of specific myeloid progenitors and M-MDSCs and their recruitment to sites of injury, inflammation, or other pathologic perturbations.

Aging Affects the Role of Myeloid-Derived Suppressor Cells in Alloimmunity.

Myeloid-derived suppressor cells (MDSC) are defined as a group of myeloid cells with potent immunoregulatory functions that have been shown to be involved in a variety of immune-related diseases including infections, autoimmune disorders, and cancer. In organ transplantation, MDSC promote tolerance by modifying adaptive immune responses. With aging, however, substantial changes occur that affect immune functions and impact alloimmunity. Since the vast majority of transplant patients are elderly, age-specific modifications of MDSC are of relevance. Furthermore, understanding age-associated changes in MDSC may lead to improved therapeutic strategies. Here, we provide a comprehensive update on the effects of aging on MDSC and discuss potential consequences on alloimmunity.

Glucocorticoid receptor modulates myeloid-derived suppressor cell function via mitochondrial metabolism in immune thrombocytopenia.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells and natural inhibitors of adaptive immunity. Intracellular metabolic changes in MDSCs exert a direct immunological influence on their suppressive activity. Our previous study demonstrated that high-dose dexamethasone (HD-DXM) corrected the functional impairment of MDSCs in immune thrombocytopenia (ITP); however, the MDSC population was not restored in nonresponders, and the mechanism remained unclear. In this study, altered mitochondrial physiology and reduced mitochondrial gene transcription were detected in MDSCs from HD-DXM nonresponders, accompanied by decreased levels of carnitine palmitoyltransferase-1 (CPT-1), a rate-limiting enzyme in fatty acid oxidation (FAO). Blockade of FAO with a CPT-1 inhibitor abolished the immunosuppressive function of MDSCs in HD-DXM responders. We also report that MDSCs from ITP patients had lower expression of the glucocorticoid receptor (GR), which can translocate into mitochondria to regulate the transcription of mitochondrial DNA (mtDNA) as well as the level of oxidative phosphorylation. It was confirmed that the expression of CPT-1 and mtDNA-encoded genes was downregulated in GR-siRNA-treated murine MDSCs. Finally, by establishing murine models of active and passive ITP via adoptive transfer of DXM-modulated MDSCs, we confirmed that GR-silenced MDSCs failed to alleviate thrombocytopenia in mice with ITP. In conclusion, our study indicated that impaired aerobic metabolism in MDSCs participates in the pathogenesis of glucocorticoid resistance in ITP and that intact control of MDSC metabolism by GR contributes to the homeostatic regulation of immunosuppressive cell function.

Immunosuppressive activity is attenuated by Astragalus polysaccharides through remodeling the gut microenvironment in melanoma mice.

Astragalus polysaccharides (APS), the main effective component of Astragalus membranaceus, can inhibit tumor growth, but the underlying mechanisms remain unclear. Previous studies have suggested that APS can regulate the gut microenvironment, including the gut microbiota and fecal metabolites. In this work, our results showed that APS could control tumor growth in melanoma‐bearing mice. It could reduce the number of myeloid‐derived suppressor cells (MDSC), as well as the expression of MDSC‐related molecule Arg‐1 and cytokines IL‐10 and TGF‐β, so that CD8+ T cells could kill tumor cells more effectively. However, while APS were administered with an antibiotic cocktail (ABX), MDSC could not be reduced, and the growth rate of tumors was accelerated. Consistent with the changes in MDSC, the serum levels of IL‐6 and IL‐1β were lowest in the APS group. Meanwhile, we found that fecal suspension from mice in the APS group could also reduce the number of MDSC in tumor tissues. These results revealed that APS regulated the immune function in tumor‐bearing mice through remodeling the gut microbiota. Next, we focused on the results of 16S rRNA, which showed that APS significantly regulated most microorganisms, such as Bifidobacterium pseudolongum, Lactobacillus johnsonii and Lactobacillus. According to the Spearman analysis, the changes in abundance of these microorganisms were related to the increase of metabolites like glutamate and creatine, which could control tumor growth. The present study demonstrates that APS attenuate the immunosuppressive activity of MDSC in melanoma‐bearing mice by remodeling the gut microbiota and fecal metabolites. Our findings reveal the therapeutic potential of APS to control tumor growth.

Tumor-related HSP70 released after cryo-thermal therapy targeted innate immune initiation in the antitumor immune response

Purpose In our previous study, a novel cryo-thermal therapy that could stimulate the maturation of innate immune cells to subsequently activate the CD4+Th1 cell-dominated antitumor response was developed. However, why cryo-thermal therapy can induce the maturation of innate immunity remains unknown. Methods In this study, western blot and ELISA were used to analyze the levels of damage-associated molecular patterns (DAMPs, including heat shock protein 70 (HSP70), calreticulin and high-mobility group box protein 1) in situ and in the peripheral blood at different times after cryo-thermal therapy or traditional radiofrequency ablation. The effects of these three DAMPs on myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs) and macrophages were investigated by antibody neutralization in vitro. The phenotypic and functional changes in MDSCs, DCs and macrophages were analyzed using FACS and qRT-PCR. An anti-HSP70 antibody was injected intravenously at 6 h after cryo-thermal therapy on days 1 and 2 and mouse survival was monitored. Results Cryo-thermal therapy could trigger the release of DAMPs in situ and in the peripheral circulation, which could downregulate the proportion and suppressive signature of MDSCs, and promote the M1 macrophages polarization and DCs maturation. Among three DAMPs, HSP70 played the most evident role in M1 macrophage polarization. In vivo neutralization of HSP70 in the early stage of treatment could significantly decrease the survival rate of cryo-thermal therapy treated mice. Conclusions Local cryo-thermal therapy not only destroyed solid tumors thermally and mechanically but also induced the release of a large amount of DAMPs to effectively trigger a systemic antitumor response.

Crosstalk between myeloid-derived suppressor cells and the immune system in prostate cancer: MDSCs and immune system in Prostate cancer.

Prostate cancer is the second most common cancer and the fifth leading cause of cancer-associated death in men. Previous studies have revealed a surprising ability for an immature population of myeloid cells called myeloid-derived suppressor cells (MDSCs) in the commencement and development of many tumors, including those of prostate cancer. Herein, the molecular and cellular changes of MDSCs in prostate cancer in both human and nonhuman models are reviewed. The suppressive function of MDSCs are also discussed with a particular focus on the role of IL-6 and JAK/STAT3 signaling pathways in the induction of their suppressive activity. Ultimately, a brief review of MDSC-targeting approaches for potential cancer therapy is presented.

Dynamic changes of myeloid-derived suppressor cells and regulatory T cells in livers of mice infected with Echinococcus granulosus

To investigate the dynamics changes of the myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells in mice infected with Echinococcus granulosus and explore the possible biological significance. Thirty female BALB/c mice of 6 weeks old were randomly divided into the infection and control groups, of 15 mice in each group. Mice in the infection group were intraperitoneally injected with 2 000 E. granulosus protoscoleces, while those in the control group were injected with the same volume of physiological saline. Mouse liver white blood cells were harvested 3 (early stage), 6 (medium stage) and 12 months (late stage) post-infection, and the proportions of MDSCs, their subpopulations (M-MDSCs and PMN-MDSCs) and Treg cells were assessed by flow cytometry. The proportions of MDSCs were (1.61 ± 0.36)%, (5.68 ± 0.69)% and (16.18 ± 0.69)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection with E. granulosus, and (2.19 ± 0.42)%, (0.99 ± 0.07) % and (4.18 ± 0.84)% in the control group, and there were significant differences in the proportion of the MDSCs in mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection (P < 0.01). The proportions of M-MDSCs were (0.69 ± 0.27)%, (5.30 ± 0.72)% and (10.75 ± 0.29)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection, and (0.42 ± 0.24)%, (0.69 ± 0.02)% and (2.12 ± 0.13)% in the control group, and there were significant differences in the proportion of the M-MDSCs in the mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection (P < 0.01). The proportions of PMN-MDSCs were (0.93 ± 0.23)%, (0.32 ± 0.02)% and (5.14 ± 1.03)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection, and (1.77 ± 0.26)%, (0.28 ± 0.05)% and (1.99 ± 0.90)% in the control group, and there were significant differences in the proportion of PMN-MDSCs in mouse liver white blood cells between the infection and control groups 3 and 12 months post-infection (P < 0.05). The proportions of Treg cells were (3.35 ± 0.14)%, (6.24 ± 0.38)% and (3.41 ± 0.07)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection, and (3.48 ± 0.46)%, (3.65 ± 0.45)% and (3.12 ± 0.12)% in the control group, and there were significant differences in the proportion of Treg cells in mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection (P < 0.01). The percentages of both MDSCs and Treg cells increase in mouse liver white blood cells 6 and 12 months post-infection with E. granulosus, and a more remarkable increase is seen in the percentage of MDSCs, which is mainly found in M-MDSCs. These findings suggest that M-MDSCs may play a major immunosuppressive role in the medium and late stages of E. granulosus infection in mice.

Characterization of myeloid-derived suppressor cells and cytokines GM-CSF, IL-10 and MCP-1 in dogs with malignant melanoma receiving a GD3-based immunotherapy

Melanoma in humans and canines is an aggressive and highly metastatic cancer. The mucosal forms in both species share genetic and histopathologic features, making dogs a valuable spontaneous disease animal model. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin with immunosuppressive capabilities, which are increased in many human cancers and contribute to tumor immune evasion. They are a possible target to improve immunotherapy outcomes. Current information regarding MDSCs in canines is minimal, limiting their use as translational model for the study of MDSCs. The objective of this study was to characterize major MDSCs subsets (monocytic and polymorphonuclear) and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10) and monocyte chemoattractant protein-1 (MCP-1) in canines with malignant melanoma and to evaluate changes in MDSCs and the cytokines over time in response to a GD3-based active immunotherapy. Whole blood and serum collected from 30 healthy controls and 33 patients enrolled in the University of Florida melanoma vaccine trial were analyzed by flow cytometry with canine specific CD11b, MHCII and anti-human CD14 antibodies to assess ostensibly polymorphonuclear-MDSC (CD11b+ MHCII− CD14−) and monocytic-MDSC (CD11b+ MHCII− CD14+) subsets. IL-10, MCP-1 and both MDSCs subsets were significantly elevated in melanoma dogs versus controls. Both MDSCs subsets decreased significantly following GD3-based immunotherapy administration but no significant changes in cytokines were seen over time. To our knowledge, this is the first report documenting increased monocytic-MDSCs in canine melanoma. This is consistent with human malignant melanoma data, supporting dogs as a valuable model for therapeutic intervention studies.

Baicalein ameliorates pristane-induced lupus nephritis via activating Nrf2/HO-1 in myeloid-derived suppressor cells

IntroductionLupus nephritis (LN) is a representative manifestation in systemic lupus erythematosus (SLE). Some studies have shown that myeloid-derived suppressor cells (MDSCs) play a vital role in the regulation of the SLE process. MDSC infiltration in the kidney as well as inflammation and oxidative stress provokes the acceleration and deterioration of LN. Nuclear factor E2-related factor 2 (Nrf2) is thought to be a major regulator of the antioxidant response. Baicalein is a flavonoid with known anti-inflammatory effects and antioxidant response. However, the effects of baicalein on MDSCs, inflammation, and oxidative stress are not evaluated in the development of pristane-induced LN in mice.MethodsThe renoprotective effect of baicalein was detected in a pristane-induced lupus mice model. NLRP3 inflammasome activation and NF-κB phosphorylation as well as reactive oxygen species (ROS) production and Nrf2 activation were examined. The percentages and function changes of MDSCs were measured. The possible mechanisms of the underlying effects of baicalein on ROS production and signaling pathways of Nrf2/heme-oxygenase (HO)-1, NLRP3 inflammasome, and NF-κB phosphorylation in lipopolysaccharide (LPS)-primed MDSCs were analyzed.ResultsBaicalein reduced proteinuria and attenuated renal function impairment and renal histopathology including intrinsic cell proliferation, cellular crescents, and podocyte injury as well as glomerulonephritis activity in lupus mice. Moreover, baicalein downregulated the activation of NLRP3 inflammasome and levels of ROS or NF-κB phosphorylation, and it enhanced Nrf2 activation. Of note, baicalein inhibited the expansion of MDSCs and improved the function of MDSCs in lupus mice. Through analyzing LPS-primed MDSCs in vitro, baicalein was found to exhibit cytoprotective effects coincident with the induction of Nrf2/HO-1 signaling and the suppression of the NLRP3 inflammasome.ConclusionThe data show that baicalein alleviates the symptoms of pristane-induced LN and suggest that the alleviation may be attributed to inhibition of MDSC expansion and regulation of the balance of the Nrf2/HO-1 signal and NLRP3 expression in MDSCs.

Abstract B191: The impact of postoperative myeloid-derived suppressor cells on prognosis of gastric cancer patients

Abstract Background and Aim: Myeloid cells such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and neutrophils have a suppressive role in the antitumor immunity. In gastric cancer (GC) patients, it has been reported that these myeloid cells in peripheral blood or tumor tissue were associated with their prognosis and increased after surgical treatments. Some reported that the tumor progression and frequent recurrence were caused by increased MDSC after surgical stress in a mouse model. However, in human GC patients, it is still unclear whether such an increase of myeloid cells after gastrectomy is related to their poor prognosis. In this study, we investigate the relationship between the increase of myeloid cells after surgery and prognosis in gastric cancer patients. Materials and Methods: For analysis of relationship between increased myeloid cells after surgery and patients’ prognosis, general archival data were obtained from 278 pStgae2-3 GC patients who received curative resection between January 2007 and December 2014. For analysis of relationship between perioperative changes of MDSCs and patients’ clinicopathologic data, peripheral blood was obtained from 75 GC patients who underwent gastrectomy between April 2016 and December 2017, and MDSCs, CD8, CD4 T-cells, and regulatory T-cells (Tregs) were analyzed by flow cytometry. For analysis of the immunosuppressive function, MDSCs were purified by FACS Aria II and the IFNγ secretion assay were performed using activated autologous T-cells as responder cells. Result and Discussion: In the analysis with 278 GC patients, short RFS was significantly correlated with increased number of monocytes after surgery (#monocytes) (P=0.0073), but not increased number of neutrophils (#neutrophils) (P=0.26). A multivariate analysis demonstrated that #monocytes (HR 1.49; 95% CI 1.01-2.21), pT (HR 2.14; 95% CI 1.26-3.83), pN (HR 2.98; 95% CI 1.81-5.18), postoperative complications (HR 1.69; 95% CI 1.03-2.68) were independently associated with RFS. In the analysis with 75 GC patients, preoperative % M-MDSCs (CD11b+ CD33+ HLA-DR- in CD14+ PBMCs) was positively associated with pathologic stages. After gastrectomy, % M-MDSCs dramatically increased in most patients. On the other hand, % Foxp3+ CD25+ in CD4+ increased but not significantly, while % CD4 and %CD8 in CD3+ were stable. By the suppression assay, smaller number of IFNγ-secreting responder cells were observed when they were co-cultured with M-MDSCs than with CD11b+ CD33+HLA-DR+ cells. Because the increase in the number of M-MDSCs (#M-MDSCs) was positively correlated with #monocytes (r2=0.57 P&amp;lt;0.0001), #M-MDSCs after surgery might be related to poor prognosis of GC patients. Conclusion: The larger number of monocytes after gastrectomy was independently associated with poorer prognosis. The immunosuppressive effect of M-MDSCs increased after surgery could be one of the reasons for the early relapse in gastric cancer patients. Citation Format: Shinya Urakawa, Hisashi Wada, Masaki Mori, Yuichiro Doki. The impact of postoperative myeloid-derived suppressor cells on prognosis of gastric cancer patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B191.

Ibrutinib Treatment Reduces Myeloid Derived Suppressor Cell Numbers and Function in Chronic Lymphocytic Leukemia

In chronic lymphocytic leukemia (CLL), bidirectional interactions of leukemic B cells with components of a complex, yet incompletely defined tumor microenvironment (TME) are critical for leukemic cell survival and proliferation. Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, blocks signals that are crucial for survival of CLL cells which are delivered by the B cell receptor (BCR) and certain other receptors. However since BTK and its family members are expressed by other cell types, ibrutinib can also affect non-leukemic cells, thereby altering their function. Here, we focused on understanding how myeloid-derived suppressor cells (MDSCs), a non-leukemic cell type within the TME, and their main target, T cells, are affected by ibrutinib therapy.Using blood cells from a set of 20 previously untreated patients receiving ibrutinib, we analyzed circulating MDSCs and their subsets 15 days before and 1, 2 and 3 months after treatment initiation. As anticipated, at the first month time point the absolute CLL B-cell count increased significantly (P=0.024), followed by a progressive reduction at consecutive time points (P<0.001). In contrast, absolute MDSC numbers slowly and continuously decreased over time, achieving a significant reduction by the third month (P=0.009). When dividing MDSCs into their cellular subsets, the same pattern was observed for granulocyte-like MDSCs (gMDSCs) (P=0.005) but not for monocyte-like MDSCs (mMDSCs); for the latter an insignificant change was found. Of note, when comparing the changes of MDSC and gMDSC numbers to that of CLL B-cell counts, MDSC and gMDSC neared statistical significance at month one (P<0.1) and achieved significance at the second month (MDSCs: P<0.001; gMDSCs: P=0.033).We also observed differences in T-cell subpopulations shortly after ibrutinib treatment began. T cell counts increased significantly at the second month compared to pre-treatment (P=0.024); this was the case for both CD4+ and CD8+ cells (P = 0.022 and 0.010, respectively). In addition, CD8+ T cells maintained significance through the third month (P = 0.033). When exploring T cell subsets defined by cytokine production, we observed a spike at the second month of CD4+IL17F+ and of CD8+IL17F+, CD8+IL17A+ and CD8+FoxP3+ T cells. However, by the 3rd month, only the IFNγ-producing subset of CD4+ and CD8+ T cells were significantly higher (P = 0.049 and 0.042, respectively).Next we analyzed the function of gMDSCs and mMDSCs in the presence of ibrutinib in vitro, addressing the two main effects of these cells on T lymphocytes: suppression of T-cell proliferation and modulation of T-cell differentiation. Specifically, ibrutinib did not directly reduce T-cell expansion in the absence of MDSCs and did not alter the effect of CLL gMDSCs nor mMDSCs on T-cell proliferation, since the significant reduction induced by gMDSCs (P=0.047) and the insignificant, variable effect of mMDSCs were unchanged by the drug. However when we analyzed the influence of gMDSCs, mMDSCs and monocytes on naïve CD4+ T-cell differentiation in the presence or absence of ibrutinib, to our surprise the only T cell subpopulation directly compromised by ibrutinib was the Th1/IFNγ-producing subset. This was opposite that observed in co-cultures with gMDSCs, mMDSCs or normal monocytes in the presence of ibrutinib where Th1 cells expanded significantly (P= 0.021, 0.010, and 0.005, respectively). In the latter co-cultures not containing ibrutinib, more IL-4-producing (Th2) cells were found. Additionally, ibrutinib had a positive effect on IL-22+ (Th22) and FoxP3+ (Treg) cell numbers in the presence of MDSCs and monocytes.In summary, opposite to what we observed for CLL B and T cells, MDSC counts fell progressively after initiating ibrutinib therapy, possibly due to a direct effect of ibrutinib on BTK in MDSCs or an indirect effect mediated by diminishing signals from CLL B cells that would normally be delivered after BCR engagement. Ibrutinib did not directly alter the T cell suppressive ability of MDSCs, but it did skew T-cell differentiation to Th1 cells when MDSCs were present, in line with our finding higher Th1 cells in the blood after 3 months of treatment. Thus over time, ibrutinib shifts the CLL TME from an immunosuppressive to a more immune effective one. DisclosuresChen:Beigene: Research Funding; Verastem: Research Funding; Pharmacyclics: Research Funding. Barrientos:Pharmacyclics, an AbbVie Company: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Janssen: Consultancy. Kolitz:Magellan Health: Consultancy, Honoraria. Rai:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

Dynamics of CD25 Positive Myeloid Derived Suppressor Cells in Solid Organ Transplanted Patients and Relationship with Rejection

Backgound Myeloid derived suppressor cells (MDSC) are immature cells with immunosuppressive capacities. Three MDSC subsets are currently defined: monocytic, early stage, and polymorphonuclear MDSC (M-MDSC, eMDSC and PMN-MDSC respectively). While MDSC increase in cancer and chronic infections and associate with poor prognosis, their role in transplantation (Tx) is unknown. Changes in MDSC in transplant patients could provide biomarkers of graft evolution, and they could be useful as immunosuppressive therapy and/or to stimulate the allograft tolerance. Methods Peripheral blood MDSC were identified in cohorts of kidney (n=164) and liver (n=30) recipients and healthy volunteers (HV) as CD33+CD11b+HLA-DRlo/-. We characterized CD14+CD15- (M-MDSC) and CD14-CD15- (eMDSC) subsets. Suppression assays and measurement of surface CD25 expression (MFI) on MDSC were performed. Results Renal recipients MDSC were able to suppress CD4 and CD8 T cell proliferation in vitro. MDSC % were similar in pre-transplant, kidney transplant recipients (KTR), than in HV. However, MDSC and M-MDSC increased, mainly at 7 and 14 days post-transplant (3.48% and 3.85% vs 1.14%; 2.47% and 2.48% vs 0.24% p≤0,001 vs pre-transplant). The increase of MDSC in KTR with basiliximab as induction therapy was lower than in patients induced with thymoglobulin or without induction, and eMDSC were particularly decreased in basiliximab-treated patients (vs no induction, p≤0.05; vs thymoglobulin, p≤0.05). We confirmed that eMDSC expressed CD25, target of basiliximab. Pre-Tx, liver transplant recipients (LTR) had higher % of CD25+ eMDSC and higher CD25 MFI than KTR and HV. In LTR and KTR without basiliximab, % and MFI of CD25 in eMDSC increased after transplant. In KTR cohort, 7% of patients suffered acute rejection (AR). Absolute numbers of MDSC at 7 days post-transplant (measured before the rejection event) were significantly lower in AR than in non-rejectors (25.84 cells/uL vs 53.18 cells/uL respectively, p≤0,05). Conclusions MDSC and M-MDSC increase post-Tx except in patients receiving basiliximab. MDSC were lower in AR patients and low MDSC counts significantly preceded rejection. Transplant recipients eMDSC express CD25 which upregulates after Tx. Whether eMDSC express a complete and functional IL-2R is under investigation.