Presence Of Myeloid-derived Suppressor Cells Research Articles

Augmentation of tumor expression of HLA-DR, CXCL9, and CXCL10 may improve olfactory neuroblastoma immunotherapeutic responses

BackgroundOlfactory neuroblastoma is a rare malignancy of the anterior skull base typically treated with surgery and adjuvant radiation. Although outcomes are fair for low-grade disease, patients with high-grade, recurrent, or metastatic disease oftentimes respond poorly to standard treatment methods. We hypothesized that an in-depth evaluation of the olfactory neuroblastoma tumor immune microenvironment would identify mechanisms of immune evasion in high-grade olfactory neuroblastoma as well as rational targetable mechanisms for future translational immunotherapeutic approaches.MethodsMultispectral immunofluorescence and RNAScope evaluation of the tumor immune microenvironment was performed on forty-seven clinically annotated olfactory neuroblastoma samples. A retrospective chart review was performed and clinical correlations assessed.ResultsA significant T cell infiltration was noted in olfactory neuroblastoma samples with a stromal predilection, presence of myeloid-derived suppressor cells, and sparse natural killer cells. A striking decrease was observed in MHC-I expression in high-grade olfactory neuroblastoma compared to low-grade disease, representing a mechanism of immune evasion in high-grade disease. Mechanistically, the immune effector stromal predilection appears driven by low tumor cell MHC class II (HLA-DR), CXCL9, and CXCL10 expression as those tumors with increased tumor cell expression of each of these mediators correlated with significant increases in T cell infiltration.ConclusionThese data suggest that immunotherapeutic strategies that augment tumor cell expression of MHC class II, CXCL9, and CXCL10 may improve parenchymal trafficking of immune effector cells in olfactory neuroblastoma and augment immunotherapeutic responses.

477 Evaluating the therapeutic efficacy of combination IL-12 and trabectedin for the treatment of triple negative breast cancer

OBJECTIVES/GOALS: IL-12 has potent immune effects but the presence of myeloid-derived suppressor cells (MDSC) can inhibit IL-12-induced NK cell cytotoxicity. Thus, we hypothesized that combining IL-12 with trabectedin, an immunosuppressive myeloid cell depleting agent, would improve its therapeutic efficacy in triple negative breast cancer (TNBC). METHODS/STUDY POPULATION: Combination IL-12 and trabectedin was tested in the 4T1 mouse model of TNBC. 4T1 cells were injected into the mammary fat pad of female BALB/cj mice. When tumors reached 50 mm3, mice were randomly divided into 4 groups and treated with PBS, IL-12 (0.5 μg/mouse 3x/wk), 0.15 mg/kg trabectedin weekly or the combination. Tumor volumes were measured by calipers. Mass cytometry was performed on spleens and tumors using a 35-antibody panel. Plasma IFN-γ levels were measured by ELISA. The role of NK cells was evaluated via depletion with anti-asialo-GM1. The Luminex Discovery Assay was used to measure plasma cytokines and immunohistochemistry was performed for CD4 and CD8a. Linear/nonlinear mixed effects modeling was used for in vivo data analysis and applicable t- or ANOVA tests were used for in vitrodata analysis. RESULTS/ANTICIPATED RESULTS: Combination IL-12 and trabectedin led to a significant reduction in tumor burden compared to single-agent IL-12, trabectedin and control treatments (all p<0.001), as well as higher levels of IFN-γ (all p<0.04). One combination treated mouse had complete tumor regression. Splenic MDSC were significantly decreased in combination treated mice. NK depletion abrogated the effects of combination therapy. Compared to mice receiving a control antibody, NK depletion of combination treated mice led to lower levels of CCL5 (p<0.01) and CXCL10 (p<0.001) and significantly higher tumor burden (p=0.001). CD8+T cell levels were significantly higher in combination treated mice compared to those receiving IL-12 (p<0.01), and these levels were decreased when mice were depleted of NK cells (p=0.01). DISCUSSION/SIGNIFICANCE: TNBC represents 15% of all breast cancer diagnoses and is associated with a worse prognosis compared to other subtypes. Black women are twice as likely to be diagnosed with TNBC and more likely to die from disease than White women. Thus, there is an increasing need to develop additional therapeutic options for this disease.

Local administration of myeloid-derived suppressor cells prevents progression of immune-mediated dry eye disease

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature hematopoietic precursors with known immunoregulatory functions. The immunosuppressive role of MDSCs has been highlighted in several inflammatory ophthalmic disorders; however, their therapeutic application in suppressing the immune-mediated changes in dry eye disease (DED) has not been studied. We observed significant reduction in antigen presenting cell (APC) frequencies and their maturation in the presence of MDSCs. Moreover, co-culturing MDSCs with T helper 17 cells (Th17) resulted in reduced Th17 frequencies and their IL-17 expression. On the contrary, MDSCs maintained regulatory T cell frequencies and enhanced their function in-vitro. Furthermore, we delineated the role of interleukin-10 (IL-10) secreted by MDSCs in their immunoregulatory functions. We confirmed these results by flow cytometry analysis and observed that treatment with MDSCs in DED mice effectively suppressed the maturation of APCs, pathogenic Th17 response, and maintained Treg function and significantly ameliorated the disease. The results in this study highlight the potential therapeutic application of MDSCs in treating refractory DED.

Abstract 6529: Multi-lineage de-differentiation (MLDD) towards the TTF1-low phenotype is a major determinant of immune response within STK11-deficient non-small cell lung cancer

Abstract Background: The loss of serine/threonine kinase 11 (STK11) in non-small cell lung cancer (NSCLC) is associated with poor prognosis and resistance to immune checkpoint blockade (ICB) in 20-25% of cases. Experimental evidence from murine models suggests that STK11 loss contributes to lineage plasticity, while clinical observations indicate its impact on differentiation phenotypes in patients. However, the intricate interplay between differentiation states, the tumor microenvironment (TME), and clinical outcomes remains poorly elucidated. This study aims to comprehensively characterize these interactions to enhance our understanding of the complex dynamics underlying STK11-deficient NSCLC pathogenesis and therapeutic responses. Methods: By leveraging large multi-omics datasets, we already demonstrated the low immune infiltration in TTF1-low and neuroendocrine MLDD subsets of LKB1-deficient tumors. In this study, we used GEMM-derived two KPL (Kras, TP53, and STK11 mutations) and one KPL-restored line to inject subcutaneously in F1 hybrid mice of B6 and Sv129 backgrounds. Tumor size was monitored at regular intervals, and upon reaching a sufficient size, tumors were harvested and partitioned for the characterization of immune cells (T-cells and NK cells) by flow cytometry and identification of the MLDD phenotypes through gene expression analysis using Nanostring technology. Results: Mice injected with the STK11 WT line (2695L) exhibited smaller tumor volumes compared to those injected with STK11 mutant lines (2695X and 2381T4). Our comprehensive analysis, combining flow cytometry and Nanostring techniques, revealed that STK11 mutant lines displayed notably low infiltration of CD8+ T-cells, an elevated proportion of exhausted T-cells, and a manifestation of the TTF1-low immune phenotype characterized by high myeloid-derived suppressor cells (MDSCs) and increased KLRC1/KLRC2 expression. The presence of MDSCs in the STK11 mutant line correlated with pro-inflammatory cytokines but exhibited an inverse correlation with MHC-2 expression. MHC-2 expression was inversely correlated with CD8+ T-cell percentage and positively correlated with naïve T-cell percentage. Conclusions: The data obtained from the study indicates that the MDSC phenotype seems to be important for immune evasion in STK11-loss tumors. Follow-up flow cytometry and gene expression experiments with combined T-cells and myeloid/PMN MDSC-related gene panels, along with further experiments involving cIAP1/PD1 inhibitors, are imperative to substantiate and expand upon our current findings. Citation Format: Rahul Shivahare, No Joon Song, Bahareh Nourmohammadi, Jemal Imam, Olivia Sanabria, Joseph M. Amann, Qin Ma, Zihai Li, David P. Carbone, Jacob Kaufman. Multi-lineage de-differentiation (MLDD) towards the TTF1-low phenotype is a major determinant of immune response within STK11-deficient non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6529.

Nanomedicine Targeting Myeloid-Derived Suppressor Cells Enhances Anti-Tumor Immunity.

Cancer immunotherapy, a field within immunology that aims to enhance the host's anti-cancer immune response, frequently encounters challenges associated with suboptimal response rates. The presence of myeloid-derived suppressor cells (MDSCs), crucial constituents of the tumor microenvironment (TME), exacerbates this issue by fostering immunosuppression and impeding T cell differentiation and maturation. Consequently, targeting MDSCs has emerged as crucial for immunotherapy aimed at enhancing anti-tumor responses. The development of nanomedicines specifically designed to target MDSCs aims to improve the effectiveness of immunotherapy by transforming immunosuppressive tumors into ones more responsive to immune intervention. This review provides a detailed overview of MDSCs in the TME and current strategies targeting these cells. Also the benefits of nanoparticle-assisted drug delivery systems, including design flexibility, efficient drug loading, and protection against enzymatic degradation, are highlighted. It summarizes advances in nanomedicine targeting MDSCs, covering enhanced treatment efficacy, safety, and modulation of the TME, laying the groundwork for more potent cancer immunotherapy.

Sphingosine 1-phosphate / Acetyl CoA Carboxylase1 Axis regulates the Differentiation and Function of Tumor-associated Myeloid-Derived Suppressor Cells

Abstract The presence of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME) poses a serious obstacle to cancer treatment. The bioactive lysophospholipid sphingosine 1-phosphate (S1P), which is produced by sphingosine kinases (SphK1 or SphK2), regulates tumor and T cell survival and activity. Thus, it has been targeted to improve the outcome of anti-cancer therapeutics. However, S1P has not yet been investigated in terms of how it regulates myeloid cell activity. The present study demonstrates that deletion of SphK2-mediated S1P in MDSCs favorably modifies the tumor microenvironment and renders infiltrating myeloid cells with antigen presenting phenotype, leading to increased ability to control tumor growth. Furthermore, in preclinical prostate and melanoma cancer models, the combination of PD1 antibody and SphK2 pharmacological inhibitor (ABC294640) dramatically reduced tumor growth and increased the survival of tumor bearing mice. Mechanistically, the S1P pulldown and subsequent MS-based proteomics analysis revealed that S1P binds to various proteins involved in metabolic and ribosomal biogenesis pathways. Notably, both the isoforms of Acetyl CoA Carboxylase, the rate-limiting fatty acid metabolic enzyme, binds to S1P. Furthermore, investigations using deletion, overexpression, and site-directed mutagenesis demonstrated that ACC1 activity is necessary to preserve the immunogenic capacity of SphK2 KO MDSCs. This study highlights the clinical value of manipulating MDSC suppressive activity by targeting the S1P/ACC1 axis to boost T cell-mediated tumor control and highlights the crucial function that S1P plays as an immunoregulatory mediator in MDSCs. Supported grants from NIH (R01 CA250458, R01 CA236379)

Immunophenotype and function of circulating myeloid derived suppressor cells in COVID-19 patients

The pathogenesis of coronavirus disease 2019 (COVID-19) is not fully elucidated. COVID-19 is due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes severe illness and death in some people by causing immune dysregulation and blood T cell depletion. Increased numbers of myeloid-derived suppressor cells (MDSCs) play a diverse role in the pathogenesis of many infections and cancers but their function in COVID-19 remains unclear. To evaluate the function of MDSCs in relation with the severity of COVID-19. 26 PCR-confirmed COVID-19 patients including 12 moderate and 14 severe patients along with 11 healthy age- and sex-matched controls were enrolled. 10 ml whole blood was harvested for cell isolation, immunophenotyping and stimulation. The immunophenotype of MDSCs by flow cytometry and T cells proliferation in the presence of MDSCs was evaluated. Serum TGF-β was assessed by ELISA. High percentages of M-MDSCs in males and of P-MDSCs in female patients were found in severe and moderate affected patients. Isolated MDSCs of COVID-19 patients suppressed the proliferation and intracellular levels of IFN-γ in T cells despite significant suppression of T regulatory cells but up-regulation of precursor regulatory T cells. Serum analysis shows increased levels of TGF-β in severe patients compared to moderate and control subjects (HC) (P = 0.003, P < 0.0001, respectively). The frequency of MDSCs in blood shows higher frequency among both moderate and severe patients and may be considered as a predictive factor for disease severity. MDSCs may suppress T cell proliferation by releasing TGF-β.

Expression of Inhibitory Molecules (Arginase-1, IDO, and PD-L1) by Myeloid-Derived Suppressor Cells in Multiple Myeloma Patients in Remission.

We studied suppressor potential of myeloid-derived suppressor cells (MDSC) in multiple myeloma patients, including before and after mobilization of hematopoietic stem cells (HSC), by evaluating the expression of arginase-1 (Arg1), indolamine-2,3-dioxygenase (IDO), and PD-L1 in MDSC subsets. The study included 20 multiple myeloma patients in remission, 5 patients with progression, as well as 10 sex-and age-matched healthy donors. The expression of Arg1, IDO, and PD-L1 in circulating granulocytic MDSC (G-MDSC, Lin-HLA-DR-CD33+CD66b+), monocytic MDSC (M-MDSC, CD14+HLA-DRlow/-), and early-stage MDSC (E-MDSC, Lin-HLA-DR-CD33+CD66b-) was evaluated by flow cytometry. Multiple myeloma patients in remission were characterized by reduced expression of Arg1 in M-MDSC in comparison with donors. The expression of Arg1 in M-MDSC depended on the number of induction therapy lines performed and was significantly lower in patients who received ⩾2 lines and responded with remission. Patients with multiple myeloma progression (resistant to therapy) showed significantly increased expression of Arg1 and PD-L1 in M-MDSC, as well as increased expression of Arg1 in E-MDSC. After G-CSF-induced mobilization of HSC, the content of circulating Arg1-expressing M-MDSC increased significantly. Considering the presence of MDSC in apheresis products, MDSC suppressive activity is discussed as a factor affecting the outcomes of autologous HSC transplantation in multiple myeloma patients.

Monocytic-Myeloid Derived Suppressor Cells Suppress T-Cell Responses in Recovered SARS CoV2-Infected Individuals.

Coronavirus disease 2019 (COVID-19) caused by SARS Coronavirus 2 (CoV2) is associated with massive immune activation and hyperinflammatory response. Acute and severe CoV2 infection is characterized by the expansion of myeloid derived suppressor cells (MDSC) because of cytokine storm, these MDSC suppress T cell functions. However, the presence of MDSC and its effect on CoV2 antigen specific T cell responses in individuals long after first detection of CoV2 and recovery from infection has not been studied. We and others have previously shown that CD11b+CD33+CD14+HLA-DR-/lo monocytic MDSC (M-MDSC) are present in individuals with clinical recovery from viral infection. In this study, we compared the frequency, functional and transcriptional signatures of M-MDSC isolated from CoV2 infected individuals after 5-months of the first detection of the virus (CoV2+) and who were not infected with CoV2 (CoV2-). Compared to CoV2- individuals, M-MDSC were present in CoV2+ individuals at a higher frequency, the level of M-MDSC correlated with the quantity of IL-6 in the plasma. Compared to CoV2-, increased frequency of PD1+, CD57+ and CX3CR1+ T effector memory (TEM) cell subsets was also present in CoV2+ individuals, but these did not correlate with M-MDSC levels. Furthermore, depleting M-MDSC from peripheral blood mononuclear cells (PBMC) increased T cell cytokine production when cultured with the peptide pools of immune dominant spike glycoprotein (S), membrane (M), and nucleocapsid (N) antigens of CoV2. M-MDSC suppressed CoV2 S- antigen-specific T cell in ROS, Arginase, and TGFβ dependent manner. Our gene expression, RNA-seq and pathway analysis studies further confirm that M-MDSC isolated from CoV2+ individuals are enriched in pathways that regulate both innate and adaptive immune responses, but the genes regulating these functions (HLA-DQA1, HLA-DQB1, HLA-B, NLRP3, IL1β, CXCL2, CXCL1) remained downregulated in M-MDSC isolated from CoV2+ individuals. These results demonstrate that M-MDSC suppresses recall responses to CoV2 antigens long after recovery from infection. Our findings suggest M-MDSC as novel regulators of CoV2 specific T cell responses, and should be considered as target to augment responses to vaccine.

Abstract 3142: BRMS1 alters the tumor inflammatory signature and immune infiltration in lung adenocarcinoma

Abstract Background: Lung adenocarcinoma (LUAD) is the leading cause of cancer-related deaths. Metastatic cancer is highly fatal which contributes to at least 90% of cancer-associated morbidities and mortalities. During metastasis, tumor cells alter their signaling cascade and interact with the immune cells in the tumor microenvironment Therefore, it is necessary to characterize and understand how these cancer cells influence tumor-infiltrating immune cells to facilitate and even enhance their ability to metastasize. BRMS1 is a metastasis suppressor gene which is frequently downregulated in LUAD. We, and others, have shown that loss of BRMS1 results in distant metastatic disease. Given the strong correlation between immune suppression, metastasis, and therapy resistance we sought to elucidate if BRMS1 contributes to these processes. Specifically, we hypothesized that LUAD cells downregulate BRMS1 which influences the immune cell composition in the tumor. This creates an immune-suppressive tumor microenvironment aiding in metastasis. Methods: To test our hypothesis, we generated KrasG12D P53fl/fl Brms1-/- mice which spontaneously develop tumors with Ad-Cre intratracheal inoculation. To elucidate the role of BRMS1 in the context of cancer, we performed RNA sequencing on tumors from Brms1 wild type and knockout background. We used MCP counter, an in silico cellular deconvolution algorithm on our bulk RNA seq data to identify various subsets of tumor-infiltrating immune cells. Furthermore, our observations were validated by immunofluorescence and flow cytometry. Results: Differential gene expression analysis using RNA seq data revealed a reduction in CXCL9, CCL5, CLEC1B, CCL9, CCL7 and other proinflammatory molecules in the Brms1-/- tumors. Gene ontology and gene set enrichment analysis highlighted a diminished immune response signature in Brms1-/- tumors. Hallmarks like interferon and IL6 signaling, complement and inflammation are highly enriched in Brms1+/+ tumors. MCP counter also suggested a significant increase in NK cells and reduction in CD8+ T cell population in the Brms1-/- tumors. To validate our observation, we performed immunofluorescence and flow cytometry to assess the number of infiltrating immune cells in the tumor microenvironment. Immunofluorescence data showed reduced cytotoxic T cells (CD8+ T cells) in Brms1-/- mice. The flow cytometric analysis also revealed a reduction in the proliferative potential of these CD8+ T cells along with the increased presence of myeloid-derived suppressor cells in the Brms1-/- mice. Conclusion: Our data suggest for the first time that reduced BRMS1 expression which is generally observed in multiple tumors not only influences metastasis but also alters tumor-infiltrating immune cell composition. Thus, BRMS1 downregulation presents as one of the potential immune evasion mechanisms that could be targeted for an improved therapy outcome in LAUD. Citation Format: Manendra B. Lankadasari, Yuan Liu, Di He, Samhita Bapat, Brooke Mastrogiacomo, Harry B. Lengel, David R. Jones. BRMS1 alters the tumor inflammatory signature and immune infiltration in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3142.

Inhibition of myeloid-derived suppressive cell function with all-trans retinoic acid enhanced anti-PD-L1 efficacy in cervical cancer

PD-1/PD-L1 inhibitor treatments are relatively inefficacious in advanced cervical cancer patients. The presence of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment may be one significant barrier to efficacy. It has been shown that all-trans retinoic acid (ATRA) can differentiate MDSCs into mature myeloid cells. However, whether ATRA suppression of MDSCs function could enhance PD-L1 blockade-mediated tumor immunotherapy remains unknown. Here, the frequency of tumor-infiltrating MDSCs in cervical cancer patients was measured. ATRA was used to target MDSCs both in vitro and in tumor-bearing mice. The impact of ATRA on the human cell line HeLa was also investigated. The frequency of MDSCs and T cells was determined by flow cytometry. The expression of immunosuppressive genes was measured with quantitative real time-PCR and infiltration of immune cells was assessed by immunohistochemical examination. We found that tumor-infiltrating PD-L1+ MDSCs were more prevalent in cervical cancer patients. Blockade of PD-L1 expression in MDSCs with anti-PD-L1 antibody cannot relieve the suppressive activity of MDSCs induced by HeLa cells, while ATRA efficiently abrogated the suppressive activity of MDSCs. Furthermore, ATRA had no effect on PD-L1 expression in HeLa cells in vitro. In in vivo treatment, ATRA decreased MDSCs accumulation and increased the frequency of CD8+ T cells in BALB/C mice with U14 cervical tumors. Importantly, a combination treatment of ATRA and anti-PD-L1 antibody further delayed U14 tumor growth and increased the proportion of CD62L−CD8+ T cells, CD62L−CD4+ T cells, CD107a+CD8+ T cells as well as IFN-γ and TNF-α levels in tumors. Our results provide a rationale for the use of ATRA to suppress MDSCs and enhance anti-PD-L1 cancer immunotherapy in cervical cancer.

Analysis of the myeloid-derived suppressor cells and annexin A1 in multibacillary leprosy and reactional episodes

BackgroundLeprosy is a chronic infectious disease caused by Mycobacterium leprae. Patients have distinct clinical forms, and the host´s immunological response regulate those manifestations. In this work, the presence of the myeloid-derived suppressor cell and the regulatory protein annexin A1 is described in patients with multibacillary leprosy and with type 1 and 2 reactions.MethodsPatients were submitted to skin biopsy for histopathological analysis to obtain a bacilloscopic index. Immunofluorescence was used to detect myeloid-derived suppressor cells and annexin A1.ResultsThe data demonstrated that the presence of granulocytic and monocytic myeloid-derived suppressor cells in leprosy patients. A high number of monocytic myeloid-derived suppressor cells were observed in lepromatous leprosy and type 2 reactional patients. The presence of annexin A1 was observed in all myeloid-derived suppressor cells. In particular, the monocytic myeloid-derived suppressor cell in the lepromatous patients has higher levels of this protein when compared to the reactional patients. This data suggest that the higher expression of this protein may be related to regulatory response against a severe infection, contributing to anergic response. In type 1 reactional patients, the expression of annexin A1 was reduced.ConclusionsMyeloid-derived suppressor cell are present in leprosy patients and annexin A1 might be regulated the host response against Mycobacterium leprae.

The Impact of NK Cell-Based Therapeutics for the Treatment of Lung Cancer for Biologics: Targets and Therapy.

Lung cancer has a dismal prognosis and novel targeted therapies leave still room for major improvements and better outcomes. Immunotherapy targeting immune checkpoint (IC) proteins, either as single agents or in combination with chemotherapy, is active but responders constitute only approximately 10–15% of non-small cell lung cancer (NSCLC) patients. Other effector immune cells such as CAR-T cells or NK cells may help to overcome the limitations of the IC inhibitor therapies for lung cancer. NK cells can kill tumor cells without previous priming and are present in the circulatory system and lymphoid organs. Tissue-residing NK cells differ from peripheral effector cells and, in case of the lung, comprise CD56bright CD16-negative populations showing high cytokine release but low cytotoxicity in contrast to the circulating CD56dim CD16-positive NK cells exhibiting high cytotoxic efficacy. This local attenuation of NK cell killing potency seems due to a specific stage of NK differentiation, immunosuppressive factors as well as presence of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (TREGs). Improved NK cell-based immunotherapies involve IL-2-stimulated effector cells, NK cells expanded with the help of cytokines, permanent NK cell lines, induced pluripotent stem cell-derived NK cells and NK cells armed with chimeric antigen receptors. Compared to CAR T cell therapy, NK cells administration is devoid of graft-versus-host disease (GvHD) and cytokine-release syndrome. Although NK cells are clearly active against lung cancer cells, the low-cytotoxicity differentiation state in lung tumors, the presence of immunosuppressive leucocyte populations, limited infiltration and adverse conditions of the microenvironment need to be overcome. This goal may be achieved in the future using large numbers of activated and armed NK cells as provided by novel methods in NK cell isolation, expansion and stimulation of cytotoxic activity, including combinations with monoclonal antibodies in antibody-dependent cytotoxicity (ADCC). This review discusses the basic characteristics of NK cells and the potential of NK cell preparations in cancer therapy.

Correlation of Myeloid Derived Suppressor Cells and Response to Standard Chemoimmunotherapy in Diffuse Large B Cell Lymphoma

INTRODUCTION: Immature myeloid (IM) cells have been shown to induce immune dysfunction(s) in non-Hodgkin's lymphoma (NHL) patients. Myeloid derived suppressor cells (MDSCs) are a subset of IM cells that play a key role in immune suppression associated with malignancies. In solid tumors, MDSCs have been implicated in promoting angiogenesis, tumor cell invasion and metastasis. The presence of MDSCs may correlate with decreased efficacy of immunotherapies. MDSCs have been associated with reduced survival in solid tumors. We aimed at identifying the patterns of immature myeloid cell subsets including MDSCs in DLBCL patients, pre and post-standard R-CHOP or R-EPOCH therapy.METHODS: Peripheral blood was collected on 31 patients with de novo diffuse large B cell lymphoma on an IRB approved study before and after frontline chemo-immunotherapy and at the time of relapse. Clinical variables at diagnosis (age, performance status, stage of the disease, number of extra nodal lesions) were obtained to calculate prognostic indices (IPI and R-IPI). Cell of origin was determined by IHC using the Hans algorithm. Response to therapy was assessed as per the Lugano criteria for response and assessment in lymphoma. Peripheral distribution of myeloid cells (CD11b+ CD33+), IM cells (HLA-DR-/low IL-4R+) and immunosuppressive IM cell subsets [CD14+ monocytic (m) and CD15+ granulocytic (g) myeloid-derived suppressor cells (MDSCs)] in fresh whole blood samples were established by flow cytometry.RESULTS: Median age was 65 years (range 24-88). Twenty-two (70.9%) patients had stage III/IV disease. Thirteen patients (41.9%) had GCB subtype, 16 patients (51.6%) had non-GCB subtype and cell of origin could not be determined in 2 patients (6.5%). Nineteen patients (61.3%) had IPI scores 0-2 and 12 (38.7%) patients had IPI scores of 3-5. Twenty-two patients (70.9%) received R-CHOP or R-mini CHOP, and 9 patients (29%) received R-EPOCH. The end of therapy ORR and CR were 100% and 96.7% respectively. One patient had a PR. Median follow up was 17.5 months. Two patients (6.4%) subsequently progressed. Both are alive after subsequent therapy. The overall survival is 100%. Prior to treatment initiation, patients presented with 0.23±0.05% circulating IM cells, of which 51.6±5.6% were mMDSC and 11.2±4.4% were gMDSC. No correlation between IM cell distribution (including MDSCs) and IPI or disease stage was observed. A trend was observed towards higher mMDSCs in GCB subtype compared to non-GCB subtype (3.49±1.4% vs. 1.44±0.7% of total myeloid cells; p=0.086). Following treatment with chemo-immunotherapy, overall distribution of circulating IM cells decreased to 0.05±0.04% of total WBC (p=0.012 compared with baseline), 31.2±5.6% of which were mMDSC and 21.3±3.7% were gMDSC. Normalization of IM cell distribution was independent of chemo-immunotherapy regimen or IPI. Compared to early stage disease (I/II), patients with advanced stage disease (stage III/IV) tended to retain more IM cells at the end of therapy (0.06±0.03 vs. 0.03±0.03% of total WBC; p=0.069), a significant proportion of which are mMDSC (0.36±0.25 vs. 0.25±0.07% of total myeloid cells; p=0.032). The two patients who relapsed after initial therapy displayed high proportions of MDSCs among total IM cells throughout the course of their disease compared with non-relapsing patients: at baseline (84.8-97.7% vs. 62.2±4.9% of total IM cells), post treatment (87.7-95.7% vs. 52.5±4.4% of total IM cells). This trend was conserved upon relapse with MDCSs representing 91-93% of total IM cells.CONCLUSIONS: To the best of our knowledge, this is the first study to prospectively examine immature myeloid cells and MDSCs in DLBCL at diagnosis, end of therapy and at the time of relapse. Circulating MDSCs, are associated with an active state of DLBCL. Response to chemo-immunotherapy was associated with a dramatic decrease of circulating IM cells, including MDSCs. Patients with advanced stage disease retained a higher titer of circulating mMDSC at end of therapy. For patients who relapsed, a disproportionate abundance of MDSCs (>85% of total IM cells) were seen at diagnosis, post treatment and at the time of relapse. Taken together, MDSCs may represent a potential biomarker and therapeutic target for relapsed and refractory DLBCL patients. [Display omitted] DisclosuresPark:Teva: Consultancy, Research Funding; Takeda: Research Funding; Cornerstone Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead: Speakers Bureau; Seattle Genetics: Research Funding. Ghosh:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Jassen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding, Speakers Bureau; TG Therapeutics: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Kite Pharma: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Speakers Bureau.

Overcoming the breast tumor microenvironment by targeting MDSCs through CAR-T cell therapy.

1032 Background: Successful targeting of solid tumors such as breast cancer (BC) using CAR T cells (CARTs) has proven challenging, largely due to the immune suppressive tumor microenvironment (TME). Myeloid derived suppressor cells (MDSCs) inhibit CART’s function and persistence within the breast TME. We generated CAR T cells targeting tumor-expressed mucin 1 (MUC1) (Bajgain P et al, 2018) for BC. To potentiate expansion and persistence of MUC1 CARTs and modulate the suppressive TME, we developed a novel chimeric co-stimulatory receptor, TR2.4-1BB, encoding a ScFv derived from a TNF-related apoptosis-inducing ligand receptor 2 (TR2) mAb followed by a 4-1BB endodomain. We hypothesize that engagement with TR2 expressed on TME-resident MDSCs, will lead to both MDSC apoptosis and CART co-stimulation, promoting T cell persistence and expansion at tumor site. Methods: Function of the novel TR2.4-1BB receptor, was assessed by exposing non-transduced (NT) and TR2.4-1BB transduced T cells to recombinant TR2 and nuclear translocation of NFκB was measured by ELISA. Functionality of in vitro generated MDSCs was determined by the suppression assay. In vitro CART/costimulatory receptor T cell function was measured by cytotoxicity assays using MUC1+ tumor targets in presence or absence of MDSCs. In vivo anti-tumor activity was assessed using MDSC enriched tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. Results: Nuclear translocation of NFκB was detected only in TR2.4-1BB T cells. MDSCs significantly attenuated T cell proliferation by 50±5% and IFNγ production by half compared with T cells cultured alone. Additionally, presence of MDSCs, diminished cytotoxic potential of MUC1 CARTs against MUC1+ BC cell lines by 25%. However, TR2.4-1BB expression on CAR.MUC1 T cells induced MDSC apoptosis thereby restoring the cytotoxic activity of CAR.MUC1 against MUC1+ BC lines in presence of TR2.4-1BB (67±8.5%). There was an approximate two-fold increase in tumor growth due enhanced angiogenesis and fibroblast accumulation in mice receiving tumors + MDSCs compared to tumors alone. Treatment of these MDSC-enriched tumors with MUC1.TR2.4-1BB CARTs led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared to CAR.MUC1 (469.79.9±81.46mm3) or TR2.4-1BB (434.86±64.25 mm3) T cells alone (Day 28 after T cell injection). The treatment also improved T cell proliferation and persistence at the tumor site. Thereby, leading to negligible metastasis demonstrating ability of CARTs to eliminate tumor and prevent dissemination. We observed similar results using HER2.TR2.4-1BB CARTs in a HER2+ BC model. Conclusions: Our findings demonstrate that CARTs co-expressing our novel TR2.4-1BB receptor have higher anti-tumor potential against BC tumors and infiltrating MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

Targeting of myeloid-derived suppressor cells by all-trans retinoic acid as host-directed therapy for human tuberculosis

Conventional anti-tuberculosis (TB) therapies comprise lengthy antibiotic treatment regimens, exacerbated by multi-drug resistant and extensively drug resistant mycobacterial strains. We assessed the ability of all-trans retinoic acid (ATRA), as repurposed compound serving as host-directed therapy (HDT), to counteract the suppressive effects of myeloid-derived suppressor cells (MDSCs) obtained from active TB cases (untreated or during week one of treatment) on T-cell responsiveness. We show for the first time that MDSCs suppress non-specific T-cell activation and production of interleukin (IL)-2, IL-4, IL-13 and GM-CSF via contact-dependent mechanisms. ATRA treatment decreases MDSC frequency, but fails to mature MDSCs to non-suppressive, terminally differentiated myeloid cells and does not restore T-cell function or cytokine production in the presence of MDSCs. The impact of ATRA treatment on improved immunity, using the concentration tested here, is likely to be minimal, but further identification and development of MDSC-targeting TB host-directed therapies are warranted.

The Role of Myeloid-Derived Suppressor Cells (MDSCs) in the Development and/or Progression of Endometriosis-State of the Art.

Endometriosis (EMS) is a common gynecological disease characterized by the presence of endometrial tissue outside the uterus. Approximately 10% of women around the world suffer from this disease. Recent studies suggest that endometriosis has potential to transform into endometriosis-associated ovarian cancer (EAOC). Endometriosis is connected with chronic inflammation and changes in the phenotype, activity, and function of immune cells. The underlying mechanisms include quantitative and functional disturbances of neutrophils, monocytes/macrophages (MO/MA), natural killer cells (NK), and T cells. A few reports have shown that immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) may promote the progression of endometriosis. MDSCs are a heterogeneous population of immature myeloid cells (dendritic cells, granulocytes, and MO/MA precursors), which play an important role in the development of immunological diseases such as chronic inflammation and cancer. The presence of MDSCs in pathological conditions correlates with immunosuppression, angiogenesis, or release of growth factors and cytokines, which promote progression of these diseases. In this paper, we review the impact of MDSCs on different populations of immune cells, focusing on their immunosuppressive role in the immune system, which may be related with the pathogenesis and/or progression of endometriosis and its transformation into ovarian cancer.

Hyal2 Expression in Tumor-Associated Myeloid Cells Mediates Cancer-Related Inflammation in Bladder Cancer.

The increased presence of myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in tumor tissue has been extensively reported. However, their role in the regulation of hyaluronan (HA) metabolism in the tumor microenvironment has not been established. Here we describe a novel function of tumor-associated myeloid cells related to the enhanced breakdown of extracellular HA in human bladder cancer tissue, leading to the accumulation of small HA fragments with molecular weight (MW) <20 kDa. Increased fragmentation of extracellular HA and accumulation of low molecular weight HA (LMW-HA) in tumor tissue was associated with elevated production of multiple inflammatory cytokines, chemokines, and angiogenic factors. The fragmentation of HA by myeloid cells was mediated by the membrane-bound enzyme hyaluronidase 2 (Hyal2). Increased numbers of Hyal2+CD11b+ myeloid cells were detected in the tumor tissue as well as in the peripheral blood of patients with bladder cancer. Coexpression of CD33 suggested that these cells belong to monocytic myeloid-derived suppressor cells. The HA-degrading function of Hyal2-expressing MDSCs could be enhanced by exposure to tumor-conditioned medium, and IL1β was identified as one of the factors involved in the stimulation of Hyal2 activity. CD44-mediated signaling played an important role in the regulation of HA-degrading activity of Hyal2-expressing myeloid cells, as the engagement of CD44 receptor with specific mAb triggered translocation of Hyal2 enzyme to the cellular surface and stimulated secretion of IL1β. Taken together, this work identifies Hyal2-expressing tumor-associated myeloid cells as key players in the accumulation of LMW-HA in the tumor microenvironment and cancer-related inflammation and angiogenesis. SIGNIFICANCE: This study identifies Hyal2-expressing tumor-associated myeloid cells of monocyte-macrophage lineage as contributors to hyaluronan degradation in bladder cancer tissue, leading to accumulation of inflammatory and proangiogenic low molecular weight hyaluronan fragments.

Immunosuppressive Myeloid Cells Induce Nitric Oxide-Dependent DNA Damage and p53 Pathway Activation in CD8+ T Cells.

Tumor-infiltrating myeloid-derived suppressor cells (MDSC) are associated with poor survival outcomes in many human cancers. MDSCs inhibit T cell-mediated tumor immunity in part because they strongly inhibit T-cell function. However, whether MDSCs inhibit early or later steps of T-cell activation is not well established. Here we show that MDSCs inhibited proliferation and induced apoptosis of CD8+ T cells even in the presence of dendritic cells (DC) presenting a high-affinity cognate peptide. This inhibitory effect was also observed with delayed addition of MDSCs to cocultures, consistent with functional data showing that T cells expressed multiple early activation markers even in the presence of MDSCs. Single-cell RNA-sequencing analysis of CD8+ T cells demonstrated a p53 transcriptional signature in CD8+ T cells cocultured with MDSCs and DCs. Confocal microscopy showed induction of DNA damage and nuclear accumulation of activated p53 protein in a substantial fraction of these T cells. DNA damage in T cells was dependent on the iNOS enzyme and subsequent nitric oxide release by MDSCs. Small molecule-mediated inhibition of iNOS or inactivation of the Nos2 gene in MDSCs markedly diminished DNA damage in CD8+ T cells. DNA damage in CD8+ T cells was also observed in KPC pancreatic tumors but was reduced in tumors implanted into Nos2-deficient mice compared with wild-type mice. These data demonstrate that MDSCs do not block early steps of T-cell activation but rather induce DNA damage and p53 pathway activation in CD8+ T cells through an iNOS-dependent pathway.

Increased Levels of Circulating Monocytic- and Early-Stage Myeloid-Derived Suppressor Cells (MDSC) in Acute Myeloid Leukemia.

To investigate the levels of circulating myeloid-derived suppressor cells (MDSC) in patients with primary acute myeloid leukemia (AML), and to explore the relationship between the number of MDSC and AML. Peripheral blood samples from 29 patients with primary AML and 30 healthy controls were collected. CD33, CD11b, HLA-DR, CD14, and CD15 were used to label cells, and flow cytometry was used to analyze the numbers of total MDSC and subgroups eMDSC (early-stage MDSC), M-MDSC (monocytic MDSCs), PMN-MDSC (polymorphonuclear-MDSCs) or G-MDSC (granulocytic-MDSC) via two gating strategies. Presence of MDSC in AML was determined after assessment of clinical data. Phenotypic analysis of MDSC by the two gating strategies was consistent. Compared with healthy controls, the numbers of total MDSC (CD33+CD11b+ HLA-DR-) and G-MDSC (CD33+CD11b+ HLA-DR-CD14¬-CD15+ or CD14¬-CD15+ CD11b+) in peripheral blood of AML patients were lower (p < 0.05), while numbers of M-MDSC (CD33+CD11b+ HLA-DR-CD14+CD15- or HLA-DR-/LOWCD14+) and eMDSC (CD33+CD11b+ HLA-DR-/LOWCD14-CD15-) were higher (p < 0.05). The levels of G-MDSC in peripheral blood of AML-M2 patients were higher than those in other subtypes, along with total MDSC, while the levels of eMDSC and M-MDSC in AML-M3 patients were higher than those in other subtypes. The high frequency of HLA-DR-/LOWCD14+M-MDSC and CD33+CD11b+ HLA-DR-/LOWCD14-CD15- eMDSC in peripheral blood of AML patients indicates potential for MDSC as a diagnostic index in AML.