Induction Of Suppressor Cells Research Articles

TMIC-87. ICAM-1 REGULATE EXTRACELLULAR VESICLE-MEDIATED IMMUNOSUPPRESSIVE MONOCYTE IN HUMAN GLIOBLASTOMA CELL

Abstract Glioblastoma is the most common malignant primary brain tumor and is universally fatal. Treatment involves maximal safe resection with adjuvant chemoradiation. Poor prognosis in glioblastoma is multifactorial, and local and systemic immunosuppression is an important factor. Extracellular vesicles (EVs) are small, membrane-bound particles released by cells into the external environment. They play a key role in intercellular communication. In glioblastomas, EVs regulate immunosuppression through induction of myeloid-derived suppressor cells (MDSCs) in a process dependent on the PD-1/PD-L1 and IL-6 axes. Primary cilia are microtubule-based organelles that play crucial roles in various cellular processes and signal transduction cascades. We recently showed that primary cilia modulate IL-6 expression and consequently, MDSC induction. Comparative proteomic analysis of EVs derived from human glioblastoma cells devoid of cilia showed loss of ICAM-1 expression compared to control glioblastoma cells. ICAM-1 (Intercellular Adhesion Molecule-1) is a cell surface glycoprotein that mediates cell-cell adhesion and immune responses. In this study, we showed that depletion of primary cilia in human glioblastoma cells result in increased PD-L1 but loss of IL-6 and ICAM-1. RNAi-meidated loss of ICAM-1 was synergistic with primary cilia depletion in preventing MDSC inductionn normal human monocytes exposed to glioblastoma-derived EVs. Exogenous IFN-g was sufficient to restore IL-6 levels and MDSC induction despite loss of primary cilia and ICAM-1 suggesting that primary cilia act upstream of IFN-g, IL-6 and ICAM-1 in glioblastoma-mediated MDSC induction. This study is the first to connect primary cilia signal transduction to IL-6 and ICAM-1 expression and to MDSC induction. The evidence suggests a potential parallel primary cilia regulatory cascade independent of the PD-L1 axis that controls tumor-mediated immunosuppression. These results may inform novel therapeutic strategies aimed at increasing efficacy of immunotherapy in the glioblastoma and other neoplastic processes.

Unzippable Siamese Nanoparticles for Programmed Two-Stage Cancer Immunotherapy.

Epigenetic drugs (epi-drugs) can destruct cancer cells and initiate both innate and adaptive immunity, yet they have achieved very limited success in solid tumors so far, partly attributing to their concurrent induction of the myeloid-derived suppressor cell (MDSC) population. Here, dissociable Siamese nanoparticles (SIANPs) are developed for tumor cell-targeted delivery of epi-drug CM-272 and MDSC-targeted delivery of small molecule inhibitor Ibrutinib. The SIANPs are assembled via interparticle DNA annealing and detached via tumor microenvironment-triggered strand separation. Such binary regulation induces endogenous retrovirus expression and immunogenic cell death in tumor cells while restraining the immunosuppressive effects of MDSCs, and synergistically promotes dendritic cell maturation and CD8+ T cell activation for tumor inhibition. Significantly, immune microenvironment remodeling via SIANPs further overcomes tumor resistance to immune checkpoint blockade therapy. This study represents a two-pronged approach for orchestrating immune responses, and paves a new way for employing epi-drugs in cancer immunotherapy.

TLR9 promotes monocytic myeloid-derived suppressor cell induction during JEV infection

Myeloid-derived suppressor cells (MDSCs) are a group of heterologous populations of immature bone marrow cells consisting of progenitor cells of macrophages, dendritic cells and granulocytes. Recent studies have revealed that the accumulation of MDSCs in the mouse spleen plays a pivotal role in suppressing the immune response following JEV infection. However, the mechanisms by which JEV induces MDSCs are poorly understood. Here, it was found that JEV infection induces mitochondrial damage and the release of mitochondrial DNA (mtDNA), which further leads to the activation of TLR9. TLR9 deficiency decreases the M-MDSCs population and their suppressive function both in vitro and in vivo. Moreover, the increase of MHCⅡ expression on antigen-presenting cells and CD28 expression on T cells in TLR9−/− mice was positively correlated with M-MDSCs reduction. Accordingly, the survival rate of TLR9−/− mice dramatically increased after JEV infection. These findings reveal the connections of mitochondrial damage and TLR9 activation to the induction of M-MDSCs during JEV infection.

CD44 expression on murine hepatic stellate cells promotes the induction of monocytic and polymorphonuclear myeloid-derived suppressor cells.

In chronic inflammation, regulatory immune cells, such as regulatory T cells and myeloid-derived suppressor cells, can develop. Local signals in the inflamed tissue, such as cytokines and eicosanoids, but also contact-dependent signals, can promote myeloid-derived suppressor cell development. In the liver, hepatic stellate cells may provide such signals via the expression of CD44. Myeloid-derived suppressor cells generated in the presence of hepatic stellate cells and anti-CD44 antibodies were functionally and phenotypically analyzed. We found that both monocytic and polymorphonuclear myeloid-derived suppressor cells generated in the presence of αCD44 antibodies were less suppressive toward T cells as measured by T-cell proliferation and cytokine production. Moreover, both monocytic and polymorphonuclear myeloid-derived suppressor cells were phenotypically altered. Monocytic myeloid-derived suppressor cells mainly changed their expression of CD80 and CD39, and polymorphonuclear myeloid-derived suppressor cells showed altered expression of CD80/86, PD-L1, and CCR2. Moreover, both polymorphonuclear and monocytic myeloid-derived suppressor cells lost expression of Nos2 messenger RNA, whereas monocytic myeloid-derived suppressor cells showed reduced expression of TGFb messenger RNA and polymorphonuclear myeloid-derived suppressor cells reduced expression of Il10 messenger RNA. In summary, the presence of CD44 in hepatic stellate cells promotes the induction of both monocytic and polymorphonuclear myeloid-derived suppressor cells, although the mechanisms by which these myeloid-derived suppressor cells may increase suppressive function due to interaction with CD44 are only partially overlapping.

Resveratrol Attenuates 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Mediated Induction of Myeloid-Derived Suppressor Cells (MDSC) and Their Functions.

Previously, we showed that 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) ligand and a potent and persistent toxicant and carcinogenic agent, induces high levels of murine myeloid-derived suppressor cell (MDSC) when injected into mice. In the current study, we demonstrate that Resveratrol (3,4,5-trihydroxy-trans-stilbene; RSV), an AhR antagonist, reduces TCDD-mediated MDSC induction. RSV decreased the number of MDSCs induced by TCDD in mice but also mitigated the immunosuppressive function of TCDD-induced MDSCs. TCDD caused a decrease in F4/80+ macrophages and an increase in CD11C+ dendritic cells, while RSV reversed these effects. TCDD caused upregulation in CXCR2, a critical molecule involved in TCDD-mediated induction of MDSCs, and Arginase-1 (ARG-1), involved in the immunosuppressive functions of MDSCs, while RSV reversed this effect. Transcriptome analysis of Gr1+ MDSCs showed an increased gene expression profile involved in the metabolic pathways in mice exposed to TCDD while RSV-treated mice showed a decrease in such pathways. The bio-energetic profile of these cells showed that RSV treatment decreased the energetic demands induced by TCDD. Overall, the data demonstrated that RSV decreased TCDD-induced MDSC induction and function by altering the dynamics of various myeloid cell populations involving their numbers, phenotype, and immunosuppressive potency. Because MDSCs play a critical role in tumor growth and metastasis, our studies also support the potential use of RSV to attenuate the immunosuppressive properties of MDSC.

Tumour-derived exosomes promote the induction of monocytic myeloid-derived suppressor cells from peripheral blood mononuclear cells by delivering miR-106a-5p and miR-146a-5p in multiple myeloma.

The shift of the tumour immune microenvironment to a suppressive state promotes not only the development and progression of the disease in multiple myeloma (MM) but also the development of resistance to immunotherapy. We previously demonstrated that myeloma cells can induce monocytic myeloid-derived suppressor cells (M-MDSCs) from healthy peripheral blood mononuclear cells (PBMCs) via the concomitant secretion of CC motif chemokine ligand 5 (CCL5) and macrophage migration inhibitory factor (MIF), but an unknown mediator also promotes M-MDSC induction. This study demonstrates that miR-106a-5p and miR-146a-5p delivered by tumour-derived exosomes (TEXs) from myeloma cells play essential roles in M-MDSC induction in MM. MiR-106a-5p and miR-146a-5p upregulate various immunosuppressive/inflammatory molecules in PBMCs, such as IDO1, CD38, programmed death-ligand 1, CCL5 or MYD88, which are involved in interferon (IFN)-α response, IFN-γ response, inflammatory response, tumour necrosis factor-α signalling and Interleukin-6-JAK-STAT3 signalling. These molecular features mirror the increases in myeloid cellular compartments of PBMCs when co-cultured with myeloma cells. MiR-106a-5p and miR-146a-5p have a compensatory relationship, and these two miRNAs collaborate with CCL5 and MIF to promote M-MDSC induction. Collectively, novel therapeutic candidates may be involved in TEX-mediated sequential cellular and molecular events underlying M-MDSC induction, potentially improving the efficacy of immunotherapy.

Abstract 6429: A surrogate to the anti-IL1RAP antibody nadunolimab induces tumor microenvironment changes to the metastatic lung and reduces metastatic lesions in mouse models of metastatic cancer

Abstract Interleukin-1 Receptor Accessory Protein (IL1RAP) is a coreceptor for the IL-1 receptor (IL1R1) and is required for IL-1α and IL-1β signaling. IL1RAP is expressed in the tumor microenvironment (TME); on cancer cells, stromal cells and on infiltrating immune cells in several types of cancers, including non-small cell lung cancer (NSCLC), pancreatic cancer (PDAC), triple-negative breast cancer (TNBC) and in metastatic lesions. Nadunolimab (CAN04) is a fully humanized ADCC-enhanced IgG1 antibody targeting IL1RAP and blocking both IL-1α and IL-1β signaling, currently evaluated in combination with chemotherapy in phase I/II clinical trials in NSCLC and PDAC (NCT03267316) and in TNBC (NCT05181462). Interim efficacy data are available for PDAC and NSCLC and are stronger than expected from chemotherapy alone based on historical controls; in a total of 73 PDAC patients, median iPFS is 7.2 months and median OS 12.7 months, while in a total of 30 NSCLC patients, a response rate of 53% is achieved, resulting in median PFS of 6.8 months. Microenvironmental IL1RAP-dependent IL-1 signaling contributes to tumorigenesis, tumor invasiveness and an immune-suppressive TME, partly driven by infiltration and induction of myeloid-derived suppressor cells (MDSCs). We have thus used the MDSC-rich 4T1 murine mammary tumor TNBC model to study metastatic lesions and TME modulation in 4T1 metastatic lungs upon treatment with a mouse surrogate antibody to nadunolimab. Infiltration of gMDSC/Ly6G+ cells was very prominent in metastatic lungs from mice with orthotopically implanted 4T1 breast cancer cells compared to lungs from naive mice. Interestingly, infiltrating Ly6G+ cells from metastatic lungs had a distinctly upregulated IL1RAP expression compared to Ly6G+ cells from naïve lungs. Treatment with the nadunolimab surrogate antibody significantly reduced the number of lung metastases and induced prominent changes in the lung microenvironment where nanostring analysis showed global changes in adhesion and migration-related genes, as well as genes associated with cell activation. The effect on metastasis was not confined to the 4T1 model since a reduction of metastatic tumor cells after treatment with the mouse surrogate antibody was also observed in the murine B16-F10-luc i.v. model. Together these data indicate that targeting IL1RAP is an effective way to modulate the TME and counteract the suppressive environment in metastatic tissue, and ultimately may reduce the potential for metastatic tumors in cancer patients. Citation Format: Elin Jaensson Gyllenbäck, Camilla Rydberg Millrud, Petter Skoog, Caitríona Grönberg, David Liberg. A surrogate to the anti-IL1RAP antibody nadunolimab induces tumor microenvironment changes to the metastatic lung and reduces metastatic lesions in mouse models of metastatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6429.

Abstract 682: Heterologous prime-boost viral vector vaccines for the treatment of a P1A-expressing BGL-1 glioblastoma model

Abstract Glioblastoma is an extremely aggressive and difficult cancer to treat, which may partly be due to its limited ability to induce T-cell responses. However, combining viral vector vaccines with other therapies to generate tumor-specific T cells may provide a meaningful benefit to patients. Here, we investigated whether heterologous prime-boost vaccination with chimpanzee-derived adenoviral vector ChAdOx1 and modified vaccinia Ankara (MVA) vaccines could generate therapeutically effective CD8+ T-cell responses against a model antigen P1A, a mouse homolog of human tumor-associated Melanoma Antigen GenE (MAGE)-type antigens, expressed by a BGL-1 mouse glioblastoma cell line. We demonstrated that heterologous prime-boost vaccination with ChAdOx1/MVA vaccines targeting P1A generated a high magnitude of CD8+ T cells specific for the P1A35-43 epitope presented by the MHC class I molecule H-2Ld. Prophylactic vaccination with ChAdOx1/MVA-P1A significantly prolonged the survival of syngeneic mice subcutaneously challenged with P1A-expressing BGL-1 tumors. Furthermore, different vaccination schedules significantly impact the magnitude of antigen-specific CD8+ T-cell responses and may impact protective efficacy. However, the substantial induction of myeloid-derived suppressor cells (MDSCs) by this tumor model presents a significant challenge in the therapeutic setting. Future work will investigate the efficacy of this vaccination strategy on intracranial P1A-expressing BGL-1 models. Citation Format: Emily E. Steffke, James McAuliffe, Vinnycius Pereira Almeida, Amanda Wicki, Laurine Noblecourt, Gary Kohanbash, Hideho Okada, Carol Sze Ki Leung, Masaki Terabe, Benoit J. Van Den Eynde. Heterologous prime-boost viral vector vaccines for the treatment of a P1A-expressing BGL-1 glioblastoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 682.

A novel interleukin-2-based fusion molecule, HCW9302, differentially promotes regulatory T cell expansion to treat atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease caused by deposition of oxidative low-density lipoprotein (LDL) in the arterial intima which triggers the innate immune response through myeloid cells such as macrophages. Regulatory T cells (Tregs) play an important role in controlling the progression or regression of atherosclerosis by resolving macrophage-mediated inflammatory functions. Interleukin-2 (IL-2) signaling is essential for homeostasis of Tregs. Since recombinant IL-2 has an unfavorable pharmacokinetic profile limiting its therapeutic use, we constructed a fusion protein, designated HCW9302, containing two IL-2 domains linked by an extracellular tissue factor domain. We found that HCW9302 exhibited a longer serum half-life with an approximately 1000-fold higher affinity for the IL-2Rα than IL-2. HCW9302 could be administered to mice at a dosing range that expanded and activated Tregs but not CD4+ effector T cells. In an ApoE-/- mouse model, HCW9302 treatment curtailed the progression of atherosclerosis through Treg activation and expansion, M2 macrophage polarization and myeloid-derived suppressor cell induction. HCW9302 treatment also lessened inflammatory responses in the aorta. Thus, HCW9302 is a potential therapeutic agent to expand and activate Tregs for treatment of inflammatory and autoimmune diseases.

The Therapeutic Effect of Tacrolimus in a Mouse Psoriatic Model is Associated with the Induction of Myeloid-derived Suppressor Cells.

Topical administration of Tacrolimus (TAC) is efective in the treatment of psoriasis in human patients and in mouse models. Previously, we showed that, though promoting the proliferative expansion of CD4+Foxp3+ regulatory T cells (Tregs), TNFR2 was protective in mouse psoriasis model. We thus examined the role of TNFR2 signal in the efect of TAC in the treatment of mouse psoriasis. To this end, psoriasis was induced in WT, or TNFR1 KO, or TNFR2 KO mice, and the psoriatic mice were treated with or without IMQ. The results showed that TAC treatment potently inhibited the development of psoriasis in WT and TNFR1 KO mice, but not in TNFR2 KO mice. However, the treatment of TAC failed to induce the expansion of Tregs in psoriatic mice. In addition to playing a decisive role in the activation of Tregs, TNFR2 stimulates the generation and activation of myeloid-derived suppressor cells (MDSCs). This led us to found that the topical treatment with TAC markedly increased the number of MDSCs in the spleen of WT and TNFR1 KO mice, but not in TNFR2 KO mice. Consequently, TAC potently decreased serum levels of IL-17A, INF-γ, and TNF and their mRNA levels in the inflamed skin lesion. Therefore, our study for the first time found that the therapeutic efect of TAC in psoriasis is associated with the expansion of MDSCs in a TNFR2-dependent manner.

Mir-106a-5p and Mir-146a-5p Delivered By Tumor-Derived Exosomes Promote the Induction of Myeloid-Derived Suppressor Cells from Normal Peripheral Blood Mononuclear Cells in Multiple Myeloma

Despite the great advance in treatment, including immunochemotherapy using monoclonal antibodies and immunomodulatory drugs, and cell therapies, such as chimeric-antigen receptor T cell therapies, multiple myeloma (MM) remains to be mostly incurable. One of the major mechanisms for the resistance to various immune-mediated treatment approaches for MM is the collapse of the tumor-immune surveillance system which is induced by the predominance of immunosuppressive cellular components over the surveillance system. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells which suppress T-cell-mediated immune response, and its increase has been associated with disease progression and treatment resistance in MM. We previously reported that the co-culture with some human-myeloma-derived cell lines (HMCLs), KMS-12-BM cells, and KMS-28-PE cells, but not others, such as AMO-1 cells and RPMI8226 cells, induced monocytic-MDSCs (M-MDSCs) from normal peripheral blood mononuclear cells (PBMCs), and showed that the combinatory secretion of CCL5, MIF, and MIP-1a, by HMCLs partly contributed to the induction of M-MDSCs in MM (Kuwahara-Ota S, Br J Haematol 2020). In addition to soluble factors, exosomes play a crucial role in cell-to-cell communication by transferring various molecules, including proteins, mRNAs, and microRNAs (miRNAs), and we here investigated the role of tumor-derived exosomes (TEXs) in the induction of M-MDSCs in MM. First, we isolated TEXs from the culture supernatant of various HMCLs. The presence of TEXs was confirmed by nanoparticle tracking analysis and flow cytometry. To examine whether TEXs promote the induction of M-MDSC, we treated PBMCs with TEXs from HMCLs. As the result, the exposure of TEXs from MDSC-inducible HMCLs, but not from MDSC-non-inducible HMCLs, induced M-MDSCs from PBMCs in a dose-dependent manner, indicating the involvement of TEXs in M-MDSC induction in MM. Next, we comparatively performed the comprehensive miRNA profiling by microarray analysis among TEXs from MDSC-inducible and non-inducible HMCLs and identified that miRNAs, including miR-16-5p, miR-17-5p, miR-20a-5p, miR-106a-5p, miR-107, and miR-146a-5p, were specifically upregulated in TEXs from MDSC-inducible HMCLs compared with MDSC-non-inducible HMCLs, which was validated by the quantitative RT-PCR. Interestingly, the expression levels of these 6 miRNAs were mostly equivalent in MDSC-inducible HMCLs and MDSC-non-inducible HMCLs, suggesting the crucial role of TEX-mediated delivery of those miRNAs. By the transfection experiments of these 6 miRNAs into PBMCs, we identified that PBMCs transfected by miR-106a-5p and miR-146a-5p exhibited differentiation into M-MDSCs. In addition, the combination of miRNA, i.e., miR-106a-5p or miR-146a-5p, and chemokine, namely, CCL5, MIP-1a, or MIF, further enhanced the induction of M-MDSC from PBMCs. Finally, to clarify the functional roles of miR-106-5p and miR-146a-5p in the TEX-mediated induction of M-MDSCs, we analyzed the comprehensive gene expression changes by the introduction of miR-106-5p or miR-146a-5p in PBMCs using gene expression microarray. As the result, the gene introduction of miR-106a-5p or miR-146a-5p was found to modulate various gene sets, including those associated with IFN-α signaling, IFN-γ signaling, inflammatory response, TNF-a signaling via NF-kB, or interleukin-6-JAK-STAT3 signaling, in CD33-positive myeloid cells, and these findings mirror the gene expression modulation induced by the co-culture with MDSC-inducible HMCLs in CD33-positive myeloid cells. In conclusion, this study for the first time identified the multi-layer functional involvement of TEX-mediated miR-106a-5p and miR-146a-5p transfer in the induction of M-MDSCs in MM.

Therapeutic Strategy for Rheumatoid Arthritis by Induction of Myeloid-Derived Suppressor Cells with High Suppressive Potential.

Combination treatment using fingolimod (FTY720), an immunomodulator, and a pathogenic antigen prevents the progression of glucose-6-phosphate isomerase (GPI)325-339-induced arthritis. In this study, we focused on myeloid-derived suppressor cells (MDSCs; CD11b+Gr-1+ cells) and investigated the effects of the combination treatment on these cells. DBA/1J mice with GPI325-339-induced arthritis were treated using FTY720 and/or GPI325-339 for five days. The expanded CD11b+Gr-1+ cell population and its inhibitory potential were examined. The percentage of CD369+CD11b+Gr-1+ cells effectively increased in the combination-treated mice. The inhibitory potential of CD369+CD11b+Gr-1+ cells was higher than that of cells not expressing CD369. Among bone marrow cells, the expression of CD369 in CD11b+Gr-1+ cells increased following stimulation with granulocyte-macrophage colony-stimulating factor, and the expression of CD11c increased accordingly. The increased CD11c expression indicated a decrease in the potential to suppress T cell proliferation based on the results of the suppression assay. The percentage of CD11c-CD369+ cells in CD11b+Gr-1+ cells that were induced by the combination treatment also increased, and these cells tended to have a higher capacity to inhibit T cell proliferation. In conclusion, the combination treatment using FTY720 and the pathogenic antigen effectively induces MDSC, which demonstrates a high potential for suppressing T cell proliferation in the lymph nodes, thereby establishing an immune-tolerant state.

Quantifying the impact of gut microbiota on inflammation and hypertensive organ damage.

Hypertension (HTN) can lead to heart and kidney damage. The gut microbiota has been linked to HTN, although it is difficult to estimate its significance due to the variety of other features known to influence HTN. In the present study, we used germ-free (GF) and colonized (COL) littermate mice to quantify the impact of microbial colonization on organ damage in HTN. 4-week-old male GF C57BL/6J littermates were randomized to remain GF or receive microbial colonization. HTN was induced by subcutaneous infusion with angiotensin (Ang) II (1.44 mg/kg/day) and 1% NaCl in the drinking water; sham-treated mice served as control. Renal damage was exacerbated in GF mice, whereas cardiac damage was more comparable between COL and GF, suggesting that the kidney is more sensitive to microbial influence. Multivariate analysis revealed a larger effect of HTN in GF mice. Serum metabolomics demonstrated that the colonization status influences circulating metabolites relevant to HTN. Importantly, GF mice were deficient in anti-inflammatory faecal short-chain fatty acids (SCFA). Flow cytometry showed that the microbiome has an impact on the induction of anti-hypertensive myeloid-derived suppressor cells and pro-inflammatory Th17 cells in HTN. In vitro inducibility of Th17 cells was significantly higher for cells isolated from GF than conventionally raised mice. The microbial colonization status of mice had potent effects on their phenotypic response to a hypertensive stimulus, and the kidney is a highly microbiota-susceptible target organ in HTN. The magnitude of the pathogenic response in GF mice underscores the role of the microbiome in mediating inflammation in HTN.

Eliminating METTL1-mediated accumulation of PMN-MDSCs prevents hepatocellular carcinoma recurrence after radiofrequency ablation.

Radiofrequency ablation (RFA) is an important curative therapy in hepatocellular carcinoma (HCC), but recurrence rate remains as high as all the other HCC therapeutic modalities. Methyltransferase 1 (METTL1), an enzyme for m 7 G tRNA modification, was reported to promote HCC development. Here, we assessed the role of METTL1 in shaping the immunosuppressive tumor microenvironment after insufficient RFA (iRFA). By immunohistochemistry and multiplex immunofluorescence (mIF) staining, we showed that METTL1 expression was enhanced in post-RFA recurrent HCC, accompanied by increased CD11b + CD15 + polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and decreased CD8 + T cells. Mechanistically, heat-mediated METTL1 upregulation enhanced TGF-β2 translation to form the immunosuppressive environment by induction of myeloid-derived suppressor cell. Liver-specific overexpression or knockdown of Mettl1 significantly affected the accumulation of PMN-MDSCs and subsequently affected CD8 + T cell infiltration. Complete RFA successfully eliminated the tumor, whereas iRFA-treated mice exhibited enhanced tumor growth and metastasis with increased PMN-MDSC accumulation and decreased CD8 + T cells compared to sham surgery. Interrupting METTL1-TGF-β2-PMN-MDSC axis by anti-Ly6G antibody, or knockdown of hepatoma-intrinsic Mettl1 or Tgfb2 , or TGF-β signaling blockade significantly mitigated tumor progression induced by iRFA and restored CD8 + T cell population. Our study sheds light on the pivotal role of METTL1 in modulating an immunosuppressive microenvironment and demonstrated that interrupting METTL1-TGF-β2-PMN-MDSC axis could be a therapeutic strategy to restore antitumor immunity and prevent HCC recurrence after RFA treatment, meriting further clinical studies.

Novel Paired Cell Lines for the Study of Lipid Metabolism and Cancer Stemness of Hepatocellular Carcinoma.

There are few well-characterized syngeneic murine models for hepatocellular carcinoma (HCC), which limits immunological studies and the development of immunotherapies for HCC. We previously established an oncogene-induced spontaneous HCC mouse model based on transposon-mediated oncogene (AKT and NRASV12) insertion into the genome of hepatocytes to induce tumorigenesis. Two tumor clones with different levels of lipid droplets (LDs) showed similar in vitro growth but distinctive in vivo phenotypes, including divergent proliferative capability and varying induction of myeloid-derived suppressor cells (MDSCs). The two clones showed distinct gene expression related to lipid metabolism, glycolysis, and cancer stemness. Endogenous fatty acid (FA) synthesis and exogenous monounsaturated fatty acid (MUFA) consumption promoted both tumor proliferation and cancer stemness, and upregulated c-Myc in the HCC cell lines. Moreover, the LDhi HCC cell line expressed a higher level of type II IL-4 receptor, which promoted tumor proliferation through binding IL-4 or IL-13. The chromosomal DNA of two tumor clones, NHRI-8-B4 (LDhi) and NHRI-1-E4 (LDlo) showed five identical AKT insertion sites in chromosomes 9, 10, 13, 16 and 18 and two NRAS integration sites in chromosomes 2 and 3. Herein, we describe two novel HCC cell lines with distinct features of lipid metabolism related to cancer stemness and differential interplay with the immune system, and present this syngeneic HCC mouse model as a practical tool for the study of cancer stemness and discovery of new therapies targeting liver cancers.

Endosomal Sequestration of TLR4 Antibody Induces Myeloid-Derived Suppressor Cells and Reverses Acute Type 1 Diabetes.

We previously showed that treating NOD mice with an agonistic monoclonal anti-TLR4/MD2 antibody (TLR4-Ab) reversed acute type 1 diabetes (T1D). Here, we show that TLR4-Ab reverses T1D by induction of myeloid-derived suppressor cells (MDSCs). Unbiased gene expression analysis after TLR4-Ab treatment demonstrated upregulation of genes associated with CD11b+Ly6G+ myeloid cells and downregulation of T-cell genes. Further RNA sequencing of purified, TLR4-Ab-treated CD11b+ cells showed significant upregulation of genes associated with bone marrow-derived CD11b+ cells and innate immune system genes. TLR4-Ab significantly increased percentages and numbers of CD11b+ cells. TLR4-Ab-induced CD11b+ cells, derived ex vivo from TLR4-Ab-treated mice, suppress T cells, and TLR4-Ab-conditioned bone marrow cells suppress acute T1D when transferred into acutely diabetic mice. Thus, the TLR4-Ab-induced CD11b+ cells, by the currently accepted definition, are MDSCs able to reverse T1D. To understand the TLR4-Ab mechanism, we compared TLR4-Ab with TLR4 agonist lipopolysaccharide (LPS), which cannot reverse T1D. TLR4-Ab remains sequestered at least 48 times longer than LPS within early endosomes, alters TLR4 signaling, and downregulates inflammatory genes and proteins, including nuclear factor-κB. TLR4-Ab in the endosome, therefore, induces a sustained, attenuated inflammatory response, providing an ideal "second signal" for the activation/maturation of MDSCs that can reverse acute T1D.

Effects of Orally Administered Cannabidiol on Neuroinflammation and Intestinal Inflammation in the Attenuation of Experimental Autoimmune Encephalomyelitis.

Cannabidiol (CBD) is a bioactive compound isolated from Cannabis plants that has garnered attention within the medical community due to its potent anti-inflammatory properties. To better understand how CBD limits excessive neuroinflammation we administered CBD via oral gavage (20 mg/kg) in a murine model of multiple sclerosis (MS) known as experimental autoimmune encephalomyelitis (EAE). Using single cell RNA sequencing (scRNA Seq) and array-based transcriptomics we were able to delineate how CBD limits excessive inflammation within the central nervous system (CNS) as well as within the intestinal lining in EAE. In-depth scRNA Seq analysis of CNS tissue demonstrated that CBD treatment resulted in a significant reduction in CXCL9, CXCL10 and IL-1β expression within the CNS, leading to inhibited infiltration of inflammatory macrophages. CBD inhibited IL-1β production independent of the classical cannabinoid receptors, CB1 and CB2. CBD treatment also led to induction of Myeloid-derived Suppressor Cells (MDSCs) both in the CNS and periphery. Interestingly, CBD treatment of EAE mice revealed significant suppression of inflammation in the gastrointestinal (GI) tract. The intestinal epithelial cells (IECs) of CBD treated mice demonstrated a transcriptional inhibition of a family of pyroptosis initiators that drive localized inflammation known as gasdermins (GSDMs). Further investigation into the GI tract via 16s sequencing of cecal and fecal contents demonstrated that oral administration of CBD resulted in no significant changes in the intestinal microbiota composition. These findings demonstrate the beneficial effect of CBD treatment on autoimmune neuroinflammation by ablating expression of pro-inflammatory chemoattractants, regulating inflammatory macrophage activity, promoting MDSC expansion, and limiting the systemic low-grade inflammation in the GI tract, culminating in the attenuation of EAE.

Artepillin C Reduces Allergic Airway Inflammation by Induction of Monocytic Myeloid-Derived Suppressor Cells

Propolis is a natural product produced by bees that is primarily used in complementary and alternative medicine and has anti-inflammatory, antibacterial, antiviral, and antitumoral biological properties. Some studies have reported the beneficial effects of propolis in models of allergic asthma. In a previous study, our group showed that green propolis treatment reduced airway inflammation and mucus secretion in an ovalbumin (OVA)-induced asthma model and resulted in increased regulatory T cells (Treg) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) frequencies in the lungs, two leukocyte populations that have immunosuppressive functions. In this study, we evaluated the anti-inflammatory effects of artepillin C (ArtC), the major compound of green propolis, in the context of allergic airway inflammation. Our results show that ArtC induces in vitro differentiation of Treg cells and monocytic MDSC (M-MDSC). Furthermore, in an OVA-induced asthma model, ArtC treatment reduced pulmonary inflammation, eosinophil influx to the airways, mucus and IL-5 secretion along with increased frequency of M-MDSC, but not Treg cells, in the lungs. Using an adoptive transfer model, we confirmed that the effect of ArtC in the reduction in airway inflammation was dependent on M-MDSC. Altogether, our data show that ArtC exhibits an anti-inflammatory effect and might be an adjuvant therapy for allergic asthma.

Rapamycin delays allograft rejection in obese graft recipients through induction of myeloid-derived suppressor cells

Obesity has become a relevant problem in transplantation medicine with steadily increasing numbers of obese graft recipients. However, the effect of immunomodulatory drugs on transplant-related outcomes among obese patients are unknown. Therefore, we evaluated the impact of rapamycin on allograft rejection and alloimmune response in a murine model of diet-induced obesity and fully-mismatched skin transplantation. Rapamycin significantly delayed allograft rejection in obese recipient mice compared to treated lean mice (14.5 days vs. 10.7 days, p = 0.005). Treatment with rapamycin increased frequencies of monocytic myeloid-derived suppressor cells (M-MDSCs), augmented the immunosuppressive activity of M-MDSCs on T cells through indoleamine 2,3-dioxygenase pathway and shifted CD4+T cells towards regulatory T cells in obese graft recipients. In summary, our results demonstrate that rapamycin delays allograft rejection in obese graft recipients by enhancing suppressive immune cell function and shifting immune cell subsets towards anti-inflammatory conditions.

Abstract PR-005: Adenosine signaling and radiation-induced immunosuppressive changes to the tumor microenvironment

Abstract Introduction/Purpose: Radiation therapy drives pro-inflammatory changes to the tumor microenvironment (TME). We have shown that this may be countered by the induction of immunosuppressive changes that drive therapy resistance. Here, we test the hypothesis that radiation therapy induces immunosuppressive myeloid cells in the TME in an adenosine-dependent fashion. Methods: Using the 4T1 orthotopic breast cancer mouse model, we tested the immunophenotypic changes to the TME by spectral flow cytometry and bulk RNASeq of tumor infiltrating leukocytes (CD45+, TIL) 72-hours after tumor-directed radiation therapy (8 Gy x 1) using the small animal radiation research platform (SARRP, Xstrahl; 220 kV). We cultured human peripheral blood mononuclear cells (PBMCs) with 0.1, 1, and 10uM of adenosine receptor agonist, 5’-N-ethylcarboxamindoadenosine (NECA), in the presence of CD3/CD28 beads. Cytokine quantification of cell-free supernatant was performed 48-hours later using the Meso Scale Discovery platform. Results: Radiation increases the abundance of CD45+CD11b+ myeloid cells (77.4% vs. 84.4%) and decreased CD45+TCRb+ lymphocytes (6.3% vs. 3.6%) in the TME, compared to untreated controls. The reduction of lymphocytes was driven by CD4+ T-cells (5.2% vs 2.7%), including a significant reduction Tregs (1.4% vs 0.63%), with no change in abundance of CD8+ T-cells (0.53% vs 0.43%). The increase in myeloid cells was predominantly influenced by an induction of granulocytic and monocytic myeloid-derived suppressor cells (PMN-MDSCs: 22% vs 34.5%; M-MSCSs: 3.0% vs 10.0%). Radiation caused a decrease in the tumor-associated macrophage population (TAM: 50% vs 31.1%) in the setting of a phenotypic shift to from pro-inflammatory M1-like to immunosuppressive M2-like TAM phenotype: M2-like TAM/M1-like TAM ratio 2.0 vs 13.3. Radiation increased the expression of adenosine 2A receptor (A2AR, log2 fold change 1.13) and A2BR (log2 fold change 0.92) by TILs, but not CD73 that metabolizes AMP to adenosine. The addition of NECA to human PBMCs resulted in significant reduction in effector T-cell cytokines (IL-2, IFN- γ, granzyme B) at all NECA concentrations tested and a dose-dependent decrease in GM-CSF, IL-12p40 and IL-23. There was a significant reduction of IL-1 β, IL-8, IL-12p70 and MCP-1 only at the highest dose (10uM) tested. IL-6 was increased at the intermediate dose (1uM) of NECA with an overall reduction with high-dose NECA. High-dose NECA alone significantly increased both IFN- β and TGF- β. Conclusion: Radiation induces an immunosuppressive TME, increasing the abundance of PMN-MDSCs, M-MDSCs and a shifting the TAM population towards an immunosuppressive phenotype. Radiation increases the expression of adenosine receptors on TILs and treatment of human PBMCs with adenosine receptor agonist resulted in loss of effector T-cell function and suppressive changes to cytokine milieu suggesting a mechanistic link between radiation and adenosine signaling. Citation Format: Shruti Bansal, Mathew Chaimowitz, Casey R. Ager, Aleksandar A. Obradovic, Charles G. Drake, Catherine S. Spina. Adenosine signaling and radiation-induced immunosuppressive changes to the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-005.