Myeloid-derived Suppressor Cells Populations Research Articles

LC-MS-based rheumatoid arthritis serum metabolomics reveals the role of deoxyinosine in attenuating collagen-induced arthritis in mice

Rheumatoid arthritis (RA) is a persistent autoimmune condition with no identified cure currently. Recently, scientists have applied metabolomics to investigate altered metabolic profiles and unique diseases-associated metabolic signatures. Herein, we applied metabolomics approach to analyze serum samples of 41 RA patients and 42 healthy controls (HC) with the aim to characterize RA patients' metabolic profile, investigate related underlying pathological processes, and identify target metabolites. By utilizing ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry, we found 168 proposed metabolites and 45 vital metabolic pathways. Our analysis revealed that deoxyinosine (DI), a metabolite of the purine metabolic pathway, was the most significant reduced metabolite in RA patients. Furthermore, through targeted detection, we confirmed lower concentration of DI in RA patients' peripheral blood. Moreover, DI inhibited lipopolysaccharide-induced inflammation both in vitro and in vivo. We further assessed DI's therapeutic potential in a collagen-induced arthritis (CIA) murine model. The results revealed that DI attenuated CIA, as evidenced by significantly lowered clinical scores of arthritis, alleviated joint swelling, and mitigated bone destruction. Moreover, we elucidated the underlying mechanism by which DI increased the population of myeloid-derived suppressor cells (MDSCs) and suppressed the proliferation of induced T cells. Collectively, these findings suggested that DI potentially ameliorated RA by inducing immunosuppressive MDSCs. The study provides key observations on RA pathogenesis and may contribute to developing novel therapeutic strategies for this debilitating condition.

Abstract 6864: Tumor suppressor function of the ubiquitin ligase Siah1a in melanoma is mediated by myeloid derived suppressor cells

Abstract Growing evidence supports the important role of myeloid-derived suppressor cells (MDSC) in melanoma progression and response to therapy. MDSC contributes to a pro-tumorigenic phenotype, yet the mechanistic basis for the immune suppressor properties acquired by MDSC remains largely unknown. Mammals have two homologous Siah genes, Siah1 (Siah1a and Siah1b in mice) and Siah2. In the context of melanoma, the ubiquitin ligases Siah1a and Siah2 have melanoma-intrinsic and -extrinsic (immune) functions. Correspondingly, ablation of Siah1a/2 in melanoma or its microenvironment attenuates melanoma development in a number of mouse melanoma models. Given that number of macrophage clusters infiltrated into melanoma when grown in Siah2 KO mice, we set to assess macrophage function in melanoma. Selective ablation of Siah1a or Siah2 in macrophages, using Lyz2Cre, revealed that Siah1a but not Siah2 elicits a tumor suppressor function in melanoma. Inoculation of mouse melanoma Yummer1.7 cells (Braf mutated and Pten deleted) in mice lacking Siah1a in macrophages, resulted in bigger melanoma tumors compared to melanoma grown in either WT or mice in which macrophages were ablated of Siah2. FACS analysis of melanoma tumors grown in mice harboring Siah1a mutant macrophages revealed a significant increase in MDSC, which coincided with a decrease in CD4+ and CD8+ cells, compared with WT mice. These observations suggest that a lack of Siah1a in macrophages leads to an expansion of MDSC that affects surrounding immune cells, including T cells. RNA-seq analysis of MDSC that were differentiated in vitro from bone marrow cells of WT or Siah1a ablated macrophage mice revealed increased expression of genes involved in proliferation and alternative macrophage activation in MDSC lacking Siah1a when compared to the WT genotype. Mapping mechanisms underlying Siah1a tumor suppressor function is expected to reveal novel regulatory cues in melanoma control by the MDSC population while mapping novel means for stratifications of melanoma to therapy, and possibly new means for therapy of this tumor type. Citation Format: Marzia Scortegagna, Yuanning Du, Yongmei Feng, Ze'ev Ronai. Tumor suppressor function of the ubiquitin ligase Siah1a in melanoma is mediated by myeloid derived suppressor cells [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 6864.

Abstract 3065: Targeting of metabolic reprogramming in lung cancer by utilizing patient-derived organoid tissue for precision medicine

Abstract In a preliminary pilot study, we reported elevated activity of indoleamine 2,3-dioxygenase 1 (IDO1) in serum from cisplatin-resistant (CR) lung cancer patients compared to cisplatin-sensitive (CS) patients. This heightened IDO1 activity was associated with increased tryptophan (TRP) catabolism, resulting in the upregulation of kynurenine (KYN) production. The detection of KYN can now be readily achieved through immunohistochemistry (IHC). We demonstrated that this upregulation contributed to increased Treg (regulatory T cell) and MDSC (myeloid-derived suppressor cell) populations resulting in an immunosuppressive tumor microenvironment. To investigate this effect, we used dual inhibition of IDO1/TDO2 (tryptophan2,3-dioxygenase 2) in an orthotopic mouse model of CR-Lewis lung carcinoma. This resulted in a significant reduction in tumor volume compared to LLC-CS (n=7, p<0.01), confirming the role of TRP catabolism in CR tumorigenesis. To determine the relevance of the data derived from this mouse model to clinical settings, we established 50 patient-derived organoid tissues (PDOT) from patients with NSCLC. PDOT is an experimental ex vivo tissue model developed to recapitulate the intricate features of lung architecture, including the lung acinus. Upon optimizing our collection and growth protocols, each PDOT successfully developed into a spherical body within 14 days. Furthermore, through bulk RNA-seq analysis, we showed that PDOT closely mirrors the expression of key molecular markers (PD-L1 and mutations), as observed in the original tumor tissues. PDOT from patients possessing high levels of PD-L1 and KYN were exquisitely sensitive to dual inhibition with 2-3-fold greater sensitivity compared to patients with undetectable KYN expression in growth inhibition assays (GI50=20±5.5μM vs. 65±7.5μM). Increased lipid peroxidation was detected via flow cytometry by accumulation of the C11-BODIPY reporter dye in the dual inhibition treatment group. Moreover, we integrated PDOT with a novel 3D microfluidic culture that can be supplied with patient-matched peripheral blood mononuclear cells (PBMC) to assess immune response. Using CyTOF, we showed that dual inhibitors with Atezolizumab (anti-PD-L1) enhanced immune effector (CD8+, NK) and suppressed immunosuppressive (Treg, MDSC) populations. We believe that our swift progress in PDOT development may bridge the divide between cancer biomarker expression and patient trials by timely complementing conventional drug studies based on cell lines and xenografts to facilitate the design of personalized therapies. As a result, the extensive reporting that we have contributed for KYN expression profiling in CR has the potential to serve as a valuable companion diagnostic tool in the future. This can assist in applying precision oncology by finding the most appropriate patients for immunotherapies. Citation Format: Chunjing Wu, Jonathan D. Nguyen, George Theodore, Estelamari Rodriguez, Sydney Spector, Emily Kim, Taranatee Khan, Pablo Puente, Yujie Wang, Dao M. Nguyen, Cheng-Bang Chen, Diane Lim, Lynn G. Feun, Niramol Savaraj, Medhi Wangpaichitr. Targeting of metabolic reprogramming in lung cancer by utilizing patient-derived organoid tissue for precision medicine [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 3065.

Abstract 1531: TIL analysis demonstrates different mechanisms underlying PD-1 resistance in animal models

Abstract Despite the tremendous success of PD-1 immunotherapy in treating cancer, resistance to the therapy is an inevitable risk. One of the major mechanisms underlying this resistance is alterations in the tumor microenvironment, including tumor-infiltrating lymphocyte (TIL) profiling changes after anti-PD1 treatment. To better understand the mechanism of PD-1 resistance, characterization of TILs in the tumor microenvironment was explored. First, immune cell profiling was performed in syngeneic tumor models established by 19 cancer cell lines without anti-PD-1 treatment. Depending on the tumor model, absolute numbers of CD45+ cells and immune cell subsets varied. Moreover, stratifying the analysis based on tumor size (100 mm3 to 3000 mm3) revealed alterations in the proportion of immune cell percentages in MC38 tumors, which implies alterations in TIL frequency during cancer progression. Next, we investigated the TIL profile in two PD-1 resistant models: a hPD-1 antibody-induced resistant model and a syngeneic model in aged mice. The hPD-1 antibody resistant model was induced by multiple rounds of hPD-1 antibody retreatment on MC38 tumor-bearing humanized B-hPD-1/hPD-L1 mice. Efficacy results from this model further validated their resistance to hPD-1 treatment (0% tumor growth inhibition (TGI)), while the control group, which was not serially treated, showed substantial tumor inhibition (82.2% TGI). Next, mPD-1 antibody resistance was evaluated in aged wild-type mice. While mPD-1 efficacy on MC38-bearing young mice was approximately 85.5% TGI, the efficacy of this treatment in aged mice yielded only 6% TGI. TIL analysis in these models was subsequently performed: in the hPD-1-treated control mice that were responsive to anti-PD1 treatment, the numbers of total T cells, cytotoxic T cells (CD8+) and MHC II positive cells were increased, while the anti-tumor effector cells in the serially treated resistant mice were either unchanged (total T cells and MHC II positive cells) or decreased (CD8+ cells); strikingly, an increase in inhibitory myeloid-derived suppressor cell (MDSC) populations was also observed in the resistant model post-treatment. In the anti-PD1 treated aged mice, the frequency of TILs was unchanged upon mPD-1 treatment, suggesting that resistance in aged mice may be due to non-responsiveness of the immune cells. In sum, our results demonstrate different mechanisms underlying anti-PD-1 resistance, and suggest that a more thorough analysis of the TILs present in the tumor microenvironment could predict the responsiveness to PD-1 therapy and inform treatment strategies. Citation Format: Sha Qiao, Yujia Liu, Wei Liu, Wenqian Pan, Xiaofei Zhou, Linlin Wang, Mengya Chai, Yujie Liu, Hao Yang, Zhaoxue Yu. TIL analysis demonstrates different mechanisms underlying PD-1 resistance in animal models [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 1531.

Abstract 179: Identification of a novel crosstalk between MDSC and NKregs in immunotherapy resistant triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive forms of breast cancer and lacks standard treatment options. Although immunotherapy shows some response in these patients along with chemotherapy, heterogeneity of tumor cells and immune cells results in significant low response to immunotherapy treatment. Based on single cell sequencing which enables identity of heterogenous population at single cell level and functional studies involving preclinical mouse models, our laboratory recently identified distinct CD56brightSOCS3highCD11b−CD27− immature NK cells subpopulations, with diminished cytotoxic granzyme signatures that promote TNBC tumor progression. However, the precise characterization and functionality of these rogue tumor promoting NK cells remains unclear in clinical samples. Here, we aimed to illustrate mechanistic insight of these novel immature NK subpopulation in context of immunotherapy resistant TNBC patient samples. The scRNA seq analysis of TNBC patient samples identified four specific subclusters of NK cells in TNBC. We further found that NK2 and NK3 subclusters are CD56dim Granzymehigh cytotoxic NK cells and the other two subclusters (NK0 and NK1) are CD56bright immature NKs. Interestingly, the NK0 and NK1 subcluster specifically exhibit distinctive characteristics and functions. Notably, CD56bright immature NK1 cells, identified as tissue-resident NK cells, were engaged in direct communication with myeloid-derived suppressor cells (MDSCs). The CD56bright NK0 possesses moderate to low SOCS3 expression and moderate to high granzyme expression with elevated early NK activation markers and inhibitory receptor expressions, suggesting its regulatory property (NKregs). scRNA seq and flow cytometry analysis showed higher NKregs and immunosuppressive MDSC population, specifically with high monocytic-MDSCs (mMDSCs) in non-responder TNBC (to adjuvant combinational treatment of chemo and immunotherapy) patient samples rather than responder patients’ tissue. Based on in silico analysis, we further found a potential crosstalk between these mMDSC & NKreg within the tumors of non-responsiveness of TNBC patients. Overall, our studies show a connection of MDSCs and NKregs in TNBC patients, who do not respond to immunotherapy, highlighting a possible mechanism for immunotherapy resistance, which could be targeted in near future for better treatment options in TNBC. Citation Format: Megha Das, Samantha Henry, Shailesh Singh, Camila Dos Santos, Rumela Chakrabarti. Identification of a novel crosstalk between MDSC and NKregs in immunotherapy resistant triple negative breast 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 179.

Abstract 105: Understanding the role of β2-adrenergic receptor signaling on type II innate lymphoid cells in cancer

Abstract Increased norepinephrine (NE) due to chronic stress can activate adrenergic receptors, specifically β2-adrenergic receptors (β2AR), to produce an immunosuppressive tumor microenvironment (TME). Previous work by our lab demonstrated that tumor burden can be reduced by blocking β2AR signaling. Immune cells express β2AR and we have shown that the increased β2AR signaling leads to increased T cell exhaustion and increased inhibitory activity of myeloid-derived suppressor cells (MDSCs). In general, the resulting immunosuppression leads to a modest but significant reduction in tumor growth. However, the lack of a dramatic reduction in tumor growth reveals the complex nature of adrenergic signaling dependent regulation of the cancer immune response. Recent literature revealed that innate lymphoid cells (ILCs), especially type II ILCs (ILC2s), express high levels of β2AR. Therefore, we evaluated whether β2AR regulates ILC2s within the TME and how that affects tumor prognosis. We generated a genetic conditional knockout murine model where β2AR expression in ILC2s was deleted by crossing IL5cre expressing mice to β2ARflox/flox mice (IL5creβ2ARf/f). AT3 breast cancer cell line was injected into the 4th mammary fat pad and tumor volume was measured every 2-3 days. Tumor infiltrating ILC2s (CD45+Lineage−CD90.2+CD127+ST2+) were analyzed by flow cytometry at D36 post-tumor implantation. Notably, IL5creβ2ARf/f mice had increased AT3 breast cancer tumor growth compared to control mice. Flow cytometric analysis of the tumor revealed an increased frequency of GATA3+ ILC2s within the TME. These GATA3+ ILC2s lacking β2AR expressed higher levels of the IL33 receptor, ST2, and a higher percentage of IL13+ cells compared to WT GATA3+ ILC2 cells. We also observe a trend toward increased MDSC population within the TME of mice lacking β2AR in ILC2s. These results reveal an anti-tumor function of β2AR signaling through the suppression of ILC2 activity within the TME that warrants further investigation. This work has been in part supported by NIH Grants F30 CA284763 (to J.C.), K99 HL155792 (to H.M.), and RO1 CA205246 (to E.R.) Citation Format: Jee Eun Choi, Cameron R. MacDonald, Saeed Daneshmandi, Jianmin Wang, Nathan T. Roberts, Caitlin M. James, Philip L. McCarthy, Hemn Mohammadpour, Elizabeth A. Repasky. Understanding the role of β2-adrenergic receptor signaling on type II innate lymphoid cells in 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 105.

Abstract 6637: The Haemophilus influenzae vaccine mitigates pancreatic tumor progression and lowers immunoregulatory cell levels in the tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease projected to become the second leading cause of cancer deaths by 2030. First-line chemotherapies gemcitabine and FOLFIRINOX provide little therapeutic benefit and are accompanied by severe adverse toxicities, precluding long-term treatment. In addition, PDAC is notoriously refractory to checkpoint inhibitor immunotherapies, due to poor immunogenicity and a tumor microenvironment dominated by suppressive cell types. Thus, there is an unmet need for innovative, immune-based strategies to combat PDAC. A previous study utilized engineered bacteria to effectively kill PDAC tumor cells by directly delivering immunogenic tetanus toxoid to the tumor. Given the potential toxicity in using live bacteria, our group utilized a less toxic alternative, an FDA-approved Haemophilus influenzae type b vaccine (HibVx) also containing a tetanus toxoid component. To determine the antitumor potential of HibVx, our group used a physiologically relevant murine model, low T cell clone murine PDAC tumor model 641c95 and clinically relevant KPC mice model for PDAC. After tumors were established, animals received three consecutive daily treatments of HibVx via intraperitoneal injection. HibVx-treated animals experienced a dramatic reduction in tumor growth compared to PBS-treated controls (p<0.001). Given that tetanus toxoid is highly immunogenic in nature, changes in intratumoral immune cell populations following HibVx treatments were of great interest. Using flow cytometry analysis, HibVx-treated tumors had a significant increase in natural killer cells compared to control tumors (p<0.05). In addition, HibVx affected myeloid-derived suppressor cell populations (MDSCs), which are heavily implicated in the suppressive nature of PDAC tumors. HibVx also significantly decreased MDSCs within the tumor (p<0.05). In all, these findings position HibVx as an effective immunostimulatory agent that can be used as an alternative to live bacteria-based therapy for the treatment of PDAC. Citation Format: Eileena F. Giurini, Sam G. Pappas, Kajal H. Gupta. The Haemophilus influenzae vaccine mitigates pancreatic tumor progression and lowers immunoregulatory cell levels in the tumor microenvironment [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 6637.

MDSC-targeting gold nanoparticles enhance PD-1 tumor immunotherapy by inhibiting NLRP3 inflammasomes

Myeloid-derived suppressor cells (MDSCs) play a crucial role in the immune escape mechanisms that limit the efficacy of immunotherapeutic strategies. In the tumor microenvironment, NLRP3 inflammasome-driven Interleukin-1β (IL-1β) production serves to dampen antitumor immune responses, promoting tumor growth, progression, and immunosuppression. In this study, we revealed that gold nanoparticles (Au NPs) with a size of 30 nm disrupted NLRP3 inflammasome, but not other inflammasomes, in bone marrow-derived macrophages through abrogating NLRP3-NEK7 interactions mediated by reactive oxygen species (ROS). Density functional theory (DFT) calculations provided insights into the mechanism underlying the exceptional ROS scavenging capabilities of Au NPs. Additionally, when coupled with H6, a small peptide targeting MDSCs, Au NPs demonstrated the capacity to effectively reduce IL-1β levels and diminish the MDSCs population in tumor microenvironment, leading to enhanced T cell activation and increased immunotherapeutic efficacy in mouse tumor models that are sensitive and resistant to PD-1 inhibition. Our findings unraveled a novel approach wherein peptide-modified Au NPs relieved the suppressive impact of the tumor microenvironment by inhibiting MDSCs-mediated IL-1β release, which is the first time reported the employing a nanostrategy at modulating MDSCs to reverse the immunosuppressive microenvironment and may hold promise as a potential therapeutic agent for cancer immunotherapy.

Abstract B087: Targeting myeloid derived suppressor cell migration decreases tumor progression in a murine model of ovarian cancer

Abstract Introduction: High grade serous ovarian, fallopian tube and primary peritoneal carcinoma (HGSC) has an immunosuppressive and tumor promoting immune microenvironment. In this study, we have targeted the migration of myeloid derived suppressor cells (MDSC) through chemical inhibition of the CXCR2 chemotactic receptor to reverse the tumor-induced immunosuppression.Methods: ID8 cells with a p53 mutation (ID8-P53null) were used as a murine model of HGSC. ID8-p53null cells were injected intraperitoneally into immune intact C57/Bl6 mice, with 10 mice per group. After 7 days, mice were treated with cisplatin (cis, 1mg/kg, i.p., weekly), a CXCR2 inhibitor (CXCR2i, 4mg/kg, i.p., daily) or a combination of the 2 treatments. Necropsy was performed on day 29. The tumors were weighed, and the number of metastatic sites was quantified. The spleen and the tumor were resected, digested and flow cytometry was performed to evaluate the effects of treatment on the tumor immune microenvironment (TIME) as well as any systemic changes (evaluated by immune cells in the spleen). Granulocytic MDSC (G-MDSC) and monocytic MDSCs (M-MDSC) were defined as CD11b+Ly6G+,Ly6Clo and CD11b+Ly6G−Ly6Chi, respectively. One-way ANOVA with Tukey multiple comparisons was used for statistics with p<0.05 as the cut off for significance. Results: Tumor weights were significantly less in cis treated vs control mice (0.064g vs 0.220g, p <0.001) and CXCR2i treated vs control mice (0.171g vs 0.220g, p=.003). There was no additive effect of the CXCR2i to the cis treatment (cis 0.064g vs cis + CXCR2i 0.049g, p=0.62). The number of metastatic sites as a measurement of dissemination also decreased with cis or CXCR2i (number of sites: control: 47 vs cis: 12.7 vs CXCR2i: 32.5 vs cis + CXCR2i: 10.2). When the TIME was evaluated by flow cytometry, there were no significant differences in the immune populations including T-cells, NK cells, myeloid cells, and macrophages. However, there was a trend for cis to increase both the G-MDSC and M-MDSC populations and this effect was inhibited with the combination treatment of cis and CXCR2i. For the G-MDSC population control: 0.6% vs cis: 0.9% vs cis+CXCR2i: 0.5% and M-MDSC population control 3.4% vs cis: 6.8% vs cis+CXCR2i: 2.6%. Conclusion: CXCR2 is a chemotactic receptor present on MDSCs. We targeted this receptor to decrease the migration and infiltration of MDSCs into the ovarian cancer TIME. The CXCR2i decreased tumor progression in the ID8-p53null model of ovarian cancer. The TIME was evaluated by flow cytometry, and there was a trend for cis to increase the suppressive MDSC populations and this increase was inhibited by the CXCR2i. These exciting data suggest that traditional chemotherapy may lead to a more immunosuppressive TIME, which could have implications for future recurrence and treatment. Blocking the migration of immunosuppressive cells into the TIME may have a long-term impact on survival in HGSC. Survival studies in this murine model of HGSC are currently underway. Citation Format: Nicole Marjon, Railey Mikeska, Elizabeth R. Woodruff, Ritsuko Iwanaga, Tomomi Yamamoto, Benjamin G. Bitler. Targeting myeloid derived suppressor cell migration decreases tumor progression in a murine model of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B087.

Decreased interleukin-17RA expression is associated with good prognosis in patients with colorectal cancer and inhibits tumor growth and vascularity in mice.

Interleukin-17 (IL-17) is a pro-inflammatory cytokine that plays a vital role in the promotion of tumorigenesis in various cancers, including colorectal cancer (CRC). Based on current evidence, IL-17 binds to interleukin-17 receptor A (IL-17RA); however, the role of IL-17RA has not been elucidated in previous studies on CRC. In this study, we explored the role of IL-17RA in human CRC tissues and the progression of CRC in humans and mice. The expressions of IL-17RA and epithelial-mesenchymal transition (EMT)-related genes were examined in CRC cells and tissue samples by quantitative real-time polymerase chain reaction. The role of IL-17RA in pathogenesis and prognosis was evaluated using a Chi-squared test, Kaplan-Meier analysis, univariate, and multivariate Cox regression analysis in 133 CRC patients. A tumor-bearing mice model was executed to evaluate the role of IL-17RA in tumor growth, vascularity and population of infiltrating immune cells. IL-17RA expression was found to be significantly higher in CRC tissues than in adjacent normal tissues. The expression of IL-17RA in Stage IV patients was significantly higher than that in Stages I and II patients. Patients with high IL-17RA expression exhibited significantly worse overall and CRC-specific survival than those with low IL-17RA expression. Functional assessment suggested that the knockdown of IL-17RA expression distinctly suppressed cellular proliferation, migration, invasion, and EMT-related gene expression. In a tumor-bearing mouse model, decreased IL-17RA expression significantly repressed tumor growth and vascularity and reduced the population of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Reduced IL-17RA expression also suppressed cellular proliferation, migration, and invasion, and the expression of EMT genes. Knockdown of IL-17RA inhibited tumor growth and vascularity and decreased the population of Tregs and MDSCs in mouse tumors. Overall, IL-17RA expression was identified to be independently associated with the prognosis of patients with CRC.

Abstract PR16: Neutrophil-intrinsic tumor necrosis factor (TNF) is a novel driver of T-cell and cancer-associated fibroblast (CAF) dysfunction in pancreatic cancer

Abstract INTRODUCTION: We have recently shown that neutrophilic myeloid-derived suppressor cells (MDSC) are the dominant source of TNF in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). Moreover, neutrophil-derived TNF amplifies stromal fibrosis and immune tolerance via transmembraneTNF-TNFR2 interactions to promote therapeutic resistance in PDAC. Here, we develop a novel tumor-bearing murine model with efficient neutrophil lineage-restricted inactivation of Tnf to mechanistically dissect the impact of neutrophil-intrinsic TNF on stromal and T-cell dysfunction in PDAC. METHODS: We generated a neutrophil-lineage restricted MRP8Cre/+Tnfflox/flox model (Neu-TnfKO) by crossing Tnfflox/flox mice with mice bearing Cre-recombinase under control of MRP8 neutrophilic promoter, and confirmed >98% Tnf reduction in circulating neutrophils. Orthotopically implanted KPC tumors in Neu-TnfKO vs. littermates (MRP8Cre+/−Tnf+/+ or MRP8Cre-/−Tnffl/fl) were subjected to single-cell RNAseq (scRNAseq), flow cytometric immunophenotyping, and tissue histology. RESULTS: Compared with littermate controls, primary tumor weights and frequency of liver metastasis were significantly reduced in Neu-TnfKO mice. ScRNAseq in CD45− fraction of d21 tumors in Neu-TnfKO vs. control mice revealed downregulation of myofibroblastic features (i.e., Ccn2, Tgfb1), hypoxia signaling, and metabolic dysregulation in CAF single-cell transcriptomes. Tissue histology in Neu-TnfKO tumors revealed striking attenuation in fibrotic collagen deposition, and myofibril organization. Moreover, Lrrc15—implicated as a central regulator of CAF-driven immunotherapy resistance—was downregulated in Neu-TnfKO CAFs. This remodeling of stromal fibrosis manifested in increased trafficking of T-cells into Neu-TnfKO vs. littermate tumors via immunostaining and flow cytometry. ScRNAseq in CD45+ fractions of Neu-TnfKO mice corroborated enrichment in effector/helper T-cell and attenuation of MDSC and macrophage populations. In investigating if neutrophil-restricted Tnf silencing invokes reprogramming of T-cell single-cell transcriptomes, we observed global skewness towards effector vs exhausted programming in Neu-TnfKO tumors; enrichment of pathways related to ribosomal activation, interferon signaling, and negative regulation of T-cell apoptosis; and overexpression of multiple effector function genes—e.g., Adgre5, Cam1kd, Dgka—together indicating functional T-cell activation. Flow cytometry validated this activated CD8+ T-cell phenotype in Neu-TnfKO tumor-infiltrating by highlighting increased antigen-experienced (PD1+CD39neg) and short-lived effector populations (P<0.0001), and also revealed reduction in terminally exhausted (CD69+Ly108− in PD1hi) CD8+ T-cell subsets (P=0.028). CONCLUSIONS: Conditional silencing of neutrophilic-restricted TNF reprograms the immunotherapy-restrictive CAF and T-cell landscapes in the PDAC TME. Compartment-specific inhibition of TNF may be a provocative strategy to overcome immunotherapy resistance in PDAC. Citation Format: Anna Bianchi, Haleh Amirian, Iago De Castro Silva, Ifeanyichukwu Ogobuiro, Karthik Rajkumar, Andrew Mark Adams, Vanessa Garrido, Samara Singh, Siddharth Mehra, Christine Rafie, Nagarj Nagathihalli, Erietta Stelekati, Nipun Merchant, Jashodeep Datta. Neutrophil-intrinsic tumor necrosis factor (TNF) is a novel driver of T-cell and cancer-associated fibroblast (CAF) dysfunction in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR16.

Myeloid Derived Suppressor Cells and Their Potential Impact on Minimal Residual Disease Status and Disease Response in a Cohort of Patients with Plasma Cell Dyscrasias

Myeloid Derived Suppressor Cells (MDSCs) are known to promote immune-suppressive and tumorigenic effects in plasma cell dyscrasias (PCDs), including multiple myeloma, and may play a role in treatment response and minimal residual disease MRD status in patients undergoing cancer-directed therapies. MDSCs are classified by their granulocyte-like effects (g-MDSCs) or monocyte-like effects (m-MDSCs). To our knowledge, there are no published studies that correlate minimal residual disease (MRD) with the presence of MDSCs and immune effector-cell populations in the blood and bone marrow of patients with PCDs. In 14 patients with PCDs, we defined immune-cell populations in the blood and marrow and correlated them with MRD status, treatment regimens, disease status (stable vs. progressive) and autologous stem cell transplantation status. In the entire cohort, there were a higher proportion of m-MDSCs in blood (blood 5.56%, aspirate 2.47%). There were a higher proportion of g-MDSCs in aspirate (blood 0.04%, aspirate 0.16%). Interestingly, in patients who remained MRD positive (3/13), the proportions were reversed, and we identified more m-MDSC in aspirate than blood, as well as a low proportion of g-MDSCs in both the aspirate and bone marrow. In patients achieving undetectable MRD (10/13) there were higher proportions of CD8 and CD4 T-cells, with no differences in MDSC populations. Not surprisingly, in patients treated with the anti-CD38 antibody daratumumab (5/14), fewer m-MDSCs were isolated in the blood and aspirate, relative to daratumumab-untreated patients (9/14).Daratumumab treatment decreased activated CD4 and CD8 T cells (HLA-DR+/CD38-, HLA-DR-/CD38+ or HLA-DR+/CD38+ CD4 and CD8 T cells) and increased the proportion of inactive HLA-DR-/CD38- CD4 and CD8 T-cells. In patients with progressive disease (3/14), there was a paucity of all immune effector-cells, including MDSCs, T-cells and monocytes with the exception of neutrophils. MDSC populations were no different in pre-transplant patients (3/14) compared to post-transplant (11/14). In this preliminary study, we have enumerated the presence of MDSCs and their potential impact in patients with PCDs undergoing cancer-directed therapy.

Platelet-Derived TGF-β1 Induces Functional Reprogramming of Myeloid-Derived Suppressor Cells in Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by isolated thrombocytopenia and varying degrees of bleeding symptoms. Platelets are anucleate cytoplasmic cell fragments that play dual physiological roles as both cellular mediators of thrombosis and immune modulatory cells. Platelets are the largest storage pool of TGF-β1, a crucial enforcer of immune homeostasis and tolerance. Studies have shown that TGF-β1 released by platelets contributes to the pathogenesis of pulmonary arterial hypertension and cervical carcinoma. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells with potent immunosuppressive functions. Our previous studies have shown that MDSCs are impaired in number and function in patients with ITP. The objective of this study was to elucidate the underlying molecular mechanisms of MDSCs reprogramming by platelet-derived TGF-β1. Methods: A transwell assay was used to determine the impact of platelets on MDSCs induced from peripheral blood mononuclear cells (PBMCs) of ITP patients and healthy controls. The percentage of circulating MDSCs in ITP patients was detected before and after administration of thrombopoietin receptor agonists (TPO-RAs). Besides, anti-CD61 immune-sensitized splenocytes were transferred into SCID mice to establish an active murine model of ITP. Subsequently, MDSCs from TPO-RA-treated ITP mice were transplanted as cell therapy to rescue treatment-free ITP mice. In specific, adeno-associated viral (AAV) vectors efficiently infected MDSCs to enable cell-specific Smad2/3 knock-down. Finally, platelet specific TGF-β1-deficient mice were used to validate the effect of platelet-derived TGF-β1 on MDSCs. Results: TGF-β1 levels in the supernatant of MDSCs co-cultured with platelets of high concentration was significantly higher than that in MDSCs co-cultured with platelets of low concentration or alone. FACS analysis showed that platelet mass enhanced the inhibitory effects of MDSCs on the proliferation of CD4 + T cells, which could be offset by TGF-β1 inhibitor (TGF-βi). Besides, TPO-RAs treatment induced significantly increased percentage of circulating MDSCs in responders with chronic ITP. Likewise, TPO-RA significantly expanded MDSC population in active ITP murine models. Furthermore, after receiving donor MDSCs from TPO-RA-treated ITP mice, arrested differentiation of plasma cells, reduced Fas ligand expression on cytotoxic T cells, and re-balanced T cell subsets were found in the recipient mice, indicating augmented inhibitory functions of MDSCs. Mechanistically, transcriptome analysis confirmed the participation of TGF-β/Smad pathway in donor MDSCs from TPO-RA-treated ITP mice, which was offset by Smad2/3 knockdown. Finally, by constructing ITP models using Pf4 cre-Tgfb1 fl/fl and Pf4 cre+Tgfb1fl/fl mice, we found that TGF-β1 inhibition and platelet TGF-β1-deficence, both, could counteract the elevated platelet count and MDSCs percentage, as well as improved immunosuppressive capacity of MDSCs trigged by TPO-RA modulation. Conclusion: To sum up, it was demonstrated that platelet TGF-β1 induced the expansion and functional reprogramming of MDSCs via TGF-β/Smad pathway. These data indicate that platelet recovery not only serves as an endpoint of remission, but also paves the way for tolerance induction in immune-mediated thrombocytopenia. Our exploratory study deciphered immunological changes induced by TPO-RAs and helped to identify molecular mechanisms responsible for long-term remission after TPO-RA discontinuation.

Multimodal Single-Cell Transcriptomic and Proteomic Correlatives of Patients Outcomes Following Anti-BCMA Cellular Therapy with Ciltacabtagene Autoleucel (Cilta-cel) in Relapsed Multiple Myeloma

Introduction Multimodal single cell technologies allow us to dissect the mechanisms of therapeutic resistance by integrating transcriptomics and proteomics through the combination of antibody labeling and next-generation sequencing. Using CITE-seq, mass cytometry (CyTOF) and quantitative multiplexed proteomics (Olink), we sought to understand the determinants of chimeric antigen receptor (CAR) T cell response and the overall survival of patients with relapsed or refractory multiple myeloma (RRMM) receiving ciltacabtagene autoleucel (Cilta-Cel) cellular therapy. Methods Twenty five patients received Cilta-Cel and had bone marrow (BM) and peripheral blood (PB) samples collected at baseline and after CAR T infusion. We isolated 262,520 BM cells and 241,657 PB cells, which were later sequenced using CITE-seq. Additionally, we submitted PB samples for CyTOF analysis and acquired 10,162,426 cells used for downstream analysis. We analyzed 92 cytokines using Olink immuno-oncology panel in PB samples. Downstream analysis was performed using the R packages Seurat, CATALYST, FlowSOM and Olink Analyze. Results Our cohort had a median progression-free survival (PFS) of 732 days. To focus our correlative analyses on patients experiencing early relapse, the initial cohort was divided into 2 groups: PFS <18 months (n = 10) and PFS >18 months (n = 15). CAR-T cell expansion was observed in week 2 post-infusion and continued up to week 4. The percentage of CD4 and CD8 CAR-T cells significantly increased between weeks 1-3 and weeks 4-6 weeks (p<0.001) and significantly decreased after week seven (p<0.05). We detected novel and significant differences among four cell populations associated with PFS longer than 18 months. These patients had a higher percentage of activated CD4 Central Memory (CM) and CD4 cytotoxic cells (p<0.05) relative to the total percentage of CAR-T cells in weeks 4-6, suggesting a key role for CD4 cells in cross priming or direct cytotoxicity in this context. In patients with a PFS longer than 18 months, the CD8 CM CART cell population had a significantly higher percentage in weeks 1-3 and 4-6 (p<0.05) when compared to their counterparts, suggesting that these were the cardinal effector population in these patients. Myeloid-derived suppressor cells (MDSCs) have been shown to be a central component of the tumor microenvironment in myeloma, with subsets being capable to mount potent suppressive activity at the tumor site. In the BM CITE-seq myeloid compartment, MDSCs were significantly increased in month 1 (p<0.05) in patients with a shorter PFS. Our PB CyTOF data confirmed this finding as the percentage of MDSCs was significantly higher in weeks 1-3 (p<0.05) in the shorter PFS group when compared to their counterparts. Using a mixed linear regression model on Olink data, we detected 26 cytokines significantly different (p<0.05) between the shorter and longer PFS groups. A pseudobulk analysis of the BM CITE-seq samples for differentially expressed genes encoding the 26 cytokines revealed 22 genes differentially expressed (p<0.05) between patients with a PFS shorter than 18 months and patients with a PFS longer than 18 months in CAR-T, T-cell, NK cell and myeloid cell populations. In the shorter PFS group, VEGFA was significantly higher in CD8 TEMRA CAR-T cells when compared to their counterparts. In the longer PFS group, we observed significantly higher levels of genes involved in T cell activation, such as CD27 and CD28, and pro-inflammatory cytokines such as TNF and IL-15 in the T cell and Myeloid cell compartments. This pattern suggests that higher production of cytotoxic and pro-inflammatory cytokines, combined with enhanced T cell activation, plays an important role in prolonging the response to CAR-T therapy. Conclusions Single cell immune profiling and transcriptomic sequencing identified subpopulations of CD4 and CD8 cells which in concert may influence long term CAR-T outcomes. Our findings demonstrate an early expansion of CART, with very few CART cells surviving after 3 months, suggesting that the efficacy of this therapy is related to early dynamics of these populations. We also provide additional evidence associating immunosuppressive MDSC populations in BM and PB patients with a shorter PFS. Ongoing studies will further analyze the role of the immune microenvironment and clonal T cell dynamics in relation to patient outcomes.

Circulating myeloid-derived suppressor cells may be a useful biomarker in the follow-up of unvaccinated COVID-19 patients after hospitalization.

SARS-CoV-2 infection is the cause of the disease named COVID-19, a major public health challenge worldwide. Differences in the severity, complications and outcomes of the COVID-19 are intriguing and, patients with similar baseline clinical conditions may have very different evolution. Myeloid-derived suppressor cells (MDSCs) have been previously found to be recruited by the SARS-CoV-2 infection and may be a marker of clinical evolution in these patients. We have studied 90 consecutive patients admitted in the hospital before the vaccination program started in the general population, to measure MDSCs and lymphocyte subpopulations at admission and one week after to assess the possible association with unfavorable outcomes (dead or Intensive Care Unit admission). We analyzed MDSCs and lymphocyte subpopulations by flow cytometry. In the 72 patients discharged from the hospital, there were significant decreases in the monocytic and total MDSC populations measured in peripheral blood after one week but, most importantly, the number of MDSCs (total and both monocytic and granulocytic subsets) were much higher in the 18 patients with unfavorable outcome. In conclusion, the number of circulating MDSCs may be a good marker of evolution in the follow-up of unvaccinated patients admitted in the hospital with the diagnosis of COVID-19.

Viral nanoparticle vaccines against S100A9 reduce lung tumor seeding and metastasis

Metastatic cancer accounts for 90% of all cancer-related deaths and continues to be one of the toughest challenges in cancer treatment. A growing body of data indicates that S100A9, a major regulator of inflammation, plays a central role in cancer progression and metastasis, particularly in the lungs, where S100A9 forms a premetastatic niche. Thus, we developed a vaccine against S100A9 derived from plant viruses and virus-like particles. Using multiple tumor mouse models, we demonstrate the effectiveness of the S100A9 vaccine candidates in preventing tumor seeding within the lungs and outgrowth of metastatic disease. The elicited antibodies showed high specificity toward S100A9 without cross-reactivity toward S100A8, another member of the S100A family. When tested in metastatic mouse models of breast cancer and melanoma, the vaccines significantly reduced lung tumor nodules after intravenous challenge or postsurgical removal of the primary tumor. Mechanistically, the vaccines reduce the levels of S100A9 within the lungs and sera, thereby increasing the expression of immunostimulatory cytokines with antitumor function [(interleukin) IL-12 and interferonγ] while reducing levels of immunosuppressive cytokines (IL-10 and transforming growth factorβ). This also correlated with decreased myeloid-derived suppressor cell populations within the lungs. This work has wide-ranging impact, as S100A9 is overexpressed in multiple cancers and linked with poor prognosis in cancer patients. The data presented lay the foundation for the development of therapies and vaccines targeting S100A9 to prevent metastasis.

Effect of External Beam Radiation Therapy (EBRT) and Brachytherapy (BT) on Circulating MDSC Populations in Patients Treated Definitively for Cervical Cancer

The immunosuppressive function of myeloid derived suppressor cells (MDSCs) has been implicated in the regulation of immune responses against cancer. MDSCs have been associated with progression and poor response to therapy in various types of cancer, including cervical cancer. Radiation treatment (RT) alters immune cell populations within the tumor and is thought to augment antitumor responses. However, whether RT also recruits immunosuppressive MDSC populations is not well understood. Here, we investigate how circulating MDSC populations change in response to RT and if changes in MDSC frequency or subsets is predictive of RT responses in cervical cancer patients. Newly diagnosed, treatment-naïve pts with locally advanced carcinoma of the cervix will be enrolled from July 2022 to July 2023. EBRT to the pelvis was delivered at a dose of 45 Gy in 25 fractions with a simultaneous integrated boost of 52-55 Gy to involved regional lymph nodes and parametria and concurrent weekly cisplatin. Gross tumor was boosted via interstitial or tandem ring BT (22.5-27.5 Gy) after completion of EBRT. Serial blood samples were collected prior to initiating therapy (T0), post-EBRT and pre-BT (T1), and one-month post-BT (T2). Treatment response was determined based on pre-treatment MRI compared to MRI post-EBRT. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using density gradient centrifugation and stained for analysis via flow cytometry. MDSC populations were identified by Live/Dead-CD11b+CD33+HLA-DR- staining. MDSC subsets were further subdivided into granulocytic (G-, CD15+CD14-), monocytic (M-, CD15-CD14+), or early-MDSCs (e-, CD15-CD14-). Blood samples were collected at indicated time points for four patients (FIGO stage IIA-IIB). Three had partial responses to chemoradiotherapy (CRT), while one had a complete response. All three patients with partial response had an increase in total frequency of circulating MDSCs in response to EBRT/BT (mean %fx MDSC 16.6 at T0 to 35.9 at T2), and an increase in total MDSCs in two of these patients occurred with EBRT alone. Interestingly, the patient that had a complete response had fewer MDSCs at T2 relative to T0 (35.8% at T0 to 27% at T2). Proportion of MDSC subsets varied considerably among the patients, and all had altered distribution of subsets in response to RT. G-MDSCs expanded the most to RT while M-MDSCs and e-MDSCs were less affected (mean fold change from T0 to T2 G-MDSC 4.75, M-MDSC 1.27, e-MDSC 0.942). In this cohort of patients, an increase in MDSC frequency occurred after RT and altered subset distribution. Only the patient with a complete response had fewer total MDSCs following completion of CRT, suggesting further studies are needed to determine if circulating MDSCs could be a biomarker for treatment response to RT in cervical cancer.

Abstract PO-088: TRPV1+ sensory neurons provide a tumor supportive environment through recruitment of MDSCs

Abstract Cancer neuroscience has become an increasing area of focus in cancer research as our understanding of the presence and function of intra-tumoral neurons continues to emerge, with studies indicating that tumor infiltrating neurons impact components of the tumor microenvironment promoting cancer initiation and progression. We have previously published that head and neck squamous cell carcinomas are innervated. We now focus on defining the contributions of intra-tumoral neurons to disease progression in HNSCC. Tumor implantation in a double transgenic mouse (TRPV1cre::DTAfl/wt) lacking the cancer-recruited TRPV1-expressing sensory neurons allows us to study the consequences of this neuronal loss on the local immune response. Importantly, comparison between wild type and TRPV1cre::DTAfl/wt mice demonstrated no significant immune population differences at baseline. Thus, wildtype and TRPV1cre::DTAfl/wt mice were orthotopically implanted with tumor and tumoral immune cell populations identified by flow cytometry. Cytokine and peptide concentrations were measured by ELISA. In vitro assays were preformed to molecularly define the neuronal signals and immune responses. Here, co-culture experiments of immortalized head and neck cancer cell lines and dorsal root ganglia (DRG) from wildtype mice were used to model tumor innervation in vitro. The media generated was applied to bone marrow derived (BMD) immune cells and the impact on immune cell phenotypes assayed by flow cytometry. Our orthotopic model demonstrates that the presence of HNSCC promotes a nerve-injury phenotype in ipsilateral trigeminal ganglion, demonstrated by increased expression of activating transcription factor 3 (ATF3). Utilizing a 12-color flow cytometry panel we show that tumors from TRPV1cre::DTAfl/wt mice harbor a significantly smaller population of myeloid derived suppressor cells (MDSCs) compared to those from wildtype animals. Further analysis demonstrates this population shift occurs primarily due to a decrease in the CD11b+ Ly6G+/Ly6Clo population. In vitro studies using BMD immune cells show that tumor cell-DRG co-culture conditioned media induces an expansion of the MDSC population compared to condition media from DRG or tumor cells alone. Additionally, transwell migration assays show that co-culture condition media also increases cellular migration compared to condition media from DRG or tumor cells alone. Moreover, ELISA analysis indicates that co-culture of tumor cells and DRG increases neuronal release of the neuropeptide, Substance P (SP), potentiating IL-6 release from tumor cells. In summary, we identified several mechanisms by which TRPV1-expressing intra-tumoral neurons promote MDSC infiltration at the tumor bed and promote disease progression. These mechanisms define targets for therapeutic intervention including SP and IL-6 signaling. Future studies will test the clinical utility of these targets. Together, our data suggests that intra-tumoral neurons contribute to cancer progression through generation of a tumor supportive microenvironment. Citation Format: Anthony C. Restaino, Maryam Ahmadi, Sebastien Talbot, Paola D. Vermeer. TRPV1+ sensory neurons provide a tumor supportive environment through recruitment of MDSCs [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-088.

The Cxcr2+ subset of the S100a8+ gastric granylocytic myeloid-derived suppressor cell population (G-MDSC) regulates gastric pathology.

Gastric myeloid-derived suppressor cells (MDSCs) are a prominent population that expands during gastric pre-neoplastic and neoplastic development in humans and mice. However, the heterogeneity of this population has circumvented the ability to study these cells or understand their functions. Aside from Schlafen-4+ (Slfn-4+) MDSCs in mouse studies, which constitute a subset of this population, limitations exist in characterizing the heterogeneity of the gastric CD11b+Ly6G+ population and targeting its different subsets. Here we identify S100a8 as a pan-specific marker for this population and utilize it to study the role of the S100a8+Cxcr2+ subset. We profiled gastric CD11b+Ly6G+ versus CD11b+Ly6G- myeloid cells by transcriptomic and single-cell RNA sequencing. We identified S100a8 as a pan-specific marker of the gastric granulocytic MDSC (G-MDSC) population, and generated S100a8CreCxcr2flox/flox to study the effects of Cxcr2 knockdown. Following 6-months of Helicobacter felis infection, gastric CD11b+Ly6G+ G-MDSCs were highly enriched for the expression of S100a8, S100a9, Slfn4, Cxcr2, Irg1, Il1f9, Hcar2, Retnlg, Wfdc21, Trem1, Csf3R, Nlrp3, and Il1b. The expression of these distinct genes following 6mo H. felis infection marked heterogeneous subpopulations, but they all represented a subset of S100a8+ cells. S100a8 was identified as a pan-marker for CD11b+Ly6G+ cells arising in chronic inflammation, but not neutrophils recruited during acute gut infection. 6mo Helicobacter felis-infected S100a8CreCxcr2flox/flox mice exhibited worsened gastric metaplastic pathology than Cxcr2flox/flox mice, which was associated with dysregulated lipid metabolism and peroxidation. S100a8 is a pan-specific marker that can be used to target gastric G-MDSC subpopulations, of which the Cxcr2+ subset regulates gastric immunopathology and associates with the regulation of lipid peroxidation.

Arachidonic acid activates NLRP3 inflammasome in MDSCs via FATP2 to promote post-transplant tumour recurrence in steatotic liver grafts

The steatotic grafts have been applied in liver transplantation frequently owing to the high incidence of non-alcoholic fatty liver disease. However, fatty livers are vulnerable to graft injury. Myeloid-derived suppressor cell (MDSC) recruitment during liver graft injury promotes tumour recurrence. Lipid metabolism exerts the immunological influence on MDSCs in tumour progression. Here, we aimed to explore the role and mechanism of inflammasome activation in MDSCs induced by lipid metabolism during fatty liver graft injury and the subsequent effects on tumour recurrence. MDSC populations and nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome levels were investigated in a clinical cohort and a rat liver transplantation model. The mechanism of NLRP3 activation by specific fatty acids was explored in mouse hepatic ischaemia/reperfusion injury (IRI) with tumour recurrence model and invitro studies. MDSC populations and NLRP3 levels were increased with higher tumour recurrent rate in patients using steatotic grafts. NLRP3 was upregulated in MDSCs with lipid accumulation post mouse fatty liver IRI. Mechanistically, arachidonic acid was discovered to activate NLRP3 inflammasome in MDSCs through fatty acid transport protein 2 (FATP2), which was identified by screening lipid uptake receptors. The mitochondrial dysfunction with enhanced reactive oxygen species bridged arachidonic acid uptake and NLRP3 activation in MDSCs, which subsequently stimulated CD4+ T cells producing more IL-17 in fatty liver IRI. Blockade of FATP2 inhibited NLRP3 activation in MDSCs, IL-17 production in CD4+ T cells, and the tumour recurrence post fatty liver IRI. During fatty liver graft injury, arachidonic acid activated NLRP3 inflammasome in MDSCs through FATP2, which subsequently stimulated CD4+ T cells producing IL-17 to promote tumour recurrence post transplantation. The high incidence of non-alcoholic fatty liver disease resulted in the frequent application of steatotic donors in liver transplantation. Our data showed that the patients who underwent liver transplantation using fatty grafts experienced higher tumour recurrence. We found that arachidonic acid activated NLRP3 inflammasome in MDSCs through FATP2 during fatty liver graft injury, which led to more IL-17 secretion of CD4+ T cells and promoted tumour recurrence post transplantation. The inflammasome activation by aberrant fatty acid metabolism in MDSCs bridged the acute-phase fatty liver graft injury and liver tumour recurrence.