Immunosuppressive Myeloid-derived Suppressor Cells Research Articles

Exportin 1 governs the immunosuppressive functions of myeloid-derived suppressor cells in tumors through ERK1/2 nuclear export.

Myeloid-derived suppressor cells (MDSCs) are a main driver of immunosuppression in tumors. Understanding the mechanisms that determine the development and immunosuppressive function of these cells could provide new therapeutic targets to improve antitumor immunity. Here, using preclinical murine models, we discovered that exportin 1 (XPO1) expression is upregulated in tumor MDSCs and that this upregulation is induced by IL-6-induced STAT3 activation during MDSC differentiation. XPO1 blockade transforms MDSCs into T-cell-activating neutrophil-like cells, enhancing the antitumor immune response and restraining tumor growth. Mechanistically, XPO1 inhibition leads to the nuclear entrapment of ERK1/2, resulting in the prevention of ERK1/2 phosphorylation following the IL-6-mediated activation of the MAPK signaling pathway. Similarly, XPO1 blockade in human MDSCs induces the formation of neutrophil-like cells with immunostimulatory functions. Therefore, our findings revealed a critical role for XPO1 in MDSC differentiation and suppressive functions; exploiting these new discoveries revealed new targets for reprogramming immunosuppressive MDSCs to improve cancer therapeutic responses.

A phase II trial to assess the impact of β2 adrenergic blockade in immune checkpoint inhibitor (ICI)– refractory metastatic triple negative breast cancer (mTNBC).

TPS1141 Background: In PD-L1+ mTNBC patients (pts), the standard of care treatment is chemotherapy and pembrolizumab (P) in the first-line setting. It has been shown that acquired resistance to P occurs as a result of immune exhaustion. Further, our group and others have demonstrated that chronic β2-adrenergic receptor (β-AR) signaling suppresses CD8+ cytotoxic T lymphocytes (CTL) function, drives their exhaustion, and increases the numbers of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the tumor microenvironment (TME), thus supporting tumor proliferation. Consequently, abrogation of β-AR signaling using the pan β-blocker propranolol or β-AR-/- knockout mice increased the intratumoral frequency of CTLs and elevated the CTL:Treg ratio (Bucsek et al. Cancer Res. 2017; PMID: 28819022). Similarly, mouse tumor models also demonstrated decreased exhaustion markers (PD1, TIM3, LAG3) on CTLs when β-AR was blocked, via propranolol (Qiao G et al. Cancer Immunol Res. 2021, PMID: 33762351). Confirming this phenomenon, we have shown, in a prospective clinical trial in metastatic melanoma, that β-AR blockade with propranolol significantly increased response to P with an objective response rate (ORR) of 78%, as opposed to 30-40% with P monotherapy (Gandhi et al, Clin Cancer Res 2021, PMID: 33127652). Therefore, we hypothesize that using propranolol in an ICI refractory population should improve responses to ICIs. Methods: This is a phase II single-arm, non-randomized single-center study for mTNBC pts. Participants will bewomen >=18 yrs with PD-L1+ mTNBC, who have progressed on treatment with chemotherapy and P in the upfront setting. They will continue P every 3 weeks in addition to 30 mg propranolol twice a day, based on our ongoing clinical trial in metastatic melanoma (NCT03384836) which has demonstrated safety with this combination. Treatment will continue until disease progression per irRECIST. The primary endpoint is ORR, defined as complete or partial response to treatment. The secondary endpoint is safety, 6-month progression-free survival and overall survival. As an exploratory endpoint, changes in tumor and blood immune markers will be assessed. In stage 1, n1=12 evaluable pts will be enrolled. If ≥ 1/12 response is observed, then the study will continue to enroll another n2=9 pts for a total of n=21, otherwise it will be suspended for futility. If ≥ 3/21 responses are observed, then the proposed therapy will be considered promising. Pre- and post-treatment tumor biopsies and sequential blood samples will be analyzed for changes in stress-induced biomarkers (epinephrine, norepinephrine, and frequency of CTL, MDSC, Treg) and expression of exhaustion markers (PD1, TIM3, LAG3). The study is currently open. Clinical trial information: NCT05741164 .

A randomized, controlled, phase 2 trial of nivolumab plus standard-dose or low-dose bevacizumab for recurrent glioblastoma (NAVAL).

2072 Background: PD-1/PD-L1 inhibition has demonstrated limited efficacy for recurrent glioblastoma (rGBM) across prior RCTs. VEGF, a proangiogenic factor upregulated in rGBM, contributes to tumor-associated immunosuppression. Preclinical and observational clinical data indicate a potential dose-dependent effect of anti-VEGF therapy on immune response. This RCT evaluated anti-PD-1 plus anti-VEGF therapy for rGBM. Methods: NAVAL was a multicenter, open label, phase 2 RCT that enrolled 90 patients (≥18 years) with GBM at first recurrence. Participants received nivolumab (240 mg IV Q2 weeks), and were 1:1 randomized to concurrent 10 mg/kg bevacizumab ( standard-dose arm) or 3 mg/kg bevacizumab ( low-dose arm) administered IV biweekly. Stratification factors included age, performance status, extent of resection, and MGMT methylation status. Primary endpoint was overall survival (OS) at 12 months (OS-12). Other outcomes were overall objective response rate (ORR), median progression-free survival (PFS), PFS at 6 months (PFS-6), and safety. Exploratory genomic and immune profiling using CITEseq was performed in 16 patients. Results: 90 patients were enrolled with median age of 60.6 years (range 27-86). 35 patients were MGMT methylated, 53 were unmethylated and 2 were indeterminate. Final analysis had 87 evaluable patients, with median follow-up of 146 days (range 7-1239). There were 3 complete responses (3.4%), 29 partial responses (33.3%), 37 stable disease (42.5%), and 18 progressive disease (20.7%). OS-12 was 41.1% and 37.7% in standard-dose and low-dose arms respectively, with PFS and ORR data in table. Post-hoc analysis demonstrated survival benefit for patients aged ≥60 years in standard-dose arm. Most frequent toxicities (>20%) included fatigue (45.6%), proteinuria (34.4 %), diarrhea (28.9%), hypertension (23.3%) and lipase increase (21.1%). Grade 3-4 toxicities included hypertension (7.8%), fatigue (5.6) and non-neurological toxicities. Differential changes across trial arms were seen in myeloid-derived suppressor cells (MDSCs) post-therapy. Network medicine identified VEGF as target to reduce MDSCs. VEGF was found expressed by MDSCs of mainly responder patients. Differential gene expression analysis identified increased pro-inflammatory gene signatures in standard-dose arm. Conclusions: Overall PFS and OS were similar for nivolumab plus standard- or low-dose bevacizumab for rGBM, with post-hoc survival benefit seen in standard-dose arm in elderly. Standard-dose bevacizumab was associated with increased systemic inflammatory response and reduced immunosuppressive MDSCs. Clinical trial information: NCT03452579 . [Table: see text]

Circadian control of tumor immunosuppression affects efficacy of immune checkpoint blockade.

The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation has an essential function in host defense and tumor immunosurveillance. Here we use a single-cell RNA sequencing approach and a genetic model of colorectal cancer to identify clock-dependent changes to the immune landscape that control the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8+ T cells. Of these immunosuppressive cell types, PD-L1-expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Disruption of the epithelial cell clock regulates the secretion of cytokines that promote heightened inflammation, recruitment of neutrophils and the subsequent development of MDSCs. We also show that time-of-day anti-PD-L1 delivery is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, these data indicate that circadian gating of tumor immunosuppression informs the timing and efficacy of immune checkpoint inhibitors.

Targeting squalene epoxidase restores anti-PD-1 efficacy in metabolic dysfunction-associated steatohepatitis-induced hepatocellular carcinoma

ObjectiveSqualene epoxidase (SQLE) promotes metabolic dysfunction-associated steatohepatitis-associated hepatocellular carcinoma (MASH-HCC), but its role in modulating the tumour immune microenvironment in MASH-HCC remains unclear.DesignWe established hepatocyte-specific Sqle transgenic (tg) and knockout...

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.

Loss of TRIM29 mitigates viral myocarditis by attenuating PERK-driven ER stress response in male mice

Viral myocarditis, an inflammatory disease of the myocardium, is a significant cause of sudden death in children and young adults. The current coronavirus disease 19 pandemic emphasizes the need to understand the pathogenesis mechanisms and potential treatment strategies for viral myocarditis. Here, we found that TRIM29 was highly induced by cardiotropic viruses and promoted protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated endoplasmic reticulum (ER) stress, apoptosis, and reactive oxygen species (ROS) responses that promote viral replication in cardiomyocytes in vitro. TRIM29 deficiency protected mice from viral myocarditis by promoting cardiac antiviral functions and reducing PERK-mediated inflammation and immunosuppressive monocytic myeloid-derived suppressor cells (mMDSC) in vivo. Mechanistically, TRIM29 interacted with PERK to promote SUMOylation of PERK to maintain its stability, thereby promoting PERK-mediated signaling pathways. Finally, we demonstrated that the PERK inhibitor GSK2656157 mitigated viral myocarditis by disrupting the TRIM29-PERK connection, thereby bolstering cardiac function, enhancing cardiac antiviral responses, and curbing inflammation and immunosuppressive mMDSC in vivo. Our findings offer insight into how cardiotropic viruses exploit TRIM29-regulated PERK signaling pathways to instigate viral myocarditis, suggesting that targeting the TRIM29-PERK axis could mitigate disease severity.

Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on HSCs, along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Additionally, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease (GvHD) and cytokine release syndrome (CRS). Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

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

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

Abstract 1331: MiNK-215, an IL-15 armored FAP-targeting CAR iNKT cell therapy, effectively treats human organoid models of treatment-refractory MSS colorectal cancer (CRC) liver metastases

Abstract The presence of liver metastases in patients with microsatellite-stable (MSS) or mismatch repair proficient (pMMR) colorectal cancer (CRC) is associated with poor response to current pharmacological treatments, including immune checkpoint blockade (ICB). The tumor microenvironment (TME) of CRC liver metastases is characterized by a highly immunosuppressive phenotype that includes poor immunogenicity, a paucity of dendritic cells, elimination of tumor-specific effector T cells, and an abundance of immunosuppressive intrahepatic Kupffer cells and myeloid-derived suppressor cells. This underscores the urgent need for innovative therapeutic approaches targeted specifically to patients with CRC liver metastases. Here we describe MiNK-215, an IL-15 armored FAP-targeting CAR iNKT cell therapy, derived from the AgenT-797 allogeneic iNKT platform, as a potential novel therapeutic approach for patients with CRC liver metastases. To better model ICB refractory human MSS CRC liver metastases, we developed an ex vivo human organoid model that recapitulates the histological and immunological features of human disease. Organoids were comprised of MSS CRC cells engineered to express nuclear GFP and HLA-A2-NY-ESO-1, hepatocytes, Kupffer cell-enriched non-parenchymal cells, and HLA-matched peripheral blood mononuclear cells (PBMCs) supplemented with NY-ESO-1-reactive T cells. Organoid models of CRC lung metastases, which are traditionally more responsive to ICB therapy were also developed to compare the models to clinical experience and further validate their usefulness. Models were treated with Fc-enhanced anti-CTLA-4 (botensilimab) and anti-PD-1 (balstilimab) antibodies, or MiNK-215 to assess tumor killing and immune activation. Botensilimab and balstilimab enhanced T cell activation and cytotoxicity of CRC tumor cells in the absence of “normal” liver cells and in lung organoids. However, they were less effective in organoid models of CRC with liver metastases consistent with clinical observations. In contrast, MiNK-215 potently enhanced tumor killing by T cells in models of CRC with liver metastases. Tumor killing in CRC liver organoid models by MiNK-215 was associated with depletion of immune suppressive FAP-expressing stellate cells and increased CD8+ T cell infiltration. Our findings demonstrate that in treatment-refractory CRC-liver metastatic organoid models, MiNK-215 overcomes the limitations of ICB therapy to home to sites of disease, reprogram the TME, recruit tumor-reactive T cells and enhance tumor killing. Ex-vivo human CRC liver and lung metastatic organoid models are a novel platform that can be leveraged to rapidly identify new therapeutic approaches for potential clinical evaluation. Citation Format: Shanmugarajan Krishnan, Bishnu Joshi, Justin Keith, Ryan Frazier, Barbara Kalinowska, Jin San Choi, Stephanie Sanders, Shannon Boi, Olivier Le Tonqueze, Nick Kushner, Kah Teong Soh, Robert Stein, Tyler J. Curiel, Marc A. van Dijk, Enoch Kim, Eleni Chantzoura, Nils-Petter Rudqvist, Dhan Chand. MiNK-215, an IL-15 armored FAP-targeting CAR iNKT cell therapy, effectively treats human organoid models of treatment-refractory MSS colorectal cancer (CRC) liver metastases [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 1331.

Abstract 542: Development of novel syngeneic tumor models with intrinsic or extrinsic resistance mechanisms to PD-1 blockade

Abstract Programmed cell death-1 (PD-1) blockade has demonstrated remarkable clinical efficacy across various tumor types. However, the current PD-1 blockade monotherapy benefits only a small proportion of patients, and primary or acquired resistance can ultimately lead to cancer progression in clinical settings. Understanding the underlying mechanisms of resistance is both necessary and urgent to enhance clinical outcomes. Increasing evidence suggests that resistance to PD-1 therapy can arise from both tumor-cell-intrinsic and tumor-cell-extrinsic factors. Loss-of-function mutations in the antigen presentation pathway and downstream elements represent intrinsic mechanisms, while tumor-infiltrating immunosuppressive cells such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) contribute to immune tolerance as extrinsic factors. Currently, the rational design of combination therapies based on these resistance mechanisms remains to be achieved. In this study, we developed two types of anti-PD-1 antibody-resistant models based on the intrinsic anti-PD-1 treatment-sensitive MC38 models. These models include genetically engineered resistant models with B2M or STK11 knockout (KO) and induced resistant models through in vivo progressive drug selection. STK11 KO mutations resulted in reduced expression of PD-L1 on tumor cells and a lower infiltration of CD8+ cytotoxic T lymphocytes in the tumor microenvironment, while B2M KO led to a lack of antigen presentation on the tumor cell surface, blocking the binding of the anti-PD-1 antibody. In terms of the induced anti-PD-1 resistant models, a higher proportion of immunosuppressive cells, including Tregs, MDSCs, and TAMs, were observed in the tumor microenvironment compared to the parental counterparts, contributing to immune escape from PD-1 blockade. These models simulate extrinsic and intrinsic mechanism-based anti-PD-1 resistance, allowing for the identification of combination therapies to overcome resistance to anti-PD-1. Our study enhances our understanding of the biological basis of anti-PD-1 resistance and supports the discovery of mechanistically-designed combination therapies to overcome resistance to anti-PD-1 therapy, potentially broadening its clinical applicability. Citation Format: Xiaomin Wang, Ziwei Zhang, Longzhou Liu, Xue Zhang, Dong Wang, Nengwei Xu, Xiangnan Qiang, Qingyang Gu, Jian Xiang, Zhixiang Zhang. Development of novel syngeneic tumor models with intrinsic or extrinsic resistance mechanisms to PD-1 blockade [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 542.

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.

Branched glycopolymer prodrug-derived nanoassembly combined with a STING agonist activates an immuno-supportive status to boost anti-PD-L1 antibody therapy

Despite the great potential of anti-PD-L1 antibodies for immunotherapy, their low response rate due to an immunosuppressive tumor microenvironment has hampered their application. To address this issue, we constructed a cell membrane-coated nanosystem (mB4S) to reverse an immunosuppressive microenvironment to an immuno-supportive one for strengthening the anti-tumor effect. In this system, Epirubicin (EPI) as an immunogenic cell death (ICD) inducer was coupled to a branched glycopolymer via hydrazone bonds and diABZI as a stimulator of interferon genes (STING) agonist was encapsulated into mB4S. After internalization of mB4S, EPI was acidic-responsively released to induce ICD, which was characterized by an increased level of calreticulin (CRT) exposure and enhanced ATP secretion. Meanwhile, diABZI effectively activated the STING pathway. Treatment with mB4S in combination with an anti-PD-L1 antibody elicited potent immune responses by increasing the ratio of matured dendritic cells (DCs) and CD8+ T cells, promoting cytokines secretion, up-regulating M1-like tumor-associated macrophages (TAMs) and down-regulating immunosuppressive myeloid-derived suppressor cells (MDSCs). Therefore, this nanosystem for co-delivery of an ICD inducer and a STING agonist achieved promotion of DCs maturation and CD8+ T cells infiltration, creating an immuno-supportive microenvironment, thus potentiating the therapy effect of the anti-PD-L1 antibody in both 4T1 breast and CT26 colon tumor mice.

Engineered Bacterial Biomimetic Vesicles Reprogram Tumor-Associated Macrophages and Remodel Tumor Microenvironment to Promote Innate and Adaptive Antitumor Immune Responses.

Tumor-associated macrophages (TAMs) are among the most abundant infiltrating leukocytes in the tumor microenvironment (TME). Reprogramming TAMs from protumor M2 to antitumor M1 phenotype is a promising strategy for remodeling the TME and promoting antitumor immunity; however, the development of an efficient strategy remains challenging. Here, a genetically modified bacterial biomimetic vesicle (BBV) with IFN-γ exposed on the surface in a nanoassembling membrane pore structure was constructed. The engineered IFN-γ BBV featured a nanoscale structure of protein and lipid vesicle, the existence of rich pattern-associated molecular patterns (PAMPs), and the costimulation of introduced IFN-γ molecules. In vitro, IFN-γ BBV reprogrammed M2 macrophages to M1, possibly through NF-κB and JAK-STAT signaling pathways, releasing nitric oxide (NO) and inflammatory cytokines IL-1β, IL-6, and TNF-α and increasing the expression of IL-12 and iNOS. In tumor-bearing mice, IFN-γ BBV demonstrated a targeted enrichment in tumors and successfully reprogrammed TAMs into the M1 phenotype; notably, the response of antigen-specific cytotoxic T lymphocyte (CTL) in TME was promoted while the immunosuppressive myeloid-derived suppressor cell (MDSC) was suppressed. The tumor growth was found to be significantly inhibited in both a TC-1 tumor and a CT26 tumor. It was indicated that the antitumor effects of IFN-γ BBV were macrophage-dependent. Further, the modulation of TME by IFN-γ BBV produced synergistic effects against tumor growth and metastasis with an immune checkpoint inhibitor in an orthotopic 4T1 breast cancer model which was insensitive to anti-PD-1 mAb alone. In conclusion, IFN-γ-modified BBV demonstrated a strong capability of efficiently targeting tumor and tuning a cold tumor hot through reprogramming TAMs, providing a potent approach for tumor immunotherapy.

Tumor-specific activation of folate receptor beta enables reprogramming of immune cells in the tumor microenvironment.

Folate receptors can perform folate transport, cell adhesion, and/or transcription factor functions. The beta isoform of the folate receptor (FRβ) has attracted considerable attention as a biomarker for immunosuppressive macrophages and myeloid-derived suppressor cells, however, its role in immunosuppression remains uncharacterized. We demonstrate here that FRβ cannot bind folate on healthy tissue macrophages, but does bind folate after macrophage incubation in anti-inflammatory cytokines or cancer cell-conditioned media. We further show that FRβ becomes functionally active following macrophage infiltration into solid tumors, and we exploit this tumor-induced activation to target a toll-like receptor 7 agonist specifically to immunosuppressive myeloid cells in solid tumors without altering myeloid cells in healthy tissues. We then use single-cell RNA-seq to characterize the changes in gene expression induced by the targeted repolarization of tumor-associated macrophages and finally show that their repolarization not only changes their own phenotype, but also induces a proinflammatory shift in all other immune cells of the same tumor mass, leading to potent suppression of tumor growth. Because this selective reprogramming of tumor myeloid cells is accompanied by no systemic toxicity, we propose that it should constitute a safe method to reprogram the tumor microenvironment.

Extracellular vesicles released by cancer-associated fibroblast-induced myeloid-derived suppressor cells inhibit T-cell function

ABSTRACT Myeloid cells are known to play a crucial role in creating a tumor-promoting and immune suppressive microenvironment. Our previous study demonstrated that primary human monocytes can be polarized into immunosuppressive myeloid-derived suppressor cells (MDSCs) by cancer-associated fibroblasts (CAFs) in a 3D co-culture system. However, the molecular mechanisms underlying the immunosuppressive function of MDSCs, especially CAF-induced MDSCs, remain poorly understood. Using mass spectrometry-based proteomics, we compared cell surface protein changes among monocytes, in vitro differentiated CAF-induced MDSCs, M1/M2 macrophages, and dendritic cells, and identified an extracellular vesicle (EV)-mediated secretory phenotype of MDSCs. Functional assays using an MDSC/T-cell co-culture system revealed that blocking EV generation in CAF-induced MDSCs reversed their ability to suppress T-cell proliferation, while EVs isolated from CAF-induced MDSCs directly inhibited T-cell function. Furthermore, we identified fructose bisphosphatase 1 (FBP1) as a cargo protein that is highly enriched in EVs isolated from CAF-induced MDSCs, and pharmacological inhibition of FBP1 partially reversed the suppressive phenotype of MDSCs. Our findings provide valuable insights into the cell surface proteome of different monocyte-derived myeloid subsets and uncover a novel mechanism underlying the interplay between CAFs and myeloid cells in shaping a tumor-permissive microenvironment.

Epitranscriptional regulation of TGF-β pseudoreceptor BAMBI by m6A/YTHDF2 drives extrinsic radioresistance.

Activation of TGF-β signaling serves as an extrinsic resistance mechanism that limits the potential for radiotherapy. Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) antagonizes TGF-β signaling and is implicated in cancer progression. However, the molecular mechanisms of BAMBI regulation in immune cells and its impact on antitumor immunity after radiation have not been established. Here, we show that ionizing radiation (IR) specifically reduces BAMBI expression in immunosuppressive myeloid-derived suppressor cells (MDSCs) in both murine models and humans. Mechanistically, YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) directly binds and degrades Bambi transcripts in an N6-methyladenosine-dependent (m6A-dependent) manner, and this relies on NF-κB signaling. BAMBI suppresses the tumor-infiltrating capacity and suppression function of MDSCs via inhibiting TGF-β signaling. Adeno-associated viral delivery of Bambi (AAV-Bambi) to the tumor microenvironment boosts the antitumor effects of radiotherapy and radioimmunotherapy combinations. Intriguingly, combination of AAV-Bambi and IR not only improves local tumor control, but also suppresses distant metastasis, further supporting its clinical translation potential. Our findings uncover a surprising role of BAMBI in myeloid cells, unveiling a potential therapeutic strategy for overcoming extrinsic radioresistance.

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.

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

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

Study on the Impact of G-CSF Mobilization on the Level of MDSCs in Donor Peripheral Blood and Its Correlation with Patient Prognosis

Introduction:MDSCs are a newly discovered and heterogeneous population of immature bone marrow cells with immunosuppressive characteristics, capable of inhibiting innate and adaptive immune responses. Granulocyte colony-stimulating factor (G-CSF) is a member of the hematopoietic growth factor cytokine family responsible for the proliferation, maturation, differentiation, and survival of neutrophil lineage. In many hospitals in China, allo-HSCT procedures often involve administering G-CSF continuously for 5 days to healthy donors before collecting the graft, to mobilize white blood cells. This study discusses the impact of G-CSF mobilization on the absolute values and functions of total immunosuppressive myeloid-derived suppressor cells (MDSCs) and their subsets, M-MDSCs and P-MDSCs, in the peripheral blood of healthy donors. Furthermore, it explores the effects of transferring MDSCs from the graft to the patient's body on their prognosis and immune reconstitution. Method：From August 2022 to December 2022, a total of 72 donors and 72 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) were selected as the subjects at Lu Daopei Hospital in Beijing. Among the donors, there were 47 males and 25 females, with a median age of 34 years (19, 48). Among the patients, there were 33 males and 39 females, with a median age of 34 years (23, 45). The patients were diagnosed as acute leukemia or MDS in CR status and further categorized into infection group, graft-versus-host disease (GVHD) group, and no-event group based on post-transplantation outcomes. Peripheral blood samples were collected from the donors before and on the 5th day after mobilization, as well as from the graft and the patients. Flow cytometry was employed to determine the absolute counts of MDSCs and their subsets. Result 1. The absolute count of total MDSCs in the donor's peripheral blood before mobilization was 3/μl (2, 8), accounting for 0.1% (0.0, 0.2) of PBMCs, significantly lower than the absolute count of total MDSCs in the donor's peripheral blood after G-CSF mobilization, which was 2283/μl (1091, 5288), accounting for 6.4% (3.2, 9.5) of PBMCs, P<0.01. In the infection group, the absolute count and percentage of total MDSCs, P-MDSCs, and LOX1+P-MDSCs in the donor's peripheral blood after mobilization were higher than those in the donor's peripheral blood corresponding to the no-event group, and significantly higher than those in the donor's peripheral blood corresponding to the GVHD group. P<0.05. Similarly, the absolute count and percentage of total MDSCs, P-MDSCs, and LOX1+P-MDSCs in the correspondingdonor's peripheral blood inthe infection group after mobilization were higher than those in the donor's peripheral blood corresponding to the no-event group, and significantly higher than those in the donor's peripheral blood corresponding to the GVHD group., P<0.05. The absolute count and percentage of M-MDSCs in the infection group were higher than the no-event group and GVHD group, but the differences among the three did not reach statistical significance. Patients who developed infections after transplantation received a higher absolute count of total MDSCs from the graft compared to the no-event group, and significantly higher than the GVHD group, P<0.05. The mortality rate in the infection group was 30% (8/26), in the GVHD group was 14% (3/21), and in the no-event group was 0% (0/25). The ROC curve area under the curve (AUC) for the absolute count of total MDSCs per kilogram of body weight from the donor in the GVHD group was 0.669, with a cutoff of 1.7×107/kg, while in the infection group, the AUC was 0.661, with a cutoff of 4.3×107/kg. Conclusion: G-CSF can induce an increase in the content and enhancement of function of MDSCs in the donor's peripheral blood Maintaining a balance of MDSCs helps ensure immune homeostasis after transplantation.