Immunosuppressive Mediators Research Articles

Abstract B007: CD163+ tumor-associated macrophage evasion contributes radiation resistance and poor prognosis in estrogen receptor-negative breast cancer

Abstract Patients with estrogen receptor-negative (ER-) breast cancer demonstrate worse clinical outcomes and poor prognosis from post-mastectomy radiation therapy (PM-RT) than those with ER-positive (ER+) breast tumors. RT modulates the tumor microenvironment (TME) dynamic and its impact on immune evasion and resistance remains unclear. In this prospective study, we explore the role of CD163, a scavenger receptor protein primarily expressed in monocytes and M2-like tumor-associated macrophages (TAM), in driving local and systemic immunosuppression and immune evasion after RT, particularly in ER- tumors. For this, ER- and ER+ biopsy breast tumor tissues (n=45) and peripheral blood samples were collected from pre-RT and post-RT exposed patients. A tumor transcriptomic profiling was performed using the NanoString platform, and peripheral blood mononuclear cells and plasma cytokines levels were analyzed through flow cytometry. Patient-derived monocytes were sorted, polarized into M2C macrophages, and co-cultured with healthy T cells to assess the immunosuppressive role of M2C-CD163, with and without the anti-CD163 monoclonal antibody (OR2805). Further, an orthotopic murine breast cancer model (4T1; ER-/TNBC cells) was developed to confirm M2-CD163's role in immunosuppression following RT and its influence on the response to checkpoint immunotherapy. The Cancer Genome Atlas (TCGA) analysis revealed that higher expression of CD163 transcripts associated with poor overall survival in ER- but not in ER+ breast cancer patients, and our data suggest that RT significantly increases CD163, the transcriptomic signature of TAM markers (MRC1, ATF3, DUSP1, PTPRC, DAB2) as well as upregulates several transcription factors (STAT1, STAT2, CEBPB, NFKB1A) that involved in TAM-mediated immunosuppression. Intriguingly, RT-induced immune evasion to ER- breast tumors, elevates CD163 in the plasma and increases monocyte plasticity toward an M2-like phenotype. These monocyte-derived macrophages suppressed T cell activity, highlighting the strong immunosuppressive potential of monocytes from irradiated ER- breast tumors. Inhibition of CD163 by monoclonal antibody reversed the RT-induced immunosuppression, reprogramming monocytes/macrophages to a pro-inflammatory M1 phenotype and restoring T cell activity. In addition, RT promoted myeloid cell differentiation into CD163+M2-like macrophages, leading to immunosuppressive changes within the tumor and systemic circulation in mice in vivo. Pharmacologic depletion of CD163+ macrophages in combination with RT improved overall survival and enhanced tumor responsiveness to checkpoint immunotherapy. In conclusion, our data suggest CD163 is a key mediator of radiation-induced immunosuppression in ER- breast cancer, and pharmacological targeting of CD163 in aggressive and advanced ER- breast tumors may improve radiation response and patient outcomes. Citation Format: Subhajit Ghosh, Suryakant Niture, Jerry Jaboin, Danushka Seneviratne. CD163+ tumor-associated macrophage evasion contributes radiation resistance and poor prognosis in estrogen receptor-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B007.

Taking a Swing at TIMP1-Armed Immunosuppressive Astrocytes Unleashes T cell Immunity against Brain Metastases.

Priego and colleagues identify a secreted glycoprotein TIMP1, expressed downstream of the transcription factor STAT3, in a subpopulation of STAT3+ reactive astrocytes as a mediator of immunosuppression in late-stage brain metastases. The STAT3 inhibitor silibinin enhances the preclinical efficacy of the combined PD-1/CTLA4 immune checkpoint blockade, providing a rationale to translate the combination therapy into clinical use for this underserved patient group with poor prognosis. See related article by Priego et al., p. 179.

FC01 Higher IL-10+ T-cell and Treg-cell counts in psoriatic skin are associated with super-response to guselkumab: data from the Phase 3b GUIDE trial

Abstract Interleukin-10 (IL-10) and regulatory T (Treg) cells are immunosuppressive mediators with key roles in autoimmunity. Their increased presence is believed to counter-regulate pathogenic tissue-resident memory (TRM) and T-helper (Th)17 cells in psoriasis. The ongoing randomized, double-blinded, controlled Phase 3b GUIDE trial (NCTO3818035) examines the clinical and immunological impact of an extended guselkumab [an interleukin (IL)-23p19 subunit inhibitor] dosing interval in patients with moderate-to-severe plaque-type psoriasis. Patients who achieved a psoriasis area and severity index (PASI) = 0 at both Week [W]20 and W28 with guselkumab treatment were defined as super-responders (SRes). In this mechanistic substudy of GUIDE, we aimed to determine whether SRes show immunological differences in skin T-cell composition vs. non-SRes, using flow cytometry analysis of non-lesional (at W0) and lesional (at W0, W4, W28 and W68) skin cells taken from 63 patients. At ­baseline, IL-10+ T-cell counts were higher in both non-lesional and lesional skin of SRes vs. non-SRes (with an SRe to non-SRe ratio of 3.1:1 in non-lesional skin, and a 3.0:1 ratio in lesional skin; P < 0.05). This trend was maintained in lesional skin after treatment with guselkumab, with higher IL-10+ T-cell counts observed in SRes than non-SRes at W4 (with an SRe to non-SRe ratio of 4.6:1; P < 0.05) and at W28 (with a ratio of 4.4:1; P < 0.05). Treg-cell (CD4+CD25+FoxP3+) counts were also higher in SRes at W4 and W28 (with an SRe to non-SRe ratio of 3.1:1 at W4, and a ratio of 2.9:1 at W28; P < 0.05). Further analysis of effector T-cell subsets showed that CD8+ TRM and IL-17A+ T-cell counts in lesional skin were reduced by guselkumab up to W68. Normalization of these T-cell populations in lesional skin to non-lesional skin levels appeared to be earlier in SRes (W28) compared with non-SRes (W68). These findings from GUIDE suggest that clinical super-response to guselkumab may be characterized by higher IL-10+ T-cell and Treg-cell counts, and faster normalization of CD8+ TRM and IL-17A+ T-cell counts. Therefore, higher IL-10+ T-cell and Treg-cell counts may act as predictive biomarkers for a better response to guselkumab in patients with moderate-to-severe plaque-type psoriasis.

Gold-siRNA supraclusters enhance the anti-tumor immune response of stereotactic ablative radiotherapy at primary and metastatic tumors.

Strategies to enhance the anti-tumor immune response of stereotactic ablative radiotherapy (SABR) at primary tumors and abscopal sites are under intensive investigation. Here we report a metabolizable binary supracluster (BSCgal) that combines gold nanoclusters as radiosensitizing adjuvants with small interfering RNA (siRNA) targeting the immunosuppressive mediator galectin-1 (Gal-1). BSCgal comprises reversibly crosslinked cationic gold nanoclusters and siRNA complexes in a polymer matrix that biodegrades over weeks, facilitating clearance (90.3% in vivo clearance at 4 weeks) to reduce toxicity. The particle size well above the renal filtration threshold facilitates passive delivery to tumors. Using mouse models of head and neck cancer, we show that BSCgal augments the radiodynamic and immunotherapeutic effects of SABR at the primary and metastatic tumors by promoting tumor-inhibitory leukocytes, upregulating cytotoxic granzyme B and reducing immunosuppressive cell populations. It outperforms SABR plus Gal-1 antagonists, chemoradiation drug cisplatin or PD-1 inhibitor. This work presents a translatable strategy to converge focal radiosensitization with targeted immune checkpoint silencing for personalized radioimmunotherapy.

Intracellular osteopontin potentiates the immunosuppressive activity of mesenchymal stromal cells

IntroductionMesenchymal stromal cell (MSC)-based cell therapy is a promising approach for various inflammatory disorders based on their immunosuppressive capacity. Osteopontin (OPN) regulates several cellular functions including tissue repair, bone metabolism and immune reaction. However, the biological function of OPN in regulating the immunosuppressive capacity of MSCs remains elusive.ObjectivesThis study aims to highlight the underlying mechanism of the proinflammatory cytokines affect the therapeutic ability of MSCs through OPN.MethodsMSCs in response to the proinflammatory cytokines were collected to determine the expression profile of OPN. In vitro T-cell proliferation assays and gene editing were performed to check the role and mechanisms of OPN in regulating the immunosuppressive capacity of MSCs. Inflammatory disease mouse models were established to evaluate the effect of OPN on improving MSC-based immunotherapy.ResultsWe observed that OPN, including its two isoforms iOPN and sOPN, was downregulated in MSCs upon proinflammatory cytokine stimulation. Interestingly, iOPN, but not sOPN, greatly enhanced the immunosuppressive activity of MSCs on T-cell proliferation and thus alleviated the inflammatory pathologies of hepatitis and colitis. Mechanistically, iOPN interacted with STAT1 and mediated its deubiquitination, thereby inducing the master immunosuppressive mediator inducible nitric oxide synthase (iNOS) in MSCs. In addition, iOPN expression was directly downregulated by activated STAT1, which formed a negative feedback loop to restrain MSC immunosuppressive capacity.ConclusionOur findings demonstrated that iOPN expression modulation in MSCs is a novel strategy to improve MSC-based immunotherapy.

Large and small extracellular vesicles from Wharton’s jelly MSCs: Biophysics, function, and strategies to improve immunomodulation

Extracellular vesicles (EVs) have emerged as mediators of immunosuppression and pro-regenerative processes, particularly through mesenchymal stromal cells (MSCs) across various disease models. Despite significant progress, there is still a need for a deeper understanding of EV content and functionality to fully harness their biomedical potential. Moreover, strategies to enhance EV production for clinical scalability are still under development. This study aimed to characterize two distinct types of EV-large EV (lgEV) and small EV (smEV)-secreted by Wharton's jelly MSCs (WJ-MSCs). Strategies were explored to augment both EV production and their immunoregulatory effects. Both lgEV and smEV displayed typical EV markers and demonstrated inhibition of human lymphocyte proliferation. Furthermore, analysis of IsomiR content revealed a pronounced immunomodulating signature within MSC-derived EVs, validated by a dual-fluorescence reporter system. MSC primed with pro-inflammatory cytokines yielded increased production of lgEV and smEV, enhancing their immunomodulatory potency. Finally, genetically engineering WJ-MSC to express CD9 resulted in lgEV and smEV with heightened efficacy in suppressing lymphocyte proliferation. This study successfully isolated, characterized, and demonstrated the potent immunosuppressive effect of WJ-MSC-derived lgEV and smEV. We propose cytokine preconditioning and genetic manipulation as viable strategies to enhance the therapeutic potential of WJ-MSC-derived EV in inflammatory conditions.

Crucial role of dendritic cells in the generation of anti-tumor T-cell responses and immunogenic tumor microenvironment to suppress tumor development.

Dendritic cells (DCs) are known as unique professional antigen (Ag)-presenting cells (APCs) to prime naïve T cells for the initiation of adaptive immunity. While DCs are believed to play a pivotal role in generating anti-tumor T-cell responses, the importance of DCs in the protection from the progression of tumors remains elusive. Here, we show how the constitutive deficiency of CD11chi DCs influences the progression of tumors with the use of binary transgenic mice with constitutive loss of CD11chi DCs. Constitutive loss of CD11chi DCs not only enhances the progression of tumors but also reduces the responses of Ag-specific T cells. Furthermore, the congenital deficiency of CD11chi DCs generates the immunosuppressive tumor microenvironment (TME) that correlates with the marked accumulation of myeloid-derived suppressor cells (MDSCs) and the prominent productions of immunosuppressive mediators. Thus, our findings suggest that CD11chi DCs are crucial for generating anti-tumor T-cell responses and immunogenic TME to suppress the development of tumors.

The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma.

While immunotherapy has shown efficacy in lung adenocarcinoma (LUAD) patients, many respond only partially or not at all. One limitation in improving outcomes is the lack of a complete understanding of immune checkpoint regulation. Here, we investigated a possible link between an environmental chemical receptor implicated in lung cancer and immune regulation, (the aryl hydrocarbon receptor/AhR), a known but counterintuitive mediator of immunosuppression (IFNγ), and regulation of two immune checkpoints (PD-L1 and IDO). AhR gene-edited LUAD cell lines, a syngeneic LUAD mouse model, bulk- and scRNA sequencing of LUADs and tumor-infiltrating leukocytes were used to map out a signaling pathway leading from IFNγ through the AhR to JAK/STAT, PD-L1, IDO, and tumor-mediated immunosuppression. The data demonstrate that: 1) IFNγ activation of the JAK/STAT pathway leading to PD-L1 and IDO1 upregulation is mediated by the AhR in murine and human LUAD cells, 2) AhR-driven IDO1 induction results in the production of Kynurenine (Kyn), an AhR ligand, which likely mediates an AhR→IDO1→Kyn→AhR amplification loop, 3) transplantation of AhR-knockout LUAD cells results in long-term tumor immunity in most recipients. 4) The 23% of AhR-knockout tumors that do grow do so at a much slower pace than controls and exhibit higher densities of CD8+ T cells expressing markers of immunocompetence, increased activity, and increased cell-cell communication. The data definitively link the AhR to IFNγ-induced JAK/STAT pathway and immune checkpoint-mediated immunosuppression and support the targeting of the AhR in the context of LUAD.

Longitudinal Intravascular Antibody Labeling Identified Regulatory T Cell Recruitment as a Therapeutic Target in a Mouse Model of Lung Cancer.

Anticancer immunity is predicated on leukocyte migration into tumors. Once recruited, leukocytes undergo substantial reprogramming to adapt to the tumor microenvironment. A major challenge in the field is distinguishing recently recruited from resident leukocytes in tumors. In this study, we developed an intravascular Ab technique to label circulating mouse leukocytes before they migrate to tissues, providing unprecedented insight into the kinetics of recruitment. This approach unveiled the substantial role of leukocyte migration in tumor progression using a preclinical mouse model of lung adenocarcinoma. Regulatory T cells (Tregs), critical mediators of immunosuppression, were continuously and rapidly recruited into tumors throughout cancer progression. Moreover, leukocyte trafficking depended on the integrins CD11a/CD49d, and CD11a/CD49d blockade led to significant tumor burden reduction in mice. Importantly, preventing circulating Treg recruitment through depletion or sequestration in lymph nodes was sufficient to decrease tumor burden, indicating that Treg migration was crucial for suppressing antitumor immunity. These findings underscore the dynamic nature of the immune compartment within mouse lung tumors and demonstrate the relevance of a temporal map of leukocyte recruitment into tumors, thereby advancing our understanding of leukocyte migration in the context of tumor development.

Immunomodulatory alpha neutrophils: The first-in-class neutrophil progenitor-based allogeneic immuno-cell therapy with cytotoxic and immunomodulatory functionality for the treatment of solid tumours.

e14523 Background: Adoptive cell therapy for the treatment of cancer has largely focused on the administration of autologous engineered αβ T cells. Although successful in some haematological malignancies, the solid tumour microenvironment (TME) poses significant challenges to the efficacy of CAR-T cells, including physical barriers, heterogeneous antigen expression or antigen escape and immunosuppressive mediators. Remodeling the immunosuppressive solid TME is pivotal to the success of immunotherapies. Recent studies have highlighted key roles for neutrophils in tumour control and in facilitating successful responses to cancer immunotherapy, with neutrophil functional heterogeneity within the TME becoming increasingly appreciated. Methods: At LIfT Biosciences we have developed the first-in-class, stem cell-derived immuno-cell therapy with anti-cancer neutrophil properties called Immunomodulatory Alpha Neutrophils (IMANs). IMANs are derived from CD34+ stem cells expanded and differentiated in proprietary media compositions in a simple and scalable GMP-compliant manufacturing process. IMANs are designed to recapitulate those neutrophil states that are both cytotoxic and capable of immunomodulating the solid TME for long-lasting tumour control. Off-the-shelf, allogeneic, innate IMANs have the potential to provide major clinical benefits in the treatment of multiple solid tumours. Results: IMANs multimodal mechanisms of action (MoA) have been demonstrated in different state-of-the-art solid tumour models, including co-culture with patient-derived xenograft organoids (PDX-O) and with 3D ex vivo patient tissues (tumour-on-a-chip). Direct antigen-independent cytotoxicity towards PDX-O from a range of solid tumour indications has been demonstrated. Additionally, and fundamental to IMANs MoA is their ability to migrate to the solid TME and recruit and activate recipients’ anti-tumour immune responses. We have demonstrated IMANs ability to rapidly migrate into the solid TME and promote recruitment of patients’ immune cells leading to enhanced tumour cell death using tumour-on-a-chip technology accompanied by daily live cell imaging. Mechanistically, co-culture with IMANs enhanced expression of co-stimulatory receptors on T and NK cells, such as OX40 and 4-1BB, as well as promoting secretion of TME-modulating factors, such as CXCL10. Furthermore, co-culture with IMANs enhanced secretion of IFN-γ by activated tumour-infiltrating lymphocytes. Conclusions: These cytotoxic and immunomodulatory functions enable IMANs to remodel the TME, leaving the solid tumour more susceptible to host immune responses.

Neuropilin-1high monocytes protect against neonatal inflammation.

Neonates are susceptible to inflammatory disorders such as necrotizing enterocolitis (NEC) due to their immature immune system. The timely appearance of regulatory immune cells in early life contributes to the control of inflammation in neonates, yet the underlying mechanisms of which remain poorly understood. In this study, we identified a subset of neonatal monocytes characterized by high levels of neuropilin-1 (Nrp1), termed Nrp1high monocytes. Compared with their Nrp1low counterparts, Nrp1high monocytes displayed potent immunosuppressive activity. Nrp1 deficiency in myeloid cells aggravated the severity of NEC, whereas adoptive transfer of Nrp1high monocytes led to remission of NEC. Mechanistic studies showed that Nrp1, by binding to its ligand Sema4a, induced intracellular p38-MAPK/mTOR signaling and activated the transcription factor KLF4. KLF4 transactivated Nos2 and enhanced the production of nitric oxide (NO), a key mediator of immunosuppression in monocytes. These findings reveal an important immunosuppressive axis in neonatal monocytes and provide a potential therapeutic strategy for treating inflammatory disorders in neonates.

Pan-Cancer Proteomics Analysis Reveals Wiskott-Aldrich Syndrome Protein as a Potential Regulator of Programmed Death-Ligand 1.

The programmed death-ligand 1 (PD-L1) is a key mediator of immunosuppression in the tumor microenvironment. The expression of PD-L1 in cancer cells is useful for the clinical determination of an immune checkpoint blockade (ICB). However, the regulatory mechanism of the PD-L1 abundance remains incompletely understood. Here, we integrated the proteomics of 52 patients with solid tumors and examined immune cell infiltration to reveal PD-L1-related regulatory modules. Wiskott-Aldrich syndrome protein (WASP) was identified as a potential regulator of PD-L1 transcription. In two independent cohorts containing 164 cancer patients, WASP expression was significantly associated with PD-L1. High WASP expression contributed to immunosuppressive cell composition, including cells positive for immune checkpoints (PD1, CTLA4, TIGIT, and TIM3), FoxP3+ Treg cells, and CD163+ tumor-associated macrophages. Overexpression of WASP increased, whereas knockdown of WASP decreased the protein level of PD-L1 in cancer cells without alteration of PD-L1 protein stability. The WASP-mediated cell migration and invasion were markedly attenuated by the silence of PD-L1. Collectively, our data suggest that WASP is a potential regulator of PD-L1 and the WASP/PD-L1 axis is responsible for cell migration and an immunosuppressive microenvironment.

Abstract 6819: Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma

Abstract Intrahepatic cholangiocarcinoma (CCA) is an aggressive biliary tract cancer that carries unfavorable prognosis. 10-20% of intrahepatic CCAs harbor a mutation in IDH1 that can be targeted with mutant IDH1-specific inhibitors. However, objective and durable responses to treatment with mutant IDH1 inhibitors are rare in CCA. Mutant IDH1 has neomorphic enzymatic activity that produces the oncometabolite D-2-hydroxyglutarate (D-2-HG). D-2-HG promotes biliary tumor formation through cancer cell-intrinsic mechanisms but is also a paracrine factor in the tumor microenvironment (TME). Our group and others have identified that IDH1-mutant CCAs are not responsive to immunotherapy treatment approaches in part due to decreased CD8+ T cell infiltration and an increase in immunosuppressive macrophage cells in the TME. To better understand the immunosuppressive features of the IDH1-mutant CCA TME, we generated an isogenic cell line panel of IDH1-mutant (MUT) and IDH1-wild type (WT) mouse cholangiocarcinoma cells in the SB1 cell line background through a CRISPR homology directed repair approach. Compared to IDH1-WT cells, IDH1-MUT cells produce high levels of D-2-HG and both cell line panels readily form tumors in immunocompetent C57BL/6 mice. Tumors formed from IDH1-mut cells have reduced CD8+ T cell infiltration compared to IDH1-WT tumors, a finding that correlates with analysis of human CCA samples. Using cytometry by time of flight (CyTOF), we identified that CD8+ T cells in IDH1-MUT tumors have decreased expression of key regulators of the antitumor immune response including EOMES, TBET, and GZMB compared to those isolated from IDH1-WT tumors. Analysis of secreted factors from these IDH1-MUT and IDH1-WT cells identified that IDH1-MUT cells release increased levels of CCL2, a chemokine shown to diminish the antitumor immune response across multiple cancer types, compared to IDH1-WT cells. Further, IDH1-MUT tumors grown in syngeneic, immunocompetent mice have increased CCL2 staining by IHC compared to IDH1-WT tumors. CCL2 expression is also increased in a preliminary analysis of IDH1-mutant human CCA primary tumors. Treatment of mice harboring orthotopic liver tumors formed from IDH1-MUT cells with a CCL2 neutralizing antibody resulted in decreased tumor size compared to isotype control. Together, our data suggest that CCL2 is a mediator of immunosuppression in the IDH1-mutant CCA TME and that modulation of CCL2 activity may improve outcomes in this rare disease subtype. Further work to understand the mechanisms through which IDH1 mutations regulate CCL2 levels in these tumors is underway. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Kiyoko Yoshima, Daniel J. Zabransky, Mark Yarchoan. Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma [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 6819.

Protective effect of FKBP12 on dextran sulfate sodium-induced ulcerative colitis in mice as a tacrolimus receptor

Ulcerative colitis (UC) is a multifactorial intestinal disease with a high incidence. In recent years, there has been an urgent need for pleiotropic drugs with a clear biosafety profile. Tacrolimus (TAC) is an immunosuppressant with stronger in vivo effects and better gastrointestinal absorption and is considered a potential treatment for UC. FKBP12 is a mediator of TAC immunosuppression; however, it is unclear whether it can participate in the development of UC in combination with TAC. The purpose of this study is to preliminarily validate the function of FKBP12 by establishing dextran sulfate sodium (DSS)-induced UC model and TAC treatment. The results revealed that TAC was effective in alleviating DSS-induced UC symptoms such as body weight and disease activity index (DAI). TAC significantly protects colonic tissue and attenuates DSS-induced histomorphological changes. In addition, FKBP12 is down-regulated in the intestinal tissue of DSS-induced UC mice and in serum samples of UC patients. In conclusion, our study revealed that FKBP12 may act as a TAC receptor to have anti-inflammatory and protective effects on DSS-induced UC in mice, which will provide a new option for the treatment of UC.

Glycolysis Induced by METTL14 Is Essential for Macrophage Phagocytosis and Phenotype in Cervical Cancer.

N 6-methyladenosine (m6A) is the most abundant mRNA modification in mammals and it plays a vital role in various biological processes. However, the roles of m6A on cervical cancer tumorigenesis, especially macrophages infiltrated in the tumor microenvironment of cervical cancer, are still unclear. We analyzed the abnormal m6A methylation in cervical cancer, using CaSki and THP-1 cell lines, that might influence macrophage polarization and/or function in the tumor microenvironment. In addition, C57BL/6J and BALB/c nude mice were used for validation invivo. In this study, m6A methylated RNA immunoprecipitation sequencing analysis revealed the m6A profiles in cervical cancer. Then, we discovered that the high expression of METTL14 (methyltransferase 14, N6-adenosine-methyltransferase subunit) in cervical cancer tissues can promote the proportion of programmed cell death protein 1 (PD-1)-positive tumor-associated macrophages, which have an obstacle to devour tumor cells. Functionally, changes of METTL14 in cervical cancer inhibit the recognition and phagocytosis of macrophages to tumor cells. Mechanistically, the abnormality of METTL14 could target the glycolysis of tumors invivo and vitro. Moreover, lactate acid produced by tumor glycolysis has an important role in the PD-1 expression of tumor-associated macrophages as a proinflammatory and immunosuppressive mediator. In this study, we revealed the effect of glycolysis regulated by METTL14 on the expression of PD-1 and phagocytosis of macrophages, which showed that METTL14 was a potential therapeutic target for treating advanced human cancers.

The transcriptomic expression pattern of immune checkpoints shows heterogeneity between and within cancer types.

Transcriptomic expression profiles of immune checkpoint markers are of interest in order to decipher the mechanisms of immunotherapy response and resistance. Overall, 514 patients with various solid tumors were retrospectively analyzed in this study. The RNA expression levels of tumor checkpoint markers (ADORA2A, BTLA, CD276, CTLA4, IDO1, IDO2, LAG3, NOS2, PD-1, PD-L1, PD-L2, PVR, TIGIT, TIM3, VISTA, and VTCN) were ranked from 0-100 percentile based on a reference population. The expression of each checkpoint was correlated with cancer type, microsatellite instability (MSI), tumor mutational burden (TMB), and programmed death-ligand 1 (PD-L1) by immunohistochemistry (IHC). The cohort included 30 different tumor types, with colorectal cancer being the most common (27%). When RNA percentile rank values were categorized as "Low" (0-24), "Intermediate" (25-74), and "High" (75-100), each patient had a distinctive portfolio of the categorical expression of 16 checkpoint markers. Association between some checkpoint markers and cancer types were observed; NOS2 showed significantly higher expression in colorectal and stomach cancer (P < 0.001). Principal component analysis demonstrated no clear association between combined RNA expression patterns of 16 checkpoint markers and cancer types, TMB, MSI or PD-L1 IHC. Immune checkpoint RNA expression varies from patient to patient, both within and between tumor types, though colorectal and stomach cancer showed the highest levels of NOS2, a mediator of inflammation and immunosuppression. There were no specific combined expression patterns correlated with MSI, TMB or PD-L1 IHC. Next generation immunotherapy trials may benefit from individual analysis of patient tumors as selection criteria for specific immunomodulatory approaches.

High hypoxia status in pancreatic cancer is associated with multiple hallmarks of an immunosuppressive tumor microenvironment.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is a particularly lethal disease that is often diagnosed late and is refractory to most forms of treatment. Tumour hypoxia is a key hallmark of PDAC and is purported to contribute to multiple facets of disease progression such as treatment resistance, increased invasiveness, metabolic reprogramming, and immunosuppression. We used the Buffa gene signature as a hypoxia score to profile transcriptomics datasets from PDAC cases. We performed cell-type deconvolution and gene expression profiling approaches to compare the immunological phenotypes of cases with low and high hypoxia scores. We further supported our findings by qPCR analyses in PDAC cell lines cultured in hypoxic conditions. First, we demonstrated that this hypoxia score is associated with increased tumour grade and reduced survival suggesting that this score is correlated to disease progression. Subsequently, we compared the immune phenotypes of cases with high versus low hypoxia score expression (HypoxiaHI vs. HypoxiaLOW) to show that high hypoxia is associated with reduced levels of T cells, NK cells and dendritic cells (DC), including the crucial cDC1 subset. Concomitantly, immune-related gene expression profiling revealed that compared to HypoxiaLOW tumours, mRNA levels for multiple immunosuppressive molecules were notably elevated in HypoxiaHI cases. Using a Random Forest machine learning approach for variable selection, we identified LGALS3 (Galectin-3) as the top gene associated with high hypoxia status and confirmed its expression in hypoxic PDAC cell lines. In summary, we demonstrated novel associations between hypoxia and multiple immunosuppressive mediators in PDAC, highlighting avenues for improving PDAC immunotherapy by targeting these immune molecules in combination with hypoxia-targeted drugs.

Correlation of Intra-tumoral and Peripheral PD-1/PD-L1 Immunity in Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) represents a heterogeneous malignant disease of the oral cavity, pharynx, and larynx. HNSCC cells evade the host immune system through alterations in their immunogenicity, production of immunosuppressive mediators, and induction of immunomodulatory cell types. The immune status of solid HNSCC can be considered as hot, cold, or excluded for each patient individually, based on the distribution of tumor infiltrating immune cells. In this context immunotherapies based on the blockade of checkpoint molecules programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) have significantly improved therapeutic outcomes in different cancer types. In HNSCC, intra-tumoral expression levels of PD-L1 are used for decision making in checkpoint inhibitor treatment. The significance of PD-L1 as a prognostic indicator is still controversial because both PD-1 and PD-L1 are also expressed in different types of circulating immune cells and the interaction of systemic and intra-tumoral cell-type-specific expression patterns of checkpoint molecules PD-1/PD-L1 has not yet been fully unveiled. Using immunohistochemical (IHC) staining and flow cytometry, we correlated the expression patterns of the checkpoint molecules PD1/PD-L1 in peripheral blood CD14/CD16 monocytes and CD4/CD8 T cells with intra-tumoral conditions in patients with head and neck cancer. Our data demonstrate significant connections between systemic and intra-tumoral PD-1/PD-L1 immune patterns, both of which may serve as promising combined biomarkers for treatment decisions in patients with head and neck cancer.

IFN-γ and TNF-α Synergistically Induce Mesenchymal Stem/Stromal Cell Death Via RIPK1-Independent Necroptosis

Interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) are two vital inflammatory factors elevated in many diseases (e.g. aplastic anemia), and an inflammatory microenvironment can cause cell damage and may be involved in the pathogenesis of certain diseases. However, the precise mechanisms of cell dysfunction or impairment during the inflammatory process are not fully understood. IFN-γ alone or in combination with TNF-α have distinct effects on the immunoregulatory properties of bone marrow derived mesenchymal stem cells (MSCs), and they have been raised to be optimal prime factors to enhance the immunosuppressive capacity of MSCs engineered in vitro. Yet controversies remain with regards to the normal function maintenance of cells as they may be impaired after exposure to inflammatory factors. Here, we find that IFN-γ and TNF-α can synergistically induce cell dysfunction and death of MSCs via a RIPK1-independent manner of necroptosis. When MSCs are exposed to both IFN-γ and TNF-α at a broad range of concentrations used in previous studies for about 24 hours or more, its morphological features, biological functions, differentiation capacity and immunoregulatory properties are totally injured. First of all, IFN-γ and TNF-α synergistically induce distinct morphological changes of MSCs. When primed with both IFN-γ and TNF-α, MSCs show a gradual change from a fibroblastic-like spindle shape to an enlarged widespread morphology and the cells grow in a disordered way. And from a high power view, the cells looked like a flat spider web and the blurred cell membrane hints that these cells may have “ruptured”, but most notably, the immunofluorescence staining shows that the nuclei were intact. Secondly, MSCs are functionally impaired after exposure in regard to their capacities of proliferation and migration, which, however, are not associated with increased cell apoptosis or senescence. Furthermore, the adipocytes, osteocytes and chondrocytes differentiation capacities of MSCs are entirely impaired and the immunosuppressive efficacies of MSCs on T cells are also attenuated by the synergistic effect of IFN-γ and TNF-α, though these impaired MSCs retain normal capacity of releasing indoleamine 2,3-dioxygenase (IDO), an immunosuppressive mediator of human MSCs. Mechanistically, we initially find that the signaling of IFN-γ might be enhanced by TNF-α through increasing the abundance of its receptor IFN-γ receptor1 on the surface of MSCs, which is possible to be the interplay mechanism of the two cytokines. And when the injured cells are subjected to RNA-Sequencing, it reveals that those MSCs have undergone a unique cell death, which is necroptosis, a form of programmed cell death characterized by a necrotic morphology including cell swelling, membrane rupture, and releasing of cellular contents, such as inflammatory cytokines, damage-associated molecular patterns (DAMPs), and chemokines, thereby leading to a variety of inflammatory responses subsequently. Compared with the control groups, IFN-γ and TNF-α synergistically increased the expression level of major necroptotic signaling cascade RIPK1, RIPK3 and MLKL into a significant level, and further analysis reveals that all other genes associated with necroptosis (TRADD, TICAM1, ZBP1, TLR3, and TLR4) are significantly upregulated as well. Finally, the rescue experiments targeting signaling pathways of necroptosis show that the necroptotic process of MSCs induced by IFN-γ &amp; TNF-α cannot be reversed by RIPK1 inhibitor (Necrostatin-1), yet RIPK3 and MLKL inhibitor (GSK872 and Necrosulfonamide) hold individual or collaborative effects of protection against it, suggesting the undergoing necroptosis in this setting is independent of RIPK1, and targeting downstream proteins of necroptotic signaling pathway is a promising strategy for inhibiting such a cell death. Collectively, our results disclose an inflammatory injury mechanism of human bone marrow derived MSCs based on its morphological and functional alterations in combination with RNA-Seq identification, and a firstly found necroptotic manner of cell death in MSCs sheds new light on revealing the MSC deficits in some inflammatory or immune-mediated diseases with expectations to innovate some potential therapeutics. Our findings also suggest that cell therapy based on MSCs primed with IFN-γ and TNF-α should take this inflammatory injury effect into consideration.

Notch Signaling Regulates Immunosuppressive Tumor-Associated Macrophage Function in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) continues to have a dismal prognosis. The poor survival of patients with PDA has been attributed to a high rate of early metastasis and low efficacy of current therapies, which partly result from its complex immunosuppressive tumor microenvironment. Previous studies from our group and others have shown that tumor-associated macrophages (TAM) are instrumental in maintaining immunosuppression in PDA. Here, we explored the role of Notch signaling, a key regulator of immune response, within the PDA microenvironment. We identified Notch pathway components in multiple immune cell types within human and mouse pancreatic cancer. TAMs, the most abundant immune cell population in the tumor microenvironment, expressed high levels of Notch receptors, with cognate ligands such as JAG1 expressed on tumor epithelial cells, endothelial cells, and fibroblasts. TAMs with activated Notch signaling expressed higher levels of immunosuppressive mediators, suggesting that Notch signaling plays a role in macrophage polarization within the PDA microenvironment. Genetic inhibition of Notch in myeloid cells led to reduced tumor size and decreased macrophage infiltration in an orthotopic PDA model. Combination of pharmacologic Notch inhibition with PD-1 blockade resulted in increased cytotoxic T-cell infiltration, tumor cell apoptosis, and smaller tumor size. Our work implicates macrophage Notch signaling in the establishment of immunosuppression and indicates that targeting the Notch pathway may improve the efficacy of immune-based therapies in patients with PDA.