Suppression Of Phenotype Research Articles

Abstract P264: Targeting oxygen metabolism reduces hypoxia in the tumor microenvironment of a syngeneic mouse model

Abstract When tumors outgrow their chaotic vasculature, limited diffusion of oxygen into remote tumor areas combined with increased oxygen consumption leads to hypoxia in most solid tumors. This oxygen scarcity is known to induce radioresistance, but may also interfere with the anti-tumor immune response. Hypoxia inhibits immune effector cell function, while immune cells with a more suppressive phenotype become more active. Using the novel mitochondrial complex I inhibitor IACS-010759, we aim to pharmacologically attenuate oxidative phosphorylation (OXPHOS) and subsequently decrease tumor oxygen consumption, hereby relieving hypoxia and sensitizing tumors to both radio- and immunotherapy. Several syngeneic murine cell lines and tumor models on a C57Bl/6 background were used (B16ova, MOC1, MC38 and GL261). In vitro oxygen consumption of these tumor cells was measured using the Agilent XF Seahorse Analyzer and cell growth was measured using the Incucyte ZOOM Live Cell Imaging system. Hypoxia and immune marker expression by these cells in vitro, and in vivo in the tumor microenvironment (TME), were determined using flow cytometry and immunohistochemistry. Tumor hypoxia was calculated as the fraction of tumor area in different zones at increasing distances around vessels on tumor sections. In vitro, IACS-010759 treatment potently inhibits oxygen consumption in a dose dependent manner. Under physiological glucose concentrations (1.5mM) IACS-010759 reduces cell growth at least 2-fold more potently than under high glucose culture conditions. On the other hand, growth is only marginally or not at all inhibited under low oxygen conditions (1.0%). This shows that cancer cells use oxygen when it is available, and tend to rely on glycolysis under hypoxia. This contradicts the longstanding notion that cancer cells primarily use glycolysis, also under aerobic conditions, and suggests that cancer cells are often metabolically plastic. Daily IACS-010759 treatment (10mg/kg) of mice bearing B16ova tumors for 4 or 9 days reduces the hypoxic fraction at increased distance from vessels, presumably by the inhibition of oxygen consumption closer to vessels, allowing oxygen to diffuse further into the tissue. No growth reduction by IACS-010759 treatment was observed in these tumors, showing they are able to use glycolysis for their energy supply. Flow cytometry analysis of these tumors showed no changes in abundance of lymphocyte and myeloid cell populations, suggesting inhibition of OXPHOS has no direct inhibitory effect on immune cell presence. We show that IACS-010759 decreases oxygen consumption in several tumor cell lines in vitro while it also causes growth inhibition if cancer cells rely on oxygen. In vivo, diffusion limited hypoxia can be reduced by the inhibition of OXPHOS, while the abundance of immune cells within the TME was not affected. The observed changes might lead to a more immune permissive TME when induced by radio- and immunotherapy, potentially increasing the efficacy of this treatment combination. Citation Format: Daan F. Boreel, Paul Span, Hans Peters, Renske J.E. van den Bijgaart, Sandra Heskamp, Gosse J. Adema, Johan Bussink. Targeting oxygen metabolism reduces hypoxia in the tumor microenvironment of a syngeneic mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P264.

Immunotherapy for cancer: effects of iron oxide nanoparticles on polarization of tumor-associated macrophages.

Cancer immunotherapy is the most promising trend in oncology, focusing on helping or activating the patient's immune system to identify and fight against cancer. In the last decade, interest in metabolic reprogramming of tumor-associated macrophages from M2-like phenotype (promoting tumor progression) to M1-like phenotypes (suppressing tumor growth) as a therapeutic strategy against cancer has increased considerably. Iron metabolism has been standing out as a target for the reprogramming of tumor-associated macrophages to M1-like phenotype with therapeutic purposes against cancer. Due to the importance of the iron levels in macrophage polarization states, iron oxide nanoparticles can be used to change the activation state of tumor-associated macrophages for a tumor suppressor phenotype and as an anti-tumor strategy.

Abstract PO-124: EZH2 blockade overcomes suppression of the proinflammatory senescence-associated secretory phenotype in the pancreas and drives NK cell-mediated pancreatic tumor responses

Abstract T cell-targeting immunotherapies that produce durable and sometimes curative responses in other malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and tumor immunogenicity. We and others have demonstrated that cellular senescence and its associated secretory phenotype (SASP), which leads to production of proinflammatory cytokines, chemokines, and growth factors, can be a powerful way to reactivate a different type of Natural Killer (NK) cell immunity. Recently, we found that RAS pathway targeting MEK and CDK4/6 inhibitors can induce senescence in KRAS mutant lung tumor and PDAC models; however, therapy-induced senescence (TIS) led to NK cell-mediated tumor regressions only in the lungs. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for PDAC immunotherapy. Syngeneic murine KRAS-driven lung tumor and PDAC cells were transplanted into lungs, pancreas, or liver of C57BL/6 mice. Following 2-week treatment with the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P) to induce senescence, immune responses were assessed by flow cytometry, and the SASP profile assessed by RNA- and ATAC-seq analysis. An NK1.1-targeted antibody was also used to deplete NK cells and evaluate treatment efficacy. shRNA-mediated EZH2 knockdown and treatment with EZH2 methyltransferase inhibitors in murine and human PDAC cells and mouse models was used to determine the role of EZH2 in SASP regulation by qPCR and cytokine array. The effects of EZH2 blockade on NK cell immunity were determined in co-culture migration and cytotoxicity assays in vitro and in PDAC transplant models in vivo by flow cytometry. The efficacy of T/P treatment in combination with EZH2-targeting shRNAs or inhibitors was measured by ultrasound tumor measurements and survival in PDAC-bearing animals. Tumors propagated in the pancreas TME display transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15,-18) and chemotaxis (CCL2,-7,-8; CXCL9,-10) following TIS, and do not undergo anti-tumor NK immune surveillance as compared to tumors grown in the lungs and liver. We identified induction of EZH2 and repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell infiltration and anti-tumor cytotoxicity in PDAC models. Remarkably, EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in PDAC-bearing mice that were reversed following NK cell depletion. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with TIS could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor control. Citation Format: Loretah Chibaya, Yvette Lopez-Diaz, Haibo Liu, Katherine C. Murphy, John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan, Amanda Kulick, Lakhena Leang, Elisa de Stanchina, Lihua J. Zhu, Scott W. Lowe, Marcus Ruscetti. EZH2 blockade overcomes suppression of the proinflammatory senescence-associated secretory phenotype in the pancreas and drives NK cell-mediated pancreatic tumor responses [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-124.

Abstract PR-016: Extrinsic KRAS signaling shapes the pancreatic microenvironment through fibroblast reprogramming

Abstract Pancreatic Ductal Adenocarcinoma (PDA) has an exceedingly poor prognosis with only 10% 5-year survival rate. Kras mutations are found in over 90% of cases of pancreatic cancer and drive the formation of pancreatic intraepithelial neoplasia (PanIN), precursor lesions to PDA. Both PanIN and PDA are characterized by dense stroma, containing fibroblasts and immune cells. The infiltrating immune cells have a suppressive phenotype and prevent anti-tumor immunity by cytotoxic T cells. The mechanisms underlying the immunosuppression in pancreatic cancer are only partially understood. Our goal was to unravel the non-cell autonomous role of oncogenic Kras (Kras*) expressing epithelial cells in driving the formation of a complex, tumor promoting microenvironment during the onset of pancreatic cancer. Our laboratory has described a mouse model (iKras*) of inducible and reversible expression of Kras* in the pancreas. Taking advantage of the reversibility of Kras* expression in this model, we conducted a thorough characterization of the immune infiltration and function upon modulation of Kras* at different stages of pancreatic carcinogenesis. iKras* mice and wild type littermates were enrolled in experiments at the age of 8-12 weeks. Kras* expression was activated, then we induced acute pancreatitis to promote the formation of pre-neoplastic lesions. After 3 weeks, a time when widespread low-grade lesions and fibrosis are observed, mice were either harvested or Kras* expression was inactivated and 3 days or 1 week later the pancreas was harvested. We performed flow cytometry, immunohistochemistry and cytometry time of flight (CyTOF) in the pancreas to analyze myeloid cell populations, as well as functional markers (Arg1, iNOS, IFN). Myeloid cells and T cells infiltrated the pancreas in presence of active Kras*. Inactivation of Kras* resulted in a relatively modest decrease in infiltrating myeloid cells. However, analysis of the functional marker Arg1, a putative immune suppressive molecule expressed in myeloid cells, indicated that its expression depends on Kras*-expressing cells. When we further studied the interactions among cell types using single cell RNA sequencing, we discovered that non-cell autonomous (extrinsic) oncogenic KRAS signaling reprograms pancreatic fibroblasts, activating an inflammatory gene expression program. As a result, fibroblasts become a hub of extracellular signaling, mediating the polarization and function of pro-tumorigenic myeloid cells while also preventing tissue repair. Citation Format: Ashley Velez-Delgado, Katelyn L. Donahue, Kristee L. Brown, Wenting Du, Valerie Irizarry-Negron, Rosa E. Menjivar, Emily L. Lasse-Opsahl, Nina G. Steele, Stephanie The, Jenny Lazarus, Veerin R. Sirihorachai, Filip Bednar, Timothy L. Frankel, Yaqing Zhang, Marina Pasca di Magliano. Extrinsic KRAS signaling shapes the pancreatic microenvironment through fibroblast reprogramming [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-016.

IMMU-05. CCR2+ MYELOID-DERIVED SUPPRESSOR CELLS FROM MURINE GLIOMAS ARE SOURCED FROM THE BONE MARROW, SUPPRESS T CELL ACTIVATION, AND MIGRATE TO CCL2

Abstract INTRODUCTION Mounting evidence suggests infiltrating immune-suppressive cells contribute to immune checkpoint inhibitor resistance and poor survival in Glioblastoma (GBM) patients. We have previously shown glioma-associated monocytic-myeloid derived suppressor cells (M-MDSCs) express chemokine receptors CCR2 and CX3CR1. Genetic and pharmacologic targeting of CCR2 promoted sequestration of M-MDSCs in the bone marrow and, in combination with PD-1 blockade, slowed progression of KR158 and 005GSC murine gliomas. This combination treatment also enhanced infiltration of IFNg-producing T cells that were less exhausted. Although CCR2+/CX3CR1+ cells display surface markers indicative of bone marrow-derived M-MDSCs, additional studies are needed to formally establish the source of these cells and to determine if they exhibit an immune-suppressive phenotype as well as migrate to the CCR2 ligands, CCL2 and/or CCL7. OBJECTIVE Evaluate the source, migration, and immune suppressive function of CCR2+/CX3CR1+ myeloid cells from glioma bearing mice. METHODS To identify the source of CCR2+/CX3CR1+ myeloid cells, chimeric wild type mice harboring bone marrow cells from transgenic CCR2WT/RFP/CX3CR1WT/GFP mice were generated. CCR2+/CX3CR1+ cells were enriched from bone marrow obtained from either wild-type or CCR2WT/RFP/CX3CR1WT/GFP naïve and glioma-bearing mice in order to evaluate their immune suppressive phenotype and ability to migrate to CCL2 and CCL7. RESULTS CCR2+/CX3CR1+ cells are present in glioma isolates from chimeric mice, indicative of a bone marrow-derived cell population, and are detectable within the tumor microenvironment as early as 3 days post orthotopic implantation of KR158 cells; these cells accumulate as tumors increase in size (r=0.7605, p=0.007). CCR2+/CX3CR1+ M-MDSCs isolated from the bone marrow of tumor bearing mice suppress CD8+ T cell production of IFNg and migrate to CCL2 more efficiently than CCL7. CONCLUSION CCR2+/CX3CR1+ cells from glioma bearing mice are derived from the bone marrow and represent an immune suppressive population that migrates to CCL2.

Remodeling tumor microenvironment by liposomal codelivery of DMXAA and simvastatin inhibits malignant melanoma progression

Anti-angiogenic therapies for melanoma have not yet been translated into meaningful clinical benefit for patients, due to the development of drug-induced resistance in cancer cells, mainly caused by hypoxia-inducible factor 1α (HIF-1α) overexpression and enhanced oxidative stress mediated by tumor-associated macrophages (TAMs). Our previous study demonstrated synergistic antitumor actions of simvastatin (SIM) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on an in vitro melanoma model via suppression of the aggressive phenotype of melanoma cells and inhibition of TAMs-mediated angiogenesis. Therefore, we took the advantage of long circulating liposomes (LCL) superior tumor targeting capacity to efficiently deliver SIM and DMXAA to B16.F10 melanoma in vivo, with the final aim of improving the outcome of the anti-angiogenic therapy. Thus, we assessed the effects of this novel combined tumor-targeted treatment on s.c. B16.F10 murine melanoma growth and on the production of critical markers involved in tumor development and progression. Our results showed that the combined liposomal therapy almost totally inhibited (> 90%) the growth of melanoma tumors, due to the enhancement of anti-angiogenic effects of LCL-DMXAA by LCL-SIM and simultaneous induction of a pro-apoptotic state of tumor cells in the tumor microenvironment (TME). These effects were accompanied by the partial re-education of TAMs towards an M1 phenotype and augmented by combined therapy-induced suppression of major invasion and metastasis promoters (HIF-1α, pAP-1 c-Jun, and MMPs). Thus, this novel therapy holds the potential to remodel the TME, by suppressing its most important malignant biological capabilities.

Deep Phenotyping Reveals Expansion of T Follicular Regulatory Cells and Triple Positive (CTLA4, TIGIT, PD1) Exhausted Effector Memory T Cells Characterizing the Immunosuppressive Microenvironment in Follicular Lymphoma

INTRODUCTIONLymphoma cells are dependent on the tumor microenvironment (TME) for their survival and proliferation. Dissection of TME composition provides insight into lymphomagenesis, better prognostication, and enhancement of therapeutic options. Flow cytometry provides a robust approach for single-cell analysis. Nonetheless lack of well-defined comparator groups and/or a robust, reproducible approaches for analyzing the multidimensional data often limited such studies. In the current study, we analyzed the T cell background on a large reference set of follicular hyperplasia (FH) lymph node samples utilizing a robust standardized high dimensionality flow cytometry and a novel reproducible analytical pipeline. We then compared this baseline reference set with tumor infiltrating T cell subsets in follicular lymphoma (FL; in treatment naïve FL-NA and relapsed/refractory FL-RR sets).METHODSOn behalf of the imCORE Network we analyzed 44 FH and 81 FL (35 FL-NA and 44 FL-RR) with 2 standardized flow cytometry panels (23 antigen/18-color) (Table 1). We evaluated the T cell subset distribution as well as immune checkpoint expression (TIGIT, TIM3, PD-1, CD96, LAG3, CTLA4, CD73) within analytically defined T cell clusters. Analysis was performed using semiautomated multi-step analytical pipeline as outlined in Figure 1. Using standardized instrument settings and an R-based algorithms (gaussNorm) to minimize technical variations in sample acquisition we analyzed the T-cells subpopulations using a dimensionality reduction technique (UMAP), combined with an unsupervised clustering algorithm (FlowSOM). Statistical analysis was performed using non-parametric Wilcoxon test.RESULTSIn the 3 cohorts, several marked differences in the composition T cells subsets were observed. Compared to FH the FL lymph nodes were depleted for CD4 & CD8 naïve subsets (Table 2) and were characterized by an immune suppressive microenvironment enriched in specific subsets of activated T regulatory cells and exhausted memory effector cells. The pool of CD8 naïve cells was restored in FL-RR cases (Table 2).FL-NA nodes showed enrichment of T follicular regulatory cells (Tfr; Table 2) (0.9% vs 2.7%, p<0.0001) expressing FoxP3, dim to negative CD25, CD45RO, TIGIT, PD1, CTLA and CXCR5 (Table 2) and activated regulatory T cells (T-Reg) characterized by a highly suppressive phenotype CD25+, CTLA4+, TIGIT+ and PD1+ (Fig. 2B). Moreover, Tfr compartment was further expanded in relapsed/refractory FL cases (2.7% vs 4.5%, p=0.02).In association with the increase of Tfr cells, the concentration of CTLA4+, TIGIT+, PD1bright T follicular helper cells (Tfh) was reduced in FL-RR compared to FL-NA (Fig. 2C).The Tfr/Treg expansion was also associated with a marked increase in exhausted memory T cells in both CD4 and CD8 compartments (CD4 EM TP and CD8 EM DP, Table 2). In FL isolates the CD4 compartment was characterized by the expression of triple positive (CTLA4, TIGIT, PD1) phenotype in EM cells while the memory CD8 cells overexpressed TIGIT and PD1 (Fig. 2D). A smaller subpopulation of memory CD8 cells, almost undetectable in FH samples, characterized by the expression of CTLA4, PD1, TIGIT and TIM3 is expanded in FL isolates (Fig. 2D).CONCLUSIONOur data suggest that change in balance between TFH and Tfr may lead to more aggressive therapy resistant disease in FL. The interplay between TFH and Tfr, has been postulated to shape the immune response in FL with TFH promoting germinal center formation and Tfr inhibiting TFH and follicular effector T cells. While both TFH and Tfr compartments are expanded in FL, in the relapsed/ refractory FL cases, the Tfr compartment is further expanded at the expense of TFH leading to more immunosuppressive background. Furthermore our study suggests a rational way of designing immune checkpoint inhibitor studies in FL. Effector memory T-cells in FL isolates show an exhausted phenotype characterized by the expression of the inhibitory receptors CTLA4, TIGIT, PD1 in the CD4 compartment, and TIGIT, PD1 in the CD8. In addition, the noteworthy expansion of TIM3+ memory CD8 cells in FL. Targeting these most highly expressed checkpoints in FL alone or in combination may provide an avenue for rational trial design. [Display omitted] DisclosuresRoshal: Celgene: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Physicians' Education Resource: Other: Provision of services. Dogan: Physicians' Education Resource: Honoraria; Peer View: Honoraria; Roche: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Seattle Genetics: Consultancy; EUSA Pharma: Consultancy.

The Bacterial Microbiota Promotes Normal Hematopoiesis Via Interferon Alpha and NOD1 Signaling Pathways

Long-term antibiotic therapy is associated with hematological side effects such as neutropenia and anemia. Our lab and others have shown that long-term antibiotic treatment in mice leads to bone marrow suppression and agranulocytosis in mice through depletion of the commensal bacteria. Our work further showed that Stat1-deficient mice phenocopy the bone marrow suppression phenotype of antibiotic-treated mice, suggesting that commensal microbiota mediate hematopoiesis via Stat1 signaling. However, the upstream actors of this pathway and the bacterial mediators required for commensal microbiota regulation of normal hematopoiesis still remain poorly understood. Such knowledge will be essential for understanding how to treat antibiotic-associated cytopenias. We hypothesize that microbial products detected by host cells trigger STAT1 signaling to potentiate normal hematopoiesis.To identify the host cells that require Stat1 for microbiota-promoted hematopoiesis, we treated conditional Stat1 knock-out mice with two weeks of antibiotic therapy. Of the four conditional knock-out mice we evaluated (LepR-Cre, Villin-Cre, Vav-iCre, LysM-Cre), only the mice deficient in STAT1 in hematopoietic cells (Vav-iCre Stat1 fl/fl) phenocopied the bone marrow suppression of antibiotic-treated mice. Our data suggest that STAT1 signaling is necessary in non-myeloid hematopoietic cells, but not intestinal epithelial cells or mesenchymal stromal cells for microbiota-promoted hematopoiesis. Non-competitive transplantation of Stat1 -/- bone marrow into wild type mice validated these findings; mice lacking STAT1 only in hematopoietic tissues phenocopied the Vav-iCre mice, consistent with a specific role for microbiota-mediated STAT1 signaling in the hematopoietic compartment.To assess the upstream mediator of STAT1 signaling in this biological context, we treated interferon (IFN) receptor knock out mice with two weeks of antibiotics. Of the three types of IFN receptor knock-outs evaluated, only mice deficient in type I IFN signaling phenocopied the bone marrow suppression of antibiotic-treated mice. These findings suggest that type I IFN signaling, and not type II or III, was required for microbiota-dependent hematopoiesis. These results were validated by showing that the administration of pegylated-IFNα was sufficient to rescue the depletion of type I IFN-STAT1 signaling in antibiotic-treated mice.To determine the microbial signals that may potentiate hematopoiesis, we evaluated two microbial products that were previously shown to alter hematopoiesis and to activate type I IFN signaling (Iwamura et al. Blood 2017 & Steed et al. Science 2017). We discovered that oral administration of these commensal-derived products, the metabolite desaminotyrosine (DAT) or NOD1 ligand (NOD1L), a motif of peptidoglycan, were each sufficient to rescue the hematopoietic defects induced by antibiotics in mice. To test whether these products rescue hematopoiesis by activating STAT1 signaling, we attempted to rescue the hematopoietic defects in Stat1 -/- mice. These studies showed that NOD1L rescues granulocyte but not progenitor counts in Stat1 -/- mice, suggesting that NOD1 and type I IFN signaling work together at the progenitor level, but independently at the downstream myeloid progenitor level to promote granulopoiesis.Overall, our studies expand our understanding of the signaling pathways by which the microbiota promotes normal hematopoiesis and identify novel therapeutic agents that can be used to ameliorate antibiotic-induced BM suppression. DisclosuresNo relevant conflicts of interest to declare.

CD103 integrin identifies a high IL-10-producing FoxP3+ regulatory T-cell population suppressing allergic airway inflammation.

Although FoxP3+ regulatory T (Treg) cells constitute a highly heterogeneous population, with different regulatory potential depending on the disease context, distinct subsets or phenotypes remain poorly defined. This hampers the development of immunotherapy for allergic and autoimmune disorders. The present study aimed at characterizing distinct FoxP3+ Treg subpopulations involved in the suppression of Th2-mediated allergic inflammation in the lung. We used an established mouse model of allergic airway disease based on ovalbumin sensitization and challenge to analyze FoxP3+ Tregs during the induction and resolution of inflammation, and identify markers that distinguish their most suppressive phenotypes. We also developed a new knock-in mouse model (Foxp3cre Cd103dtr ) enabling the specific ablation of CD103+ FoxP3+ Tregs for functional studies. We found that during resolution of allergic airway inflammation in mice >50% of FoxP3+ Treg cells expressed the integrin CD103 which marks FoxP3+ Treg cells of high IL-10 production, increased expression of immunoregulatory molecules such as KLRG1, ICOS and CD127, and enhanced suppressive capacity for Th2-mediated inflammatory responses. CD103+ FoxP3+ Tregs were essential for keeping allergic inflammation under control as their specific depletion in Foxp3cre Cd103dtr mice lead to severe alveocapillary damage, eosinophilic pneumonia, and markedly reduced lifespan of the animals. Conversely, adoptive transfer of CD103+ FoxP3+ Tregs effectively treated disease, attenuating Th2 responses and allergic inflammation in an IL-10-dependent manner. Our study identifies a novel regulatory T-cell population, defined by CD103 expression, programmed to prevent exuberant type 2 inflammation and keep homeostasis in the respiratory tract under control. This has important therapeutic implications.

Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines.

The adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited due to their low frequency in peripheral blood and the scarce development of efficient protocols to ensure their purity, expansion, and stability. Here, we describe a new experimental protocol that allows the long-term expansion of highly purified allospecific natural Tregs (nTregs) from both healthy controls and chronic kidney disease (CKD) patients, which maintain their phenotype and suppressive function under inflammatory conditions. Firstly, we co-cultured CellTrace Violet (CTV)-labeled Tregs from CKD patients or healthy individuals with allogeneic monocyte-derived dendritic cells in the presence of interleukin 2 (IL-2) and retinoic acid. Then, proliferating CD4+CD25hiCTV− Tregs (allospecific) were sorted by fluorescence-activated cell sorting (FACS) and polyclonally expanded with anti-CD3/CD28-coated beads in the presence of transforming growth factor beta (TGF-β), IL-2, and rapamycin. After 4 weeks, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded alloTregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of inflammatory cytokines (IFN-γ, IL-4, IL-6, or TNF-α). Unexpectedly, the long-term expansion resulted in an increased methylation of the specific demethylated region of Foxp3. Interestingly, alloTregs from both normal individuals and CKD patients maintained their immunosuppressive phenotype and function after being expanded for two additional weeks under an inflammatory microenvironment. Finally, phenotypic and functional evaluation of cryopreserved alloTregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients.

Differential dynamics of peripheral immune responses to acute SARS-CoV-2 infection in older adults.

In this study, peripheral blood mononuclear cells from young and old patients with COVID-19 were examined phenotypically, transcriptionally and functionally to reveal age-, time- and severity-specific adaptations. Gene signatures within memory B cells and plasmablasts correlated with reduced frequency of antigen-specific B cells and neutralizing antibodies in older patients with severe COVID-19. Moreover, these patients exhibited exacerbated T cell lymphopenia, which correlated with lower plasma interleukin-2, and diminished antigen-specific T cell responses. Single-cell RNA sequencing revealed augmented signatures of activation, exhaustion, cytotoxicity and type I interferon signaling in memory T and natural killer cells with age. Although cytokine storm was evident in both age groups, older individuals exhibited elevated levels of myeloid cell recruiting factors. Furthermore, we observed redistribution of monocyte and dendritic cell subsets and emergence of a suppressive phenotype with severe disease, which was reversed only in young patients over time. This analysis provides new insights into the impact of aging on COVID-19.

Adolescent Binge-Type Ethanol Exposure in Rats Mirrors Age-Related Cognitive Decline by Suppressing Cholinergic Tone and Hippocampal Neurogenesis.

Heavy alcohol consumption followed by periods of abstinence (i.e., binge drinking) during adolescence is a concern for both acute and chronic health issues. Persistent brain damage after adolescent intermittent ethanol exposure in rodents, a model of binge drinking, includes reduced hippocampal neurogenesis and a loss of neurons in the basal forebrain that express the cholinergic phenotype. The circuit formed between those regions, the septohippocampal pathway, is critical for learning and memory. Furthermore, this circuit is also altered during the aging process. Thus, we examined whether pathology in septohippocampal circuit and impairments in spatial behaviors are amplified during aging following adolescent intermittent ethanol exposure. Female and male rats were exposed to intermittent intragastric gavage of water (control) or 20% ethanol (dose of 5 g/kg) for a 2 days on/off cycle from postnatal days 25–55. Either 2 (young adult) or 12–14 (middle-age) months post exposure, rats were tested on two spatial tasks: spontaneous alternation and novel object in place. Acetylcholine efflux was assessed in the hippocampus during both tasks. There was no adolescent ethanol-induced deficit on spontaneous alternation, but middle-aged male rats displayed lower alternation rates. Male rats exposed to ethanol during adolescence had blunted behavioral evoked acetylcholine during spontaneous alternation testing. All ethanol-exposed rats displayed suppression of the cholinergic neuronal phenotype. On the novel object in place task, regardless of sex, ethanol-exposed rats performed significantly worse than control-treated rats, and middle aged-rats, regardless of sex or ethanol exposure, were significantly impaired relative to young adult rats. These results indicate that male rats display earlier age-related cognitive impairment on a working memory task. Furthermore, male rats exposed to ethanol during adolescence have blunted behavior-evoked hippocampal acetylcholine efflux. In addition, middle-aged and ethanol-exposed rats, regardless of sex, are impaired at determining discrete spatial relationship between objects. This type of pattern separation impairment was associated with a loss of neurogenesis. Thus, binge-type adolescent ethanol exposure does affect the septohippocampal circuit, and can accelerate age-related cognitive impairment on select spatial tasks.

ROLE OF GLYCODELIN IN REGULATION OF MYELOIDDERIVED SUPPRESSOR CELL DIFFERENTIATION

Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with molecular weight of 42 to 56 kDa) is considered as a reproductive tissue receptivity marker. Despite that glycodelin immunosuppressive effects are well-known there still remains uncovered its role in myeloid suppressor cell (MDSC) regulation. MDSC represent the heterogeneous population of immature myeloid cells that acquire suppressor phenotype while inhibiting the immune response under the pathological states. MDSC are known to play an essential role in supporting the immune tolerance in pregnancy and at transplantation. Our hypothesis suggests that glycodelin is capable of inducing the MDSC formation as the level of these cells is elevated during the successful pregnancy, whereas the spontaneous abortion and progression of eclampsia are associated with low circulating glycodelin. Therefore, the aim of the work was to analyze the role of recombinant glycodelin in physiological concentrations in regulation of MDSC differentiation. Peripheral blood mononuclear cells of donor volunteers were separated via centrifugation on density gradient of 1,077 g/cm3 (Ficoll-Hypaque, Sigma-Aldrich) to obtain MDSC generation in vitro. Then cells obtained were cultured in 24-well plate at a concentration of 1 × 106 cell/ml in complete medium with cytokines IL-6 (20 ng/ml), GM-CSF (40 ng/ml) therein for 14 days at 37 °C and 5% CO2. Medium replacement was made by 7th day in culture followed by cytokine re-introduction, and on the 11th day recombinant glycodelin in physiological concentrations (0,2; 2 mkg/ml) was applied while the pharmacological concentration was 50 mkg/ml. The M-MDSC (LinHLA-DRCD33+CD11b+CD14+CD66b- ) and PMN-MDSC (LinHLA-DRCD33+CD11b+CD14- CD66b+) level was evaluated in cultures using flow cytometry (СytoFlexS (Beckman Coulter)) and “R&amp;D Systems” antibodies according to standard protocol. Statistical data processing was realized with GraphPad Prizm software using Friedman test. It was found that glycodelin did not significantly affect cell viability being assessed with flow cytometry (PI). It was revealed that high GdA concentration (50 mkg/ml) being pharmacological did not render significant effect on MDSC differentiation. Meanwhile, glycodelin in concentrations correspanding the healthy pregnancy (0,2; 2 mkg/ml) was stated to increase the MDSC percentage in induced cultures of human mononuclear cells. When analyzing the subsets it was disclosed that this effect was conditioned by the increase in PMN-MDSC level while the M-MDSC level remained significantly unchanged. This result could be interpreted as glycodelin fetoprotective effect as the increase of the PMN-MDSC level is associated with the suppression of the immune response to paternal antigens. The PMN-MDSC level is known to be elevated in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to nonpregnant subjects whereas the M-MDSC amount remains unaltered. Meanwhile, patients with miscarriage demonstrated more that by 30% lowering in the MDSC amount in blood and endometrium and in I trimester, in particular. During the physiological pregnancy PMN-MDSC accumulate in placenta, but at spontaneous abortion their number is found to be declined. Placental PMN-MDSC efficiently suppress the T-cell response while concurrently polarizing the CD4+ lymphocytes in Th2 phenotype. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens that allows considering these as promising target of therapeutical manipulation in pregnancy complications. As a whole, we have originally demonstrated the GdA effect on MDSC differentiation.

K15 promoter-driven enforced expression of NKIRAS exhibits tumor suppressive activity against the development of DMBA/TPA-induced skin tumors

NKIRAS1 and NKIRAS2 (also called as κB-Ras) were identified as members of the atypical RAS family that suppress the transcription factor NF-κB. However, their function in carcinogenesis is still controversial. To clarify how NKIRAS acts on cellular transformation, we generated transgenic mice in which NKIRAS2 was forcibly expressed using a cytokeratin 15 (K15) promoter, which is mainly activated in follicle bulge cells. The ectopic expression of NKIRAS2 was mainly detected in follicle bulges of transgenic mice with NKIRAS2 but not in wild type mice. K15 promoter-driven expression of NKIRAS2 failed to affect the development of epidermis, which was evaluated using the expression of K10, K14, K15 and filaggrin. However, K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA). This observation suggested that NKIRAS seemed to function as a tumor suppressor in follicle bulges. However, in the case of oncogenic HRAS-driven cellular transformation of murine fibroblasts, knockdown of NKIRAS2 expression drastically suppressed HRAS-mutant-provoked cellular transformation, suggesting that NKIRAS2 was required for the cellular transformation of murine fibroblasts. Furthermore, moderate enforced expression of NKIRAS2 augmented oncogenic HRAS-provoked cellular transformation, whereas an excess NKIRAS2 expression converted its functional role into a tumor suppressive phenotype, suggesting that NKIRAS seemed to exhibit a biphasic bell-shaped enhancing effect on HRAS-mutant-provoked oncogenic activity. Taken together, the functional role of NKIRAS in carcinogenesis is most likely determined by not only cellular context but also its expression level.

Association between delay in intensive care unit admission and the host response in patients with community-acquired pneumonia

BackgroundA delay in admission to the intensive care unit (ICU) of patients with community-acquired pneumonia (CAP) has been associated with an increased mortality. Decisions regarding interventions and eligibility for immune modulatory therapy are often made at the time of admission to the ICU. The primary aim of this study was to compare the host immune response measured upon ICU admission in CAP patients admitted immediately from the emergency department (direct ICU admission) with those who were transferred within 72 h after admission to the general ward (delayed ICU admission).MethodsSixteen host response biomarkers providing insight in pathophysiological mechanisms implicated in sepsis and blood leukocyte transcriptomes were analysed in patients with CAP upon ICU admission in two tertiary hospitals in the Netherlands.ResultsOf 530 ICU admissions with CAP, 387 (73.0%) were directly admitted and 143 (27.0%) had a delayed admission. Patients with a delayed ICU admission were more often immunocompromised (35.0 versus 21.2%, P = .002) and had more malignancies (23.1 versus 13.4%, P = .011). Shock was more present in patients who were admitted to the ICU directly (46.6 versus 33.6%, P = .010). Delayed ICU admission was not associated with an increased hospital mortality risk (hazard ratio 1.25, 95% CI 0.89–1.78, P = .20). The plasma levels of biomarkers (n = 297) reflecting systemic inflammation, endothelial cell activation and coagulation activation were largely similar between groups, with exception of C-reactive protein, soluble intercellular adhesion molecule-1 and angiopoietin-1, which were more aberrant in delayed admissions compared to direct ICU admissions. Blood leukocyte transcriptomes (n = 132) of patients with a delayed ICU admission showed blunted innate and adaptive immune response signalling when compared with direct ICU admissions, as well as decreased gene expression associated with tissue repair and extracellular matrix remodelling pathways.ConclusionsBlood leukocytes of CAP patients with delayed ICU admission show evidence of a more immune suppressive phenotype upon ICU admission when compared with blood leukocytes from patients directly transferred to the ICU.Trial registration: Molecular Diagnosis and Risk Stratification of Sepsis (MARS) project, ClinicalTrials.gov identifier NCT01905033.

The PUB4 E3 Ubiquitin Ligase Is Responsible for the Variegated Phenotype Observed upon Alteration of Chloroplast Protein Homeostasis in Arabidopsis Cotyledons.

During a plant’s life cycle, plastids undergo several modifications, from undifferentiated pro-plastids to either photosynthetically-active chloroplasts, ezioplasts, chromoplasts or storage organelles, such as amyloplasts, elaioplasts and proteinoplasts. Plastid proteome rearrangements and protein homeostasis, together with intracellular communication pathways, are key factors for correct plastid differentiation and functioning. When plastid development is affected, aberrant organelles are degraded and recycled in a process that involves plastid protein ubiquitination. In this study, we have analysed the Arabidopsis gun1-102 ftsh5-3 double mutant, lacking both the plastid-located protein GUN1 (Genomes Uncoupled 1), involved in plastid-to-nucleus communication, and the chloroplast-located FTSH5 (Filamentous temperature-sensitive H5), a metalloprotease with a role in photosystem repair and chloroplast biogenesis. gun1-102 ftsh5-3 seedlings show variegated cotyledons and true leaves that we attempted to suppress by introgressing second-site mutations in genes involved in: (i) plastid translation, (ii) plastid folding/import and (iii) cytosolic protein ubiquitination. Different phenotypic effects, ranging from seedling-lethality to partial or complete suppression of the variegated phenotype, were observed in the corresponding triple mutants. Our findings indicate that Plant U-Box 4 (PUB4) E3 ubiquitin ligase plays a major role in the target degradation of damaged chloroplasts and is the main contributor to the variegated phenotype observed in gun1-102 ftsh5-3 seedlings.

Neutrophils and lymphopenia, an unknown axis in severe COVID-19 disease.

The Coronavirus Disease 2019 (COVID-19) is caused by the betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus that can mediate asymptomatic or fatal infections characterized by pneumonia, acute respiratory distress syndrome (ARDS), and multi-organ failure. Several studies have highlighted the importance of B and T lymphocytes, given that neutralizing antibodies and T cell responses are required for an effective immunity. In addition, other reports have described myeloid cells such as macrophages and monocytes play a major role in the immunity against SARS-CoV-2 as well as dysregulated pro-inflammatory signature that characterizes severe COVID-19. During COVID-19, neutrophils have been defined as a heterogeneous group of cells, functionally linked to severe inflammation and thrombosis triggered by degranulation and NETosis, but also to suppressive phenotypes. The physiological role of suppressive neutrophils during COVID-19 and their implications in severe disease have been poorly studied and is not well understood. Here, we discuss the current evidence regarding the role of neutrophils with suppressive properties such as granulocytic myeloid-derived suppressor cells (G-MDSCs) and their possible role in suppressing CD4+ and CD8+ T lymphocytes expansion and giving rise to lymphopenia in severe COVID-19 infection.

Melatonin and its derivative disrupt cancer stem-like phenotypes of lung cancer cells via AKT downregulation.

Cancer stem cells (CSCs), a small subpopulation of tumour cells, have properties of self-renewal and multipotency, which drive cancer progression and resistance to current treatments. Compounds potentially targeting CSCs have been recently developed. This study shows how melatonin, an endogenous hormone synthesised by the pineal gland, and its derivative suppress CSC-like phenotypes of human non-small cell lung cancer (NSCLC) cell lines, H460, H23, and A549. The effects of MLT and its derivative, acetyl melatonin (ACT), on CSC-like phenotypes were investigated using assays for anchorage-independent growth, three-dimensional spheroid formation, scratch wound healing ability, and CSC marker and upstream protein signalling expression. Enriched CSC spheroids were used to confirm the effect of both compounds on lung cancer cells. MLT and ACT inhibited CSC-like behaviours by suppression of colony and spheroid formation in NSCLC cell lines. Their effects on spheroid formation were confirmed in CSC-enriched H460 cells. CSC markers, CD133 and ALDH1A1, were depleted by both compounds. The behaviour and factors associated to epithelial-mesenchymal transition, as indicated by cell migration and the protein vimentin, were also decreased by MLT and ACT. Mechanistically, MLT and ACT decreased the expression of stemness proteins Oct-4, Nanog, and β-catenin by reducing active AKT (phosphorylated AKT). Suppression of the AKT pathway was not mediated through melatonin receptors. This study demonstrates a novel role, and its underlying mechanism, for MLT and its derivative ACT in suppression of CSC-like phenotypes in NSCLC cells, indicating that they are potential candidates for lung cancer treatment.

Molecular and Microenvironmental Basis of Immunodysfunction in CLL

Context Representing the major cause of morbidity and mortality for chronic lymphocytic leukemia (CLL) patients, immunosuppression is a common feature of the disease.1 Effectors of the innate and the adaptive immune response show marked dysfunction and skewing toward the generation of a tolerant environment that favors disease expansion. Major deregulations have been described in the T lymphocyte compartment, with inhibition of CD8+ cytotoxic and CD4+ activated effector T cells replaced by exhausted and tolerogenic subsets. Likewise, differentiation of monocytes toward a suppressive M2-like phenotype is induced at the expense of pro-inflammatory subpopulations. Thanks to their B-regulatory phenotype, leukemic cells play a central role in driving immunosuppression, progressively inhibiting immune responses. A number of signaling cascades triggered by soluble mediators and cell-cell contacts contribute to immunomodulation in CLL, fostered also by local environmental conditions, such as hypoxia and derived metabolic acidosis.2

The Fibronectin-ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells.

Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.