Coupled scRNA-Seq and Intracellular Protein Activity Reveal an Immunosuppressive Role of TREM2 in Cancer.

Cell function and activity are regulated through integration of signaling, epigenetic, transcriptional, and metabolic pathways. Here, we introduce INs-seq, an integrated technology for massively parallel recording of single-cell RNA sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for discovering new immune subsets by profiling different intracellular signatures of immune signaling, transcription factor combinations, and metabolic activity. Comprehensive mapping of Arginase 1-expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers novel Arg1+ Trem2+ regulatory myeloid (Mreg) cells and identifies markers, metabolic activity, and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8+ T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps and identifies the molecular signature of myeloid suppressive cells in tumors.

LXR/ApoE Activation Restricts Innate Immune Suppression in Cancer.

Therapeutic harnessing of adaptive immunity via checkpoint inhibition has transformed the treatment of many cancers. Despite unprecedented long-term responses, most patients do not respond to these therapies. Immunotherapy non-responders often harbor high levels of circulating myeloid-derived suppressor cells (MDSCs)-an immunosuppressive innate cell population. Through genetic and pharmacological approaches, we uncovered a pathway governing MDSC abundance in multiple cancer types. Therapeutic liver-X nuclear receptor (LXR) agonism reduced MDSC abundance in murine models and in patients treated in a first-in-human dose escalation phase 1 trial. MDSC depletion was associated with activation of cytotoxic T lymphocyte (CTL) responses in mice and patients. The LXR transcriptional target ApoE mediated these effects in mice, where LXR/ApoE activation therapy elicited robust anti-tumor responses and also enhanced T cell activation during various immune-based therapies. We implicate the LXR/ApoE axis in the regulation of innate immune suppression and as a target for enhancing the efficacy of cancer immunotherapy in patients.

Activation of p53 in Immature Myeloid Precursor Cells Controls Differentiation into Ly6c+CD103+ Monocytic Antigen-Presenting Cells in Tumors.

CD103+ dendritic cells are critical for cross-presentation of tumor antigens. Here we have shown that during immunotherapy, large numbers of cells expressing CD103 arose in murine tumors via direct differentiation of Ly6c+ monocytic precursors. These Ly6c+CD103+ cells could derive from bone-marrow monocytic progenitors (cMoPs) or from peripheral cells present within the myeloid-derived suppressor cell (MDSC) population. Differentiation was controlled by inflammation-induced activation of the transcription factor p53, which drove upregulation of Batf3 and acquisition of the Ly6c+CD103+ phenotype. Mice with a targeted deletion of p53 in myeloid cells selectively lost the Ly6c+CD103+ population and became unable to respond to multiple forms of immunotherapy and immunogenic chemotherapy. Conversely, increasing p53 expression using a p53-agonist drug caused a sustained increase in Ly6c+CD103+ cells in tumors during immunotherapy, which markedly enhanced the efficacy and duration of response. Thus, p53-driven differentiation of Ly6c+CD103+ monocytic cells represents a potent and previously unrecognized target for immunotherapy.

Platelet-activating factor (PAF) promotes immunosuppressive neutrophil differentiation within tumors.

Chronic inflammatory milieu in the tumor microenvironment (TME) leads to the recruitment and differentiation of myeloid-derived suppressor cells (MDSCs). Polymorphonuclear (PMN)-MDSCs, which are phenotypically and morphologically defined as a subset of neutrophils, cause major immune suppression in the TME, posing a significant challenge in the development of effective immunotherapies. Despite recent advances in our understanding of PMN-MDSC functions, the mechanism that gives rise to immunosuppressive neutrophils within the TME remains elusive. Both in vivo and in vitro, newly recruited neutrophils into the tumor sites remained activated and highly motile for several days and developed immunosuppressive phenotypes, as indicated by increased arginase 1 (Arg1) and dcTrail-R1 expression and suppressed anticancer CD8 T cell cytotoxicity. The strong suppressive function was successfully recapitulated by incubating naive neutrophils with cancer cell culture supernatant in vitro. Cancer metabolite secretome analyses of the culture supernatant revealed that both murine and human cancers released lipid mediators to induce the differentiation of immunosuppressive neutrophils. Liquid chromatography-mass spectrometry (LC-MS) lipidomic analysis identified platelet-activation factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) as a common tumor-derived lipid mediator that induces neutrophil differentiation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2), the PAF biosynthetic enzyme, is up-regulated in human pancreatic ductal adenocarcinoma (PDAC) and shows an unfavorable correlation with patient survival across multiple cancer types. Our study identifies PAF as a lipid-driven mechanism of MDSC differentiation in the TME, providing a potential target for cancer immunotherapy.

N1 versus N2 and PMN-MDSC: A critical appraisal of current concepts on tumor-associated neutrophils and new directions for human oncology.

Research on tumor-associated neutrophils (TAN) currently surges because of the well-documented strong clinical relevance of tumor-infiltrating neutrophils. This relevance is illustrated by strong correlations between high frequencies of intratumoral neutrophils and poor outcome in the majority of human cancers. Recent high-dimensional analysis of murine neutrophils provides evidence for unexpected plasticity of neutrophils in murine models of cancer and other inflammatory non-malignant diseases. New analysis tools enable deeper insight into the process of neutrophil differentiation and maturation. These technological and scientific developments led to the description of an ever-increasing number of distinct transcriptional states and associated phenotypes in murine models of disease and more recently also in humans. At present, functional validation of these different transcriptional states and potential phenotypes in cancer is lacking. Current functional concepts on neutrophils in cancer rely mainly on the myeloid-derived suppressor cell (MDSC) concept and the dichotomous and simple N1-N2 paradigm. In this manuscript, we review the historic development of those concepts, critically evaluate these concepts against the background of our own work and provide suggestions for a refinement of current concepts in order to facilitate the transition of TAN research from experimental insight to clinical translation.

The DNA Methylcytosine Dioxygenase Tet2 Sustains Immunosuppressive Function of Tumor-Infiltrating Myeloid Cells to Promote Melanoma Progression.

Ten-Eleven-Translocation-2 (Tet2) is a DNA methylcytosine dioxygenase that functions as a tumor suppressor in hematopoietic malignancies. We examined the role of Tet2 in tumor-tissue myeloid cells and found that Tet2 sustains the immunosuppressive function of these cells. We found that Tet2 expression is increased in intratumoral myeloid cells both in mouse models of melanoma and in melanoma patients and that this increased expression is dependent on an IL-1R-MyD88 pathway. Ablation of Tet2 in myeloid cells suppressed melanoma growth in vivo and shifted the immunosuppressive gene expression program in tumor-associated macrophages to a proinflammatory one, with a concomitant reduction of the immunosuppressive function. This resulted in increased numbers of effector T cells in the tumor, and T cell depletion abolished the reduced tumor growth observed upon myeloid-specific deletion of Tet2. Our findings reveal a non-cell-intrinsic, tumor-promoting function for Tet2 and suggest that Tet2 may present a therapeutic target for the treatment of non-hematologic malignancies.

The TRAIL-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for specifically killing cancer cells, whereas in resistant cancers, TRAIL/TRAIL-R can promote metastasis via Rac1 and PI3K. It remains unknown, however, whether and to what extent TRAIL/TRAIL-R signaling in cancer cells can affect the immune microenvironment. Here we show that TRAIL-triggered cytokine secretion from TRAIL-resistant cancer cells is FADD dependent and identify the TRAIL-induced secretome to drive monocyte polarization to myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. TRAIL-R suppression in tumor cells impaired CCL2 production and diminished both lung MDSC presence and tumor growth. In accordance, the receptor of CCL2, CCR2, is required to facilitate increased MDSC presence and tumor growth. Finally, TRAIL and CCL2 are co-regulated with MDSC/M2 markers in lung adenocarcinoma patients. Collectively, endogenous TRAIL/TRAIL-R-mediated CCL2 secretion promotes accumulation of tumor-supportive immune cells in the cancer microenvironment, thereby revealing a tumor-supportive immune-modulatory role of the TRAIL/TRAIL-R system in cancer biology.

G-CSF, the guardian of granulopoiesis.

A considerable amount of continuous proliferation and differentiation is required to produce daily a billion new neutrophils in an adult human. Of the few cytokines and factors known to control neutrophil production, G-CSF is the guardian of granulopoiesis. G-CSF/CSF3R signaling involves the recruitment of non-receptor protein tyrosine kinases and their dependent signaling pathways of serine/threonine kinases, tyrosine phosphatases, and lipid second messengers. These pathways converge to activate the families of STAT and C/EBP transcription factors. CSF3R mutations are associated with human disorders of neutrophil production, including severe congenital neutropenia, neutrophilia, and myeloid malignancies. More than three decades after their identification, cloning, and characterization of G-CSF and G-CSF receptor, fundamental questions remain about their physiology.

Myeloid-derived suppressor cells and tolerogenic dendritic cells are distinctively induced by PI3K and Wnt signaling pathways.

Imbalanced immune responses are a prominent hallmark of cancer and autoimmunity. Myeloid cells can be overly suppressive, inhibiting protective immune responses or inactive not controlling autoreactive immune cells. Understanding the mechanisms that induce suppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs) and tolerogenic dendritic cells (TolDCs), can facilitate the development of immune-restoring therapeutic approaches. MDSCs are a major barrier for effective cancer immunotherapy by suppressing antitumor immune responses in cancer patients. TolDCs are administered to patients to promote immune tolerance with the intent to control autoimmune disease. Here, we investigated the development and suppressive/tolerogenic activity of human MDSCs and TolDCs to gain insight into signaling pathways that drive immunosuppression in these different myeloid subsets. Moreover, monocyte-derived MDSCs (M-MDSCs) generated in vitro were compared to M-MDSCs isolated from head-and-neck squamous cell carcinoma patients. PI3K-AKT signaling was identified as being crucial for the induction of human M-MDSCs. PI3K inhibition prevented the downregulation of HLA-DR and the upregulation of reactive oxygen species and MerTK. In addition, we show that the suppressive activity of dexamethasone-induced TolDCs is induced by ß-catenin-dependent Wnt signaling. The identification of PI3K-AKT and Wnt signal transduction pathways as respective inducers of the immunomodulatory capacity of M-MDSCs and TolDCs provides opportunities to overcome suppressive myeloid cells in cancer patients and optimize therapeutic application of TolDCs. Lastly, the observed similarities between generated- and patient-derived M-MDSCs support the use of in vitro-generated M-MDSCs as powerful model to investigate the functionality of human MDSCs.

Host ANGPTL2 establishes an immunosuppressive tumor microenvironment and resistance to immune checkpoint therapy.

Use of immune checkpoint inhibitors (ICIs) as cancer immunotherapy has advanced rapidly in the clinic; however, mechanisms underlying resistance to ICI therapy, including impaired T cell infiltration, low immunogenicity, and tumor "immunophenotypes" governed by the host, remain unclear. We previously reported that in some cancer contexts, tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) has tumor-promoting functions. Here, we asked whether ANGPTL2 deficiency could enhance antitumor ICI activity in two inflammatory contexts: a murine syngeneic model of colorectal cancer and a mouse model of high-fat diet (HFD)-induced obesity. Systemic ANGPTL2 deficiency potentiated ICI efficacy in the syngeneic model, supporting an immunosuppressive role for host ANGPTL2. Relevant to the mechanism, we found that ANGPTL2 induces pro-inflammatory cytokine production in adipose tissues, driving generation of myeloid-derived suppressor cells (MDSCs) in bone marrow and contributing to an immunosuppressive tumor microenvironment and resistance to ICI therapy. Moreover, HFD-induced obese mice showed impaired responsiveness to ICI treatment, suggesting that obesity-induced chronic inflammation facilitated by high ANGPTL2 expression blocks ICI antitumor effects. Our findings overall provide novel insight into protumor ANGPTL2 functions and illustrate the essential role of the host system in ICI responsiveness.

MAPK signaling pathway induced LOX-1+ polymorphonuclear myeloid-derived suppressor cells in biliary atresia.

Biliary atresia (BA) is a severe pediatric liver disease characterized by progressive bile duct destruction and fibrosis, leading to significant liver damage and frequently necessitating liver transplantation. This study elucidates the role of LOX-1+ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in BA pathogenesis and assesses their potential as non-invasive early diagnostic biomarkers. Using flow cytometry, immunofluorescence, and molecular profiling, we analyzed the expression and activity of these cells in peripheral blood and liver tissues from BA patients and controls. Our findings reveal a significant increase in the frequencies and function of LOX-1+PMN-MDSCs in BA patients, along with MAPK signaling pathway upregulation, indicating their involvement in disease mechanisms. Additionally, the frequencies of LOX-1+PMN-MDSC in peripheral blood significantly positively correlate with liver function parameters in BA patients, demonstrating diagnostic performance comparable to traditional serum markers. These findings suggest that LOX-1+PMN-MDSCs contribute to the immunosuppressive environment in BA and could serve as potential diagnostic targets.

HIF-1α targeted deletion in myeloid cells decreases MDSC accumulation and alters microbiome in neonatal mice.

The newborn's immune system is faced with the challenge of having to learn quickly to fight off infectious agents, but tolerating the colonization of the body surfaces with commensals without reacting with an excessive inflammatory response. Myeloid-derived suppressor cells (MDSC) are innate immune cells with suppressive activity on other immune cells that regulate fetal-maternal tolerance during pregnancy and control intestinal inflammation in neonates. Until now, nothing is known about the role of MDSC in microbiome establishment. One of the transcription factors regulating MDSC homeostasis is the hypoxia-inducible factor 1α (HIF-1α). We investigated the impact of HIF-1α on MDSC accumulation and microbiome establishment during the neonatal period in a mouse model with targeted deletion of HIF-1α in myeloid cells (Hif1a loxP/loxP LysMCre+). We show that in contrast to wildtype mice, where an extensive expansion of MDSC was observed, MDSC expansion in neonatal Hif1a loxP/loxP LysMCre+ mice was dramatically reduced both systemically and locally in the intestine. This was accompanied by an altered microbiome composition and intestinal T-cell homeostasis. Our results point toward a role of MDSC in inflammation regulation in the context of microbiome establishment and thus reveal a new aspect of the biological role of MDSC during the neonatal period.

VEGFA contributes to tumor property of glioblastoma cells by promoting differentiation of myeloid-derived suppressor cells.

BACKGROUND: Glioblastoma (GBM) is a malignant astrocytic tumor and its progression involves the regulation of vascular endothelial growth factor-A (VEGFA). However, the mechanism of VEGFA in regulating GBM progression remains unclear. METHODS: VEGFA mRNA expression was analyzed by quantitative real-time polymerase chain reaction. Protein expression of VEGFA, cluster of differentiation 9 (CD9), CD81, and transforming growth factor-ß1 (TGF-ß1) was detected by western blotting assay. Flow cytometry assay was conducted to assess cell proliferation, cell apoptosis and myeloid-derived suppressor cell (MDSC) differentiation. TUNEL cell apoptosis detection kit was utilized to analyze cell apoptosis of tumors. Angiogenic capacity was investigated by tube formation assay. Transwell assay was used to assess cell migration and invasion. The effect of VEGFA on tumor formation was determined by a xenograft mouse model assay. Immunohistochemistry assay was used to analyze positive expression rate of VEGFA in tumor tissues. TGF-ß1 level was detected by enzyme-linked immunosorbent assay. RESULTS: VEGFA expression was upregulated in GBM tissues, GBM cells, and exosomes from GBM patients and GBM cells. VEGFA silencing led to decreased cell proliferation, tube formation, migration and invasion and increased cell apoptosis. Moreover, VEGFA knockdown also delayed tumor formation. VEGFA promoted MDSC differentiation and TGF-ß1 secretion by MDSCs by being packaged into exosomes. In addition, TGF-ß1 knockdown displayed similar effects with VEGFA silencing on GBM cell phenotypes, and MDSCs attenuated VEGFA knockdown-induced effects by secreting TGF-ß1 in A172 and U251 cells. CONCLUSION: VEGFA contributed to tumor property of GBM cells by promoting MDSC differentiation and TGF-ß1 secretion by MDSCs, providing potential targets for GBM treatment.

Local administration of myeloid-derived suppressor cells prevents progression of immune-mediated dry eye disease.

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature hematopoietic precursors with known immunoregulatory functions. The immunosuppressive role of MDSCs has been highlighted in several inflammatory ophthalmic disorders; however, their therapeutic application in suppressing the immune-mediated changes in dry eye disease (DED) has not been studied. We observed significant reduction in antigen presenting cell (APC) frequencies and their maturation in the presence of MDSCs. Moreover, co-culturing MDSCs with T helper 17 cells (Th17) resulted in reduced Th17 frequencies and their IL-17 expression. On the contrary, MDSCs maintained regulatory T cell frequencies and enhanced their function in-vitro. Furthermore, we delineated the role of interleukin-10 (IL-10) secreted by MDSCs in their immunoregulatory functions. We confirmed these results by flow cytometry analysis and observed that treatment with MDSCs in DED mice effectively suppressed the maturation of APCs, pathogenic Th17 response, and maintained Treg function and significantly ameliorated the disease. The results in this study highlight the potential therapeutic application of MDSCs in treating refractory DED.

Targeting tumorous Circ-E-Cadherinencoded C-E-Cad inhibits the recruitment and function of breast cancer-associated myeloid-derived suppressor cells.

We previously demonstrated that the C-E-cad protein encoded by circ-E-cadherin promotes the self-renewal of glioma stem cells. The expression pattern of C-E-cad in breast cancer and its potential function in the tumor microenvironment are unclear. The expression of circ-E-cadherin and C-E-cad was detected in breast cancer specimens. The influence of C-E-cad expression on MDSCs was assessed using FACS and in vivo tumorigenesis experiments. The synergistic effect of anti-C-E-cad and anti-PD-1 antibodies was validated in vivo. circ-E-cadherin and the encoded protein C-E-cad were found to be upregulated in breast cancer vs. normal samples. C-E-cad promotes the recruitment of MDSCs, especially PMN-MDSCs. C-E-cad activates EGFR signaling in tumor cells and promotes the transcription of CXCL8; moreover, C-E-cad binds to MDSCs and maintains glycolysis in PMN-MDSCs. Targeting C-E-cad enhanced anti-PD-1 efficiency. Our data suggested that C-E-cad is markedly overexpressed in breast cancer and promotes MDSC recruitment and survival. Targeting C-E-cad increases the efficacy of immune checkpoint inhibitor therapy.

Clinicopathological significance of microsatellite instability and immune escape mechanism in patients with gastric solid-type poorly differentiated adenocarcinoma.

BACKGROUND: In gastric solid-type poorly differentiated adenocarcinoma (PDA), the role of microsatellite instability and immune escape mechanism remains unclear. The current study aimed to elucidate the clinical significance of mismatch repair (MMR) status, genome profile, C-X-C motif chemokine receptor 2 (CXCR2) expression, and myeloid-derived suppressor cell (MDSC) infiltration in solid-type PDA. METHODS: In total, 102 primary solid-type PDA cases were retrieved, and classified into 46 deficient-MMR (dMMR) and 56 proficient-MMR (pMMR) cases based on immunohistochemistry (IHC) and polymerase chain reaction-based molecular testing results. The mRNA expression profiles (NanoString nCounter Assay) of stage-matched dMMR (n = 6) and pMMR (n = 6) cases were examined. The CXCR2 expression and MDSC infiltration (CD11b- and CD33-positive cells) were investigated via IHC in all solid-type PDA cases. RESULTS: mRNA analysis revealed several differentially expressed genes and differences in biological behavior between the dMMR (n = 46) and pMMR (n = 56) groups. In the multivariate analysis, the dMMR status was significantly associated with a longer disease-free survival (hazard ratio = 5.152, p = 0.002) and overall survival (OS) (hazard ratio = 5.050, p = 0.005). CXCR2-high expression was significantly correlated with a shorter OS in the dMMR group (p = 0.018). A high infiltration of CD11b- and CD33-positive cells was significantly correlated with a shorter OS in the pMMR group (p = 0.022, 0.016, respectively). CONCLUSIONS: dMMR status can be a useful prognostic predictor, and CXCR2 and MDSCs can be novel therapeutic targets in patients with solid-type PDA.

Dual regulation effect and mechanism of human myeloid-derived suppressor cells on anticolorectal cancer cells activity of Vγ9Vδ2 T cells.

Myeloid-derived suppressor cells (MDSC) are a group of immature inhibitory cells of bone marrow origin. Human γδ T cells (mainly Vγ9Vδ2 T cells) have emerged as dominant candidates for cancer immunotherapy because of their unique recognition pattern and broad killing activity against tumor cells. Intestinal mucosal intraepithelial lymphocytes are almost exclusively γδ T cells, so it plays an important role in inhibiting the development of colorectal cancer. In this study, we investigated the effects and molecular mechanism of human MDSC on anticolorectal cancer cells activity of Vγ9Vδ2 T cells. Our results suggested that MDSC can reduce the NKG2D expression of Vγ9Vδ2 T cells through direct cell-cell contact, which is associated with membrane-type transforming growth factor-ß. In contrast, MDSC can increase Vγ9Vδ2 T cells activation and production of IFN-γ, perforin, Granzyme B through direct cell-cell contact. This may be related to the upregulation of T-bet in Vγ9Vδ2 T cells by MDSC. However, MDSC had a dominant negative regulatory effect on the anticolorectal cancer cells activity of Vγ9Vδ2 T cells. Our study provides a theoretical basis for the immune regulatory function of human MDSC on γδ T cells. This will be conducive to the clinical development of a new antitumor therapy strategy.

Myeloid-derived suppressor cells impair CD4+ T cell responses during chronic Staphylococcus aureus infection via lactate metabolism.

Staphylococcus aureus is an important cause of chronic infections resulting from the failure of the host to eliminate the pathogen. Effective S. aureus clearance requires CD4+ T cell-mediated immunity. We previously showed that myeloid-derived suppressor cells (MDSC) expand during staphylococcal infections and support infection chronicity by inhibiting CD4+ T cell responses. The aim of this study was to elucidate the mechanisms underlying the suppressive effect exerted by MDSC on CD4+ T cells during chronic S. aureus infection. It is well known that activated CD4+ T cells undergo metabolic reprogramming from oxidative metabolism to aerobic glycolysis to meet their increased bioenergetic requirements. In this process, pyruvate is largely transformed into lactate by lactate dehydrogenase with the concomitant regeneration of NAD+, which is necessary for continued glycolysis. The by-product lactate needs to be excreted to maintain the glycolytic flux. Using SCENITH (single-cell energetic metabolism by profiling translation inhibition), we demonstrated here that MDSC inhibit CD4+ T cell responses by interfering with their metabolic activity. MDSC are highly glycolytic and excrete large amount of lactate in the local environment that alters the transmembrane concentration gradient and prevent removal of lactate by activated CD4+ T. Accumulation of endogenous lactate impedes the regeneration of NAD+, inhibit NAD-dependent glycolytic enzymes and stop glycolysis. Together, the results of this study have uncovered a role for metabolism on MDSC suppression of CD4+ T cell responses. Thus, reestablishment of their metabolic activity may represent a mean to improve the functionality of CD4+ T cells during chronic S. aureus infection.

Sex-Dependent T Cell Dysregulation in Mice with Diet-Induced Obesity.

Obesity is an emerging public health problem. Chronic low-grade inflammation is considered a major promotor of obesity-induced secondary diseases such as cardiovascular and fatty liver disease, type 2 diabetes mellitus, and several cancer entities. Most preliminary studies on obesity-induced immune responses have been conducted in male rodents. Sex-specific differences between men and women in obesity-induced immune dysregulation have not yet been fully outlined but are highly relevant to optimizing prevention strategies for overweight-associated complications. In this study, we fed C57BL/6 female vs. male mice with either standard chow or an obesity-inducing diet (OD). Blood and spleen immune cells were isolated and analyzed by flow cytometry. Lean control mice showed no sex bias in systemic and splenic immune cell composition, whereas the immune responses to obesity were significantly distinct between female and male mice. While immune cell alterations in male OD mice were characterized by a significant reduction in T cells and an increase in myeloid-derived suppressor cells (MDSC), female OD mice displayed preserved T cell numbers. The sex-dependent differences in obesity-induced T cell dysregulation were associated with varying susceptibility to body weight gain and fatty liver disease: Male mice showed significantly more hepatic inflammation and histopathological stigmata of fatty liver in comparison to female OD mice. Our findings indicate that sex impacts susceptibility to obesity-induced T cell dysregulation, which might explain sex-dependent different incidences in the development of obesity-associated secondary diseases. These results provide novel insights into the understanding of obesity-induced chronic inflammation from a sex-specific perspective. Given that most nutrition, exercise, and therapeutic recommendations for the prevention of obesity-associated comorbidities do not differentiate between men and women, the data of this study are clinically relevant and should be taken into consideration in future trials and treatment strategies.

Tumor Cell-Associated IL-1α Affects Breast Cancer Progression and Metastasis in Mice through Manipulation of the Tumor Immune Microenvironment.

IL-1α is a dual function cytokine that affects inflammatory and immune responses and plays a pivotal role in cancer. The effects of intracellular IL-1α on the development of triple negative breast cancer (TNBC) in mice were assessed using the CRISPR/Cas9 system to suppress IL-1α expression in 4T1 breast cancer cells. Knockout of IL-1α in 4T1 cells modified expression of multiple genes, including downregulation of cytokines and chemokines involved in the recruitment of tumor-associated pro-inflammatory cells. Orthotopical injection of IL-1α knockout (KO) 4T1 cells into BALB/c mice led to a significant decrease in local tumor growth and lung metastases, compared to injection of wild-type 4T1 (4T1/WT) cells. Neutrophils and myeloid-derived suppressor cells were abundant in tumors developing after injection of 4T1/WT cells, whereas more antigen-presenting cells were observed in the tumor microenvironment after injection of IL-1α KO 4T1 cells. This switch correlated with increased infiltration of CD3+CD8+ and NKp46+cells. Engraftment of IL-1α knockout 4T1 cells into immunodeficient NOD.SCID mice resulted in more rapid tumor growth, with increased lung metastasis in comparison to engraftment of 4T1/WT cells. Our results suggest that tumor-associated IL-1α is involved in TNBC progression in mice by modulating the interplay between immunosuppressive pro-inflammatory cells vs. antigen-presenting and cytotoxic cells.