技术资料

Regulatory T cells (Tregs) are capable of inhibiting the proliferation,activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study,using the Eµ-TCL1 mouse model of CLL,we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly,we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation,the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment,supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far,however,above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific,described in this study Tregs subpopulation. In addition,functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover,inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit,both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether,activation of Tregs appears to be crucial for CLL progression. View Publication

BACKGROUND Bispecific T-cell engager (BiTE) molecules induce redirected lysis of cancer cells by T cells and are an emerging modality for solid tumor immunotherapy. While signs of clinical activity have been demonstrated,efficacy of T-cell engagers (TCEs) in solid tumors settings,molecular determinants of response,and underlying mechanisms of resistance to BiTE therapy require more investigation. METHODS To uncover cancer cell-intrinsic genetic modifiers of TCE-mediated cytotoxicity,we performed genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loss-of-function and CRISPRa (CRISPR activation) gain-of-function screens using TCEs against two distinct tumor-associated antigens (TAAs). By using in vitro T-cell cytotoxicity assays and in vivo efficacy studies,we validated the roles of two common pathways identified in our screen,T-cell costimulation pathway and apoptosis pathway,as key modifiers of BiTE activity. RESULTS Our genetic screens uncovered TAAs-independent cancer cell-intrinsic genes with functions in autophagy,T-cell costimulation,the apoptosis pathway,chromatin remodeling,and cytokine signaling that altered responsiveness to BiTE-mediated killing. Notably,loss of CD58 (the ligand of the CD2 T-cell costimulatory receptor),a gene frequently altered in cancer,led to decreased TCE-mediated cytotoxicity,T-cell activation and antitumor efficacy in vitro and in vivo. Moreover,the effects of CD58 loss were synergistically compounded by concurrent loss of CD80/CD86 (ligands for the CD28 T-cell costimulatory receptor),whereas joint CD2 and CD28 costimulation additively enhanced TCE-mediated killing,indicating non-redundant costimulatory mechanisms between the two pathways. Additionally,loss of CFLAR (Caspase-8 and FADD Like Apoptosis Regulator),BCL2L1,and BID (BH3 Interacting Domain Death Agonist) induced profound changes in sensitivity to TCEs,indicating that key regulators of apoptosis,which are frequently altered in cancer,impact tumor responsiveness to BiTE therapy. CONCLUSIONS This study demonstrates that genetic alterations central to carcinogenesis and commonly detected in cancer samples lead to significant modulation of BiTE antitumor activity in vitro and in vivo,findings with relevance for a better understanding of patient responses to BiTE therapy and novel combinations that enhance TCE efficacy. View Publication

BACKGROUND Myeloid-derived suppressor cells (MDSCs) represent a negative prognostic factor in malignant melanoma. These cells are generated under chronic inflammatory conditions typical of cancer. The transcription factor signal transducer and activator of transcription 3 (STAT3) orchestrates MDSC accumulation and acquisition of immunosuppressive properties. Here we studied STAT3 inhibition by Napabucasin as a way to block MDSC accumulation and activity and its potential to treat malignant melanoma. METHODS In vitro generated murine MDSC and primary MDSC from melanoma-bearing mice were used to investigate the effects of Napabucasin on MDSC in vitro. The RET transgenic mouse model of malignant melanoma was used to examine Napabucasin therapy efficiency and its underlying mechanisms in vivo. Furthermore,STAT3 activation and its correlation with survival were explored in MDSC from 19 patients with malignant melanoma and human in vitro generated monocytic myeloid-derived suppressor cell (M-MDSC) were used to evaluate the effects of Napabucasin. RESULTS Napabucasin was able to abrogate the capacity of murine MDSC to suppress CD8+ T-cell proliferation. The STAT3 inhibitor induced apoptosis in murine MDSC,significantly increased expression of molecules associated with antigen processing and presentation,as well as slightly decreased expression of immunosuppressive factors on these cells. RET transgenic mice treated with Napabucasin showed prolonged survival accompanied by a strong accumulation of tumor-infiltrating antigen-presenting cells and activation of CD8+ and CD4+ T cells. Interestingly,patients with malignant melanoma with high expression of activated STAT3 in circulating M-MDSC showed significantly worse progression-free survival (PFS) than patients with low levels of activated STAT3. In addition,Napabucasin was able to abrogate suppressive capacity of human in vitro generated M-MDSC. CONCLUSION Our findings demonstrate that STAT3 inhibitor Napabucasin completely abrogated the immunosuppressive capacity of murine MDSC and human M-MDSC and improved melanoma-bearing mouse survival. Moreover,patients with malignant melanoma with high expression levels of activated STAT3 in M-MDSC displayed shorter PFS,indicating its role as a promising therapeutic target in patients with malignant melanoma and a predictive marker for their clinical outcome. View Publication

BACKGROUND Epidemiological surveys have revealed that low serum vitamin D level was correlated with increased risk of tumors. Dysfunctional T cells in patients with tumor are characterized as exhausted with high levels of immune checkpoint receptors (ICRs). However,whether the reduced level of vitamin D in patients with cancer correlates with cytotoxic T-cell exhaustion is unknown. METHODS Periphery blood samples from 172 patients with non-small cell lung cancer (NSCLC) were prospectively collected. Patients with NSCLC received one course of intravenous docetaxel (75 mg/m2) followed by treatment with or without rocaltrol at a dose of 0.5-2.0 µg/day for total of 3 weeks. We performed phenotypical and functional analysis of T-cell through flow cytometry. Vitamin D receptor (VDR) knockout and overexpression CD8+ and V$\delta$2+ T cells were constructed using Cas9-gRNA targeted and overexpressing approaches to identify 1$\alpha$,25(OH)2D3/VDR-mediated transcription regulation for ICRs or antitumor activity in T cells. RESULTS We show that serum level of vitamin D is negatively correlated with expression of programmed cell death-1 (PD-1),T-cell immunoreceptor with Ig and ITIM domains (TIGIT),and T-cell immunoglobulin and mucin-domain containing-3 (Tim-3),but positively correlated with CD28 expression on CD8+ and V$\gamma$9V$\delta$2+ T cells in patients with NSCLC. 1$\alpha$,25(OH)2D3,the active form of vitamin D,promotes the nuclear translocation of VDR,which binds to the promoter region of Pdcd1,Tim3,and Tigit genes and inhibits their expression. Besides,1$\alpha$,25(OH)2D3 pretreatment also promotes the methylation of CpG island in the promoter region of the Pdcd1 gene and increases H3K27 acetylation at the promoter region of the Cd28 gene,which leads to surface PD-1 downregulation and CD28 upregulation,respectively. We further reveal that VDR-mediated Ca2+ influx enhanced expression of Th1 cytokines via T-cell receptor activation. Functionally,1$\alpha$,25(OH)2D3 pretreated CD8+ T cells or V$\gamma$9V$\delta$2+ T cells showed increased Th1 cytokine production and enhanced antitumor immunity. Finally,oral 1$\alpha$,25(OH)2D3 could also decrease expression of PD-1,Tim-3,TIGIT and increase expression of CD28,resulting in cytokine production (associated with antitumor immunity) by cytotoxic T cells of patients with NSCLC. CONCLUSIONS Our findings uncover the pleiotropic effects of 1$\alpha$,25(OH)2D3 in rescuing the exhausted phenotype of human cytotoxic T cells in patients with tumor and in promoting their antitumor immunity. TRIAL REGISTRATION NUMBER ChiCTR2100051135. View Publication

Chronic decreases in the second messenger cyclic AMP (cAMP) occur in numerous settings,but how cells compensate for such decreases is unknown. We have used a unique system-murine dendritic cells (DCs) with a DC-selective depletion of the heterotrimeric GTP binding protein G$\alpha$s-to address this issue. These mice spontaneously develop Th2-allergic asthma and their DCs have persistently lower cAMP levels. We found that phosphodiesterase 4B (PDE4B) is the primary phosphodiesterase expressed in DCs and that its expression is preferentially decreased in G$\alpha$s-depleted DCs. PDE4B expression is dynamic,falling and rising in a protein kinase A-dependent manner with decreased and increased cAMP concentrations,respectively. Treatment of DCs that drive enhanced Th2 immunity with a PDE4B inhibitor ameliorated DC-induced helper T cell response. We conclude that PDE4B is a homeostatic regulator of cellular cAMP concentrations in DCs and may be a target for treating Th2-allergic asthma and other settings with low cellular cAMP concentrations. View Publication

BACKGROUND The Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3,which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered,unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man. METHODS We have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons. RESULTS AZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs,reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO,which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs,reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo,strongly modulated Treg effector molecules (eg,ICOS,CTLA-4,CD25 and 4-1BB),and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy. CONCLUSIONS Antisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669). View Publication

Alveolar macrophages (AMs) are functionally important innate cells involved in lung homeostasis and immunity and whose diversity in health and disease is a subject of intense investigations. Yet,it remains unclear to what extent conditions like smoking or chronic obstructive pulmonary disease (COPD) trigger changes in the AM compartment. Here,we aimed to explore heterogeneity of human AMs isolated from healthy nonsmokers,smokers without COPD,and smokers with COPD by analyzing BAL fluid cells by flow cytometry and bulk and single-cell RNA sequencing. We found that subpopulations of BAL fluid CD206+ macrophages could be distinguished based on their degree of autofluorescence in each subject analyzed. CD206+ autofluorescenthigh AMs were identified as classical,self-proliferative AM,whereas autofluorescentlow AMs were expressing both monocyte and classical AM-related genes,supportive of a monocytic origin. Of note,monocyte-derived autofluorescentlow AMs exhibited a functionally distinct immunoregulatory profile,including the ability to secrete the immunosuppressive cytokine IL-10. Interestingly,single-cell RNA-sequencing analyses showed that transcriptionally distinct clusters of classical and monocyte-derived AM were uniquely enriched in smokers with and without COPD as compared with healthy nonsmokers. Of note,such smoking-associated clusters exhibited gene signatures enriched in detoxification,oxidative stress,and proinflammatory responses. Our study independently confirms previous reports supporting that monocyte-derived macrophages coexist with classical AM in the airways of healthy subjects and patients with COPD and identifies smoking-associated changes in the AM compartment that may favor COPD initiation or progression. View Publication

Impaired T cell immunity with aging increases mortality from infectious disease. The branching of Asparagine-linked glycans is a critical negative regulator of T cell immunity. Here we show that branching increases with age in females more than males,in na{\{i}}ve more than memory T cells and in CD4+ more than CD8+ T cells. Female sex hormones and thymic output of na{\"{i}}ve T cells (TN) decrease with age however neither thymectomy nor ovariectomy altered branching. Interleukin-7 (IL-7) signaling was increased in old female more than male mouse TN cells and triggered increased branching. N-acetylglucosamine a rate-limiting metabolite for branching increased with age in humans and synergized with IL-7 to raise branching. Reversing elevated branching rejuvenated T cell function and reduced severity of Salmonella infection in old female mice. These data suggest sex-dimorphic antagonistic pleiotropy where IL-7 initially benefits immunity through TN maintenance but inhibits TN function by raising branching synergistically with age-dependent increases in N-acetylglucosamine." View Publication

Resistance mechanisms and heterogeneity in HER2-positive gastric cancers (GC) limit Trastuzumab benefit in 32% of patients,and other targeted therapies have failed in clinical trials. Using patient samples,patient-derived xenografts (PDXs),partially humanized biological models,and HER2-targeted imaging technologies we demonstrate the role of caveolin-1 (CAV1) as a complementary biomarker in GC selection for Trastuzumab therapy. In retrospective analyses of samples from patients enrolled on Trastuzumab trials,the CAV1-high profile associates with low membrane HER2 density and low patient survival. We show a negative correlation between CAV1 tumoral protein levels - a major protein of cholesterol-rich membrane domains - and Trastuzumab-drug conjugate TDM1 tumor uptake. Finally,CAV1 depletion using knockdown or pharmacologic approaches (statins) increases antibody drug efficacy in tumors with incomplete HER2 membranous reactivity. In support of these findings,background statin use in patients associates with enhanced antibody efficacy. Together,this work provides preclinical justification and clinical evidence that require prospective investigation of antibody drugs combined with statins to delay drug resistance in tumors. View Publication

The family of hexokinases (HKs) catalyzes the first step of glycolysis,the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed,the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system,we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead,loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death,oxidative stress,and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing,showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns,we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM,which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival,possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation. View Publication

Fibroblasts are the most abundant stromal constituents of the tumour microenvironment in primary as well as metastatic colorectal cancer (CRC). Their supportive effect on tumour cells is well established. There is growing evidence that stromal fibroblasts also modulate the immune microenvironment in tumours. Here,we demonstrate a difference in fibroblast-mediated immune modulation between primary CRC and peritoneal metastasis. Cancer-associated fibroblasts (CAFs) were isolated from primary cancer and from peritoneal metastases (MAFs) from a total of 17 patients. The ectoenzyme CD38 was consistently expressed on the surface of all MAFs,while it was absent from CAFs. Furthermore,MAFs secreted higher levels of IGFBP2,CXCL2,CXCL6,CXCL12,PDGF-AA,FGFb,and IL-6. This was associated with a decreased activation of macrophages and a suppression of CD25 expression and proliferation of co-cultivated T-cells. Downregulation of IGFBP2 abolished these immunosuppressive effects of MAFs. Taken together,these results show that MAFs contribute to an immunosuppressive tumour microenvironment in CRC metastases by modulating the phenotype of immune cells through an IGFBP2-dependent mechanism. View Publication

Metabolic reprogramming is associated with myeloid-derived suppressor cell (MDSC) immunosuppressive function. Here,we outline the process for acquiring MDSCs from human and murine sources for subsequent analysis of fatty acid oxidation,oxidative phosphorylation,and glycolysis using the Seahorse XFe 96 Analyzer. Murine MDSCs can be isolated directly from tumor-bearing mice or derived through IL-6 and GM-CSF culture of bone marrow cells from non-tumor-bearing mice. To generate human MDSCs,peripheral blood mononuclear cells (PBMCs) can be cultured with IL-6 and GM-CSF. For complete details on the use and execution of this protocol,please refer to Mohammadpour et al. (2021). View Publication