技术资料

Most cancer vaccines target peptide antigens,necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore,tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells,but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding,enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably,this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary,highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations. View Publication

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients but can be limited by poor T-cell expansion and persistence in vivo. Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses,and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1,attenuate differentiation,and enhance proliferation and antitumor efficacy in vivo. Here,we used a cell-free culture system to provide an agonistic NOTCH1 signal during na{\{i}}ve CD4+ T-cell activation and CAR-T production and studied the effects on differentiation transcription factor expression cytokine production and responses to tumor. NOTCH1 agonism efficiently induced a stem cell memory phenotype in CAR-T derived from na{\"{i}}ve but not memory CD4+ T cells and upregulated expression of AhR and c-MAF driving heightened production of interleukin-22 interleukin-10 and granzyme B. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to cotransferred CD8+ CAR-T driving improved expansion and curative antitumor responses in vivo at low CAR-T doses. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior and demonstrate that activating NOTCH1 signaling during genetic modification ex vivo is a potential strategy for enhancing the function of T cells engineered with tumor-targeting receptors." 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

BACKGROUND The success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here,we targeted an intracellular inhibiting protein 'cytokine inducible SH2-containing protein' (CISH) in NK cells to evaluate the impact on their functions and antitumor properties. METHODS To further understand CISH functions in NK cells,we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+ ). NK cells cytokine expression,signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line,metastasis evaluation was performed. Then,orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally,we targeted CISH in human NK-92 or primary NK cells,using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions. RESULTS In Cishfl/flNcr1Ki/+ mice,we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways,triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor,optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression,limiting NK cell exhaustion. Finally,using CRISPRi,we then targeted CISH in human NK-92 or primary NK cells. In human NK cells,CISH deletion also favors NCR signaling and antitumor functions. CONCLUSION This study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy. 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

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

BACKGROUND Recent investigations of the meninges have highlighted the importance of the dura layer in central nervous system immune surveillance beyond a purely structural role. However,our understanding of the meninges largely stems from the use of pre-clinical models rather than human samples. METHODS Single-cell RNA sequencing of seven non-tumor-associated human dura samples and six primary meningioma tumor samples (4 matched and 2 non-matched) was performed. Cell type identities,gene expression profiles,and T cell receptor expression were analyzed. Copy number variant (CNV) analysis was performed to identify putative tumor cells and analyze intratumoral CNV heterogeneity. Immunohistochemistry and imaging mass cytometry was performed on selected samples to validate protein expression and reveal spatial localization of select protein markers. RESULTS In this study,we use single-cell RNA sequencing to perform the first characterization of both non-tumor-associated human dura and primary meningioma samples. First,we reveal a complex immune microenvironment in human dura that is transcriptionally distinct from that of meningioma. In addition,we characterize a functionally diverse and heterogenous landscape of non-immune cells including endothelial cells and fibroblasts. Through imaging mass cytometry,we highlight the spatial relationship among immune cell types and vasculature in non-tumor-associated dura. Utilizing T cell receptor sequencing,we show significant TCR overlap between matched dura and meningioma samples. Finally,we report copy number variant heterogeneity within our meningioma samples. CONCLUSIONS Our comprehensive investigation of both the immune and non-immune cellular landscapes of human dura and meningioma at single-cell resolution builds upon previously published data in murine models and provides new insight into previously uncharacterized roles of human dura. 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

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

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

Myeloid Derived Suppressor Cells (MDSCs) play important roles in constituting the immune suppressive environment promoting cancer development and progression. They are consisted of a heterogeneous population of immature myeloid cells including polymorphonuclear MDSC (PMN-MDSC) and monocytes MDSC (M-MDSC) that are found in both the systemic circulation and in the tumor microenvironment (TME). While previous studies had shown that all-trans retinoic acid (ATRA) could induce MDSC differentiation and maturation,the very poor solubility and fast metabolism of the drug limited its applications as an immune-modulator for cancer immunotherapy. We aimed in this study to develop a drug encapsulated liposome formulation L-ATRA with sustained release properties and examined the immuno-modulation effects. We showed that the actively loaded L-ATRA achieved stable encapsulation and enabled controlled drug release and accumulation in the tumor tissues. In vivo administration of L-ATRA promoted the remodeling of the systemic immune homeostasis as well as the tumor microenvironment. They were found to promote MDSCs maturation into DCs and facilitate immune responses against cancer cells. When used as a single agent treatment,L-ATRA deterred tumor growth,but only in immune-competent mice. In mice with impaired immune functions,L-ATRA at the same dose was not effective. When combined with checkpoint inhibitory agents,L-ATRA resulted in greater anti-cancer activities. Thus,L-ATRA may present a new IO strategy targeting the MDSCs that needs be further explored for improving the immunotherapy efficacy in cancer. View Publication

To determine whether antigen presentation by HLA-DR on hematopoietic stem progenitor cells (HSPCs) is involved in the development of acquired aplastic anemia (AA),we studied the HLA-DR expression on CD45dimCD34+CD38+ cells in the peripheral blood of 61 AA patients including 23 patients possessing HLA-class I allele-lacking (HLA-class I[-]) leukocytes. HLA-DR-lacking (DR[-]) cells accounted for 13.0-57.1% of the total HSPCs in seven (11.5%) patients with HLA-DR15 who did not possess HLA-class I(-) leukocytes. The incubation of sorted DR(-) HSPCs in the presence of IFN-$\gamma$ for 72??h resulted in the full restoration of the DR expression. A comparison of the transcriptome profile between DR(-) and DR(+) HSPCs revealed the lower expression of immune response-related genes including co-stimulatory molecules (e.g.,CD48,CD74,and CD86) in DR(-) cells,which was not evident in HLA-class I(-) HSPCs. DR(-) cells were exclusively detected in GPI(+) HSPCs in four patients whose HSPCs could be analyzed separately for GPI(+) and GPI(-) HSPCs. These findings suggest that CD4+ T cells specific to antigens presented by HLA-DR15 on HSPCs may contribute to the development of AA as well as the immune escape of GPI(-) HSPCs in a distinct way from CD8+ T cells recognizing HLA-class I-restricted antigens. View Publication