技术资料

Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However,the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations,therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC),we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-?. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion,we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation. View Publication

New approaches beyond PD-1/PD-L1 inhibition are required to target the immunologically diverse tumor microenvironment (TME) in high-grade serous ovarian cancer (HGSOC). In this study,we explored the immunosuppressive effect of B7-H3 (CD276) via the CCL2-CCR2-M2 macrophage axis and its potential as a therapeutic target. Transcriptome analysis revealed that B7-H3 is highly expressed in PD-L1-low,nonimmunoreactive HGSOC tumors,and its expression negatively correlated with an IFN$\gamma$ signature,which reflects the tumor immune reactivity. In syngeneic mouse models,B7-H3 (Cd276) knockout (KO) in tumor cells,but not in stromal cells,suppressed tumor progression,with a reduced number of M2 macrophages and an increased number of IFN$\gamma$+CD8+ T cells. CCL2 expression was downregulated in the B7-H3 KO tumor cell lines. Inhibition of the CCL2-CCR2 axis partly negated the effects of B7-H3 suppression on M2 macrophage migration and differentiation,and tumor progression. In patients with HGSOC,B7-H3 expression positively correlated with CCL2 expression and M2 macrophage abundance,and patients with B7-H3-high tumors had fewer tumoral IFN$\gamma$+CD8+ T cells and poorer prognosis than patients with B7-H3-low tumors. Thus,B7-H3 expression in tumor cells contributes to CCL2-CCR2-M2 macrophage axis-mediated immunosuppression and tumor progression. These findings provide new insights into the immunologic TME and could aid the development of new therapeutic approaches against the unfavorable HGSOC phenotype. View Publication

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained,and the molecular programmes defining the autoimmune T cell state,are unknown. In type 1 diabetes,$\beta$-cell-specific CD8 T cells destroy insulin-producing $\beta$-cells. Here we followed the fate of $\beta$-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN),which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas,where they differentiate further and destroy $\beta$-cells. Whereas transplantation of as few as 20 ?»¿autoimmune progenitors induced type 1 diabetes,as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived,and stem-like ?»¿autoimmune progenitors must continuously seed the pancreas to sustain $\beta$-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes. View Publication

Oxidative stress,caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses,is one of the characteristic features of cancer. Here,we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo,when compared with blood or normal subcutaneous fluid. Therefore,we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells,and by comparing T,B,and NK cells' sensitivities to redox stress and their antioxidant capacities,we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However,the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore,we genetically modified NK cells to stably overexpress PRDX1,which led to increased survival and NK cell activity in redox stress conditions. Finally,we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide,at concentrations detected in the TME,suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors. View Publication

The atypical cannabinoid Abn-CBD improves the inflammatory status in preclinical models of several pathologies,including autoimmune diseases. However,its potential for modulating inflammation in autoimmune type 1 diabetes (T1D) is unknown. Herein we investigate whether Abn-CBD can modulate the inflammatory response during T1D onset using a mouse model of T1D (non-obese diabetic- (NOD)-mice) and of beta cell damage (streptozotocin (STZ)-injected mice). Six-week-old female NOD mice were treated with Abn-CBD (0.1-1 mg/kg) or vehicle during 12 weeks and then euthanized. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1 mg/kg of body weight) or vehicle for 1 week,following STZ challenge,and euthanized 1 week later. Blood,pancreas,pancreatic lymph nodes (PLNs) and T cells were collected and processed for analysis. Glycemia was also monitored. In NOD mice,treatment with Abn-CBD significantly reduced the severity of insulitis and reduced the pro-inflammatory profile of CD4+ T cells compared to vehicle. Concomitantly,Abn-CBD significantly reduced islet cell apoptosis and improved glucose tolerance. In STZ-injected mice,Abn-CBD decreased circulating proinflammatory cytokines and ameliorated islet inflammation reducing intra-islet phospho-NF-$\kappa$B and TXNIP. Abn-CBD significantly reduced 2 folds intra-islet CD8+ T cells and reduced Th1/non-Th1 ratio in PLNs of STZ-injected mice. Islet cell apoptosis and intra-islet fibrosis were also significantly reduced in Abn-CBD pre-treated mice compared to vehicle. Altogether,Abn-CBD reduces circulating and intra-islet inflammation,preserving islets,thus delaying the progression of insulitis. Hence,Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat the early stages of T1D. View Publication

The intradermal lipopolysaccharide (LPS) challenge in healthy volunteers has proven to be a valuable tool to study local inflammation in vivo. In the current study the inhibitory effects of oral and topical corticosteroid treatment on intradermal LPS responses were evaluated to benchmark the challenge for future investigational drugs. Twenty-four healthy male volunteers received a two-and-a-half-day twice daily (b.i.d.) pretreatment with topical clobetasol propionate 0.05% and six healthy volunteers received a two-and-a-half-day b.i.d. pretreatment with oral prednisolone at 0.25 mg/kg body weight per administration. Participants received one injection regimen of either 0,2,or 4 intradermal LPS injections (5 ng LPS in 50 µL 0.9% sodium chloride solution). The LPS response was evaluated by noninvasive (perfusion,skin temperature,and erythema) and invasive assessments (cellular and cytokine responses) in suction blister exudate. Both corticosteroids significantly suppressed the clinical inflammatory response (erythema P = 0.0001 for clobetasol and P = 0.0016 for prednisolone; heat P = 0.0245 for clobetasol,perfusion P < 0.0001 for clobetasol and P = 0.0036 for prednisolone). Clobetasol also significantly reduced the number of monocytes subsets,dendritic cells,natural killer cells,and T cells in blister exudate. A similar effect was observed for prednisolone. No relevant corticosteroid effects were observed on the cytokine response to LPS. We successfully demonstrated that the anti-inflammatory effects of corticosteroids can be detected using our intradermal LPS challenge model,validating it for evaluation of future investigational drugs,as an initial assessment of the anti-inflammatory effects of such compounds in a minimally invasive manner. View Publication

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific,mAb targeting CD28 homolog (CD28H),a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells,with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation,induced NK-cell cytotoxicity of PD-L1-expressing tumor cells,and activated tissue-resident memory CD8+ T cells. Mechanistically,the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells,including CD8+ T cells,tissue-resident memory T cells,and NK cells,in a tumor-specific manner that may lead to induction of durable,therapeutic antitumor responses. View Publication

PURPOSE Three observations drove this study. First,2'-5'-oligoadenylate synthetase-like protein (OASL) is a negative regulator of type I interferon (IFN). Second,type I IFN plays a central role during virus infections and the pathogenesis of various diseases,including asthma. Third,influenza A virus (IAV) causes non-eosinophilic asthma. To evaluate the potential relationships between OASL,type I IFN,and pulmonary innate immune cells in IAV-induced acute airway inflammation by using Oasl1-/- mice. METHODS Asthma was induced in wild-type (WT) and Oasl1-/- mice with IAV or ovalbumin (OVA). Airway hyperreactivity (AHR) and immune cell infiltration in the bronchoalveolar lavage (BAL) fluids were measured. The immune cells in the lungs were analyzed by flow cytometry. To investigate the ability of type I IFN to shape the response of lung type 2 innate lymphoid cells (ILC2s),IFN-$\alpha$ was treated intratracheally. Plasmacytoid dendritic cells (pDCs) sorted from bone marrow and ILC2s sorted from lungs of naive mice were co-cultured with/without interferon-alpha receptor subunit 1 (IFNAR-1)-blocking antibodies. RESULTS In the IAV-induced asthma model,Oasl1-/- mice developed greater AHR and immune cell infiltration in the BAL fluids than WT mice. This was not observed in OVA-induced asthma,a standard model of allergen-induced asthma. The lungs of infected Oasl1-/- mice also had elevated DC numbers and Ifna expression and depressed IAV-induced ILC2 responses,namely,proliferation and type 2 cytokine and amphiregulin production. Intratracheal administration of type I IFN in na{\{i}}ve mice suppressed lung ILC2 production of type 2 cytokines and amphiregulin. Co-culture of ILC2s with pDCs showed that pDCs inhibit the function of ILC2s by secreting type I IFN. CONCLUSIONS OASL1 may impede the IAV-induced acute airway inflammation that drives AHR by inhibiting IAV-induced type I IFN production from lung DCs thereby preserving the functions of lung ILC2s including their amphiregulin production." View Publication

BACKGROUND Adoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies,yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients,but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo,circumventing TLR-related toxicity. METHODS In this study we investigated how tumor-specific murine CD8+ T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG. RESULTS Herein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+ T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist,CpG,commonly used in the clinic,to bolster T cell-B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination,which are classically required for effective ACT therapy. CpG-conditioned CD8+ T cells acquired a unique proteomic signature hallmarked by an IL-2R$\alpha$highICOShighCD39low phenotype and an altered metabolic profile,all reliant on B cells transiently present in the culture. Likewise,human TILs benefitted from expansion with CpG ex vivo,as they also possessed the IL-2R$\alpha$highICOShighCD39low phenotype. CpG fostered the expansion of potent CD8+ T cells with the signature phenotype and antitumor ability via empowering a direct B-T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture. CONCLUSIONS Our results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+ T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors. View Publication

Low-density neutrophils (LDNs) have been described in tumors and various autoimmune diseases,where they exhibit immune dysfunction and alter disease progression. Nevertheless,LDNs have been rarely reported in sepsis. We studied sepsis patients admitted to the intensive care unit. Wright-Giemsa stain assay and Transmission electron microscopy were performed to detect the morphology of neutrophils. Flow cytometry was used to analyze the number and function of LDNs. Concentration of cytokines was measured using ELISA. Neutrophil chemotaxis was examined using an under-agarose chemotaxis model. We found that LDNs were significantly elevated in patients with sepsis. Phenotypes and morphological characteristics suggest that LDNs may be formed by mixtures of neutrophils at various maturation stages. In vitro experiments showed that LDN formation was closely associated with neutrophil degranulation. We preliminarily discussed changes in immune function in LDNs. Compared with high-density neutrophils,expression levels of CXC chemokine receptor 4 on LDN surfaces were increased,phagocytotic capacity was decreased,and life span was prolonged. The chemotactic ability of LDNs was significantly reduced,possibly related to the increased expression of P2X1. These data suggest that LDNs are essential components of neutrophils in sepsis. To clarify the source and dysfunction mechanism of LDN in sepsis may be helpful for the diagnosis and treatment of sepsis in the future. View Publication

Oncogenic activated RAS mutations have been detected in 50% of de novo and 70% of relapsed multiple myeloma (MM) patients. Translocation t(11;14) involving IgH/CCDN1 and overexpression of cyclin-Ds are early events in MM pathogenesis,enhancing uncontrolled MM cell growth. We hypothesized that targeting both RAS/MAPK pathway molecules including Erk1/2 along with cyclin-Ds enhances MM cytotoxicity and minimizes side effects. Recent studies have demonstrated the high potency of Erk1/2 and CDK4/6 inhibitors in metastatic relapsed cancers,and here we tested anti-MM effects of the Erk1/2??+??CDK4/6 inhibitor combination. Our studies showed strong synergistic (IC?? View Publication

Regulatory T cells (Tregs) suppress adaptive immunity and inflammation. Although they play a role in suppressing anti-tumor responses,development of therapeutics that target Tregs is limited by their low abundance,heterogeneity,and lack of specific cell surface markers. We isolated human PBMC-derived CD4+ CD25high Foxp3+ Tregs and demonstrate they suppress stimulated CD4+ PBMCs in a cell contact-dependent manner. Because it is not possible to functionally characterize cells after intracellular Foxp3 staining,we identified a human T cell line,MoT,as a model of human Foxp3+ Tregs. Unlike Jurkat T cells,MoT cells share common surface markers consistent with human PBMC-derived Tregs such as: CD4,CD25,GITR,LAG-3,PD-L1,CCR4. PBMC-derived Tregs and MoT cells,but not Jurkat cells,inhibited proliferation of human CD4+PBMCs in a ratio-dependent manner. Transwell membrane separation prevented suppression of stimulated CD4+PBMC proliferation by MoT cells and Tregs,suggesting cell-cell contact is required for suppressive activity. Blocking antibodies against PD-L1,LAG-3,GITR,CCR4,HLA-DR,or CTLA-4 did not reverse the suppressive activity.We show that human PBMC-derived Tregs and MoT cells suppress stimulated CD4+PBMCs in a cell contact-dependent manner,suggesting that a Foxp3+Treg population suppresses immune responses by an uncharacterized cell contact-dependent mechanism. View Publication