技术资料

Interleukin-4 (IL-4) is a signature cytokine pivotal in Type 2 helper T cell (Th2) immune response,particularly in allergy and hypersensitivity. Interestingly,IL-4 increases endogenous levels of prostaglandin D2 (PGD2 ) and its metabolites,$\Delta$12 -prostaglandin J2 ($\Delta$12 -PGJ2 ) and 15-deoxy-$\Delta$12,14 -prostaglandin J2 (15d-PGJ2 ),collectively called cyclopentenone PGs (CyPGs). However,the therapeutic role of IL-4 in hematologic malignancies remains unclear. Here,we employed a murine model of acute myeloid leukemia (AML),where human MLL-AF9 fusion oncoprotein was expressed in hematopoietic progenitor cells,to test the effect of IL-4 treatment in vivo. Daily intraperitoneal treatment with IL-4 at 60 µg/kg/d significantly alleviated the severity of AML,as seen by decreased leukemia-initiating cells (LICs). The effect of IL-4 was mediated,in part,by the enhanced expression of hematopoietic- PGD2  synthase (H-PGDS) to effect endogenous production of CyPGs,through autocrine and paracrine signaling mechanisms. Similar results were seen with patient-derived AML cells cultured ex vivo with IL-4. Use of GW9662,a peroxisome proliferator-activated receptor gamma (PPAR$\gamma$) antagonist,suggested endogenous CyPGs-PPAR$\gamma$ axis mediated p53-dependent apoptosis of LICs by IL-4. Taken together,our results reveal a beneficial role of IL-4 treatment in AML suggesting a potential therapeutic regimen worthy of clinical trials in patients with AML. View Publication

Alloantigen-specific regulatory T cell (Treg) therapy is a promising approach for suppressing alloimmune responses and minimizing immunosuppression after solid organ transplantation. Chimeric antigen receptor (CAR) targeting donor alloantigens can confer donor reactivity to Tregs. However,CAR Treg therapy has not been evaluated in vascularized transplant or multi-MHC mismatched models. Here,we evaluated the ability of CAR Tregs targeting HLA-A2 (A2-CAR) to prolong the survival of heterotopic heart transplants in mice. After verifying the in vitro activation,proliferation,and enhanced suppressive function of A2-CAR Tregs in the presence of A2-antigen,we analyzed the in vivo function of Tregs in C57BL/6 (B6) mice receiving A2-expressing heart allografts. A2-CAR Treg infusion increased the median survival of grafts from B6.HLA-A2 transgenic donors from 23 to 99 days,whereas median survival with polyclonal Treg infusion was 35 days. In a more stringent model of haplo-mismatched hearts from BALB/cxB6.HLA-A2 F1 donors,A2-CAR Tregs slightly increased median graft survival from 11 to 14 days,which was further extended to >100 days when combined with a 9-day course of rapamycin treatment. These findings demonstrate the efficacy of CAR Tregs,alone or in combination with immunosuppressive agents,toward protecting vascularized grafts in fully immunocompetent recipients. View Publication

Acute myeloid leukemia (AML) is a malignant disorder derived from neoplastic myeloid progenitor cells characterized by abnormal proliferation and differentiation. Although novel therapeutics have recently been introduced,AML remains a therapeutic challenge with insufficient cure rates. In the last years,immune-directed therapies such as chimeric antigen receptor (CAR)-T cells were introduced,which showed outstanding clinical activity against B-cell malignancies including acute lymphoblastic leukemia (ALL). However,the application of CAR-T cells appears to be challenging due to the enormous molecular heterogeneity of the disease and potential long-term suppression of hematopoiesis. Here we report on the generation of CD33-targeted CAR-modified natural killer (NK) cells by transduction of blood-derived primary NK cells using baboon envelope pseudotyped lentiviral vectors (BaEV-LVs). Transduced cells displayed stable CAR-expression,unimpeded proliferation,and increased cytotoxic activity against CD33-positive OCI-AML2 and primary AML cells in vitro. Furthermore,CD33-CAR-NK cells strongly reduced leukemic burden and prevented bone marrow engraftment of leukemic cells in OCI-AML2 xenograft mouse models without observable side effects. View Publication

Mutations causing loss of the NF-$\kappa$B regulator I$\kappa$BNS,result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming,we sought to investigate processes important for this transition using the bumble mouse strain,deficient for I$\kappa$BNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels,mitochondrial accumulation,increased OXPHOS and mROS production,along with a reduced capacity for autophagy,compared to wildtype B cells. Overall,our results demonstrate that PC differentiation in the absence of I$\kappa$BNS is characterized by excessive activation during early rounds of B cell division,increased mitochondrial metabolism and decreased autophagic capacity,thus improving our understanding of the role of I$\kappa$BNS in PC differentiation. View Publication

Monocytes undergo phenotypic and functional changes in response to inflammatory cues,but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes,which are preferentially recruited,dwell within glomerular capillaries,and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically,the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells,findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2-activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients,and their frequency correlates with clinical disease. In conclusion,CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2-activated vasculature and may establish a CCR2-based autocrine,feed-forward loop that amplifies renal inflammation. View Publication

Human norovirus (HNoV) is a global health and socioeconomic burden,estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole blood were infected with HNoV-positive stool samples and harvested at 3??days postinfection (dpi) to assess the viral RNA yield by reverse transcriptase quantitative PCR (RT-qPCR). A 3- to 18-fold increase in the HNoV RNA yield was observed in 50 to 60% of donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsy specimens. Next,we characterized infection of whole-blood-derived B cells by flow cytometry in specific functional B cell subsets (naive CD27- IgD+,memory-switched CD27+ IgD-,memory-unswitched CD27+ IgD+,and double-negative CD27- IgD- cells). While the susceptibilities of the subsets were similar,changes in the B cell subset distribution upon infection were observed,which were also noted after treatment with HNoV virus-like particles and the predicted recombinant NS1 protein. Importantly,primary B cell stimulation with the predicted recombinant NS1 protein triggered B cell activation and induced metabolic changes. These data demonstrate that primary B cells are susceptible to HNoV infection and suggest that the NS1 protein can alter B cell activation and metabolism in vitro,which could have implications for viral pathogenesis and immune responses in vivo. IMPORTANCE Human norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised,the elderly,and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socioeconomic burdens associated with HNoV infections. Moreover,HNoV does not elicit lifelong immunity as repeat infections are common,presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity,we investigated the susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood,spleen,and lymph node specimens,while the nonstructural protein NS1 can activate B cells. Because of the secreted nature of NS1,we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential impacts on systemic immune responses. View Publication

Several unique waves of ?? T cells are generated solely in the fetal/neonatal thymus,whereas additional ?? T cell subsets are generated in adults. One intriguing feature of ?? T cell development is the coordination of differentiation and acquisition of effector function within the fetal thymus; however,it is less clear whether this paradigm holds true in adult animals. In this study,we investigated the relationship between maturation and thymic export of adult-derived ?? thymocytes in mice. In the Rag2pGFP model,immature (CD24+) ?? thymocytes expressed high levels of GFP whereas only a minority of mature (CD24-) ?? thymocytes were GFP+ Similarly,most peripheral GFP+ ?? T cells were immature. Analysis of ?? recent thymic emigrants (RTEs) indicated that most ?? T cell RTEs were CD24+ and GFP+,and adoptive transfer experiments demonstrated that immature ?? thymocytes can mature outside the thymus. Mature ?? T cells largely did not recirculate to the thymus from the periphery; rather,a population of mature ?? thymocytes that produced IFN-? or IL-17 remained resident in the thymus for at least 60 d. These data support the existence of two populations of ?? T cell RTEs in adult mice: a majority subset that is immature and matures in the periphery after thymic emigration,and a minority subset that completes maturation within the thymus prior to emigration. Additionally,we identified a heterogeneous population of resident ?? thymocytes of unknown functional importance. Collectively,these data shed light on the generation of the ?? T cell compartment in adult mice. View Publication

Identifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study,hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration,indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed,HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters na{\{i}}ve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME." 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

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

Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells that recognize specific sialic acid modifications on cell surface glycans. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered immune responses and increased tumor growth. In this study,we examined the role of ST8Sia6 on immune cells in regulating antitumor immunity. ST8Sia6 knockout mice had an enhanced immune response to tumors. The loss of ST8Sia6 promoted an enhanced intratumoral activation of macrophages and dendritic cells,including upregulation of CD40. Intratumoral regulatory T cells exhibited a more inflammatory phenotype in ST8Sia6 knockout mice. Using adoptive transfer studies,the change in regulatory T cell phenotype was not cell intrinsic and depended on the loss of ST8Sia6 expression in APCs. Thus,ST8Sia6 generates ligands for Siglecs that dampen antitumor immunity. View Publication

Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis,with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN,NK cells and ILC3 were the prominent subpopulations. Among the ILC3s,we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients,NK cells were prominent,and proportions of ILC3 subsets were low. In tumors and TD-LN,NK cells display reduced levels of NCR (Natural cytotoxicity receptors),despite high transcript levels and included a small subset CD127- CD56- NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN,all cytokine activated ILC populations produced TNF$\alpha$ in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR,but cytokine stimulation restored their functionality. View Publication