技术资料

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent antitumor effects in B-cell malignancies including acute lymphoblastic leukemia (ALL),but antigen loss remains the major cause of treatment failure. To mitigate antigen escape and potentially improve the durability of remission,we developed a dual-targeting approach using an optimized,bispecific CAR construct that targets both CD19 and BAFF-R. CD19/BAFF-R dual CAR T cells exhibited antigen-specific cytokine release,degranulation,and cytotoxicity against both CD19-/- and BAFF-R-/- variant human ALL cells in vitro. Immunodeficient mice engrafted with mixed CD19-/- and BAFF-R-/- variant ALL cells and treated with a single dose of CD19/BAFF-R dual CAR T cells experienced complete eradication of both CD19-/- and BAFF-R-/- ALL variants,whereas mice treated with monospecific CD19 or BAFF-R CAR T cells succumbed to outgrowths of CD19-/BAFF-R+ or CD19+/BAFF-R- tumors,respectively. Further,CD19/BAFF-R dual CAR T cells showed prolonged in vivo persistence,raising the possibility that these cells may have the potential to promote durable remissions. Together,our data support clinical translation of BAFF-R/CD19 dual CAR T cells to treat ALL. View Publication

BACKGROUND Adoptive T-cell transfer has become an attractive therapeutic approach for hematological malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) exhibits potent antitumor efficacy against solid tumors,but its clinical application has been stalled because of toxicity. Here,we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors. METHODS We generated a cell membrane-anchored IL-12 (aIL12),a tumor-targeted IL-12 (ttIL12),and a cell membrane-anchored and ttIL-12 (attIL12) and a cell membrane-anchored and tumor-targeted ttIL-12 (attIL12) armed T cells,chimeric antigen receptor-T cells,and T cell receptor-T (TCR-T) cells with each. We compared the safety and efficacy of these armed T cells in treating osteosarcoma patient-derived xenograft tumors and mouse melanoma tumors after intravenous infusions of the armed T cells. RESULTS attIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically,attIL12-T cells targeted tumor cells expressing cell-surface vimentin,enriching effector T cell and interferon $\gamma$ production in tumors,which in turn stimulates dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects. CONCLUSIONS This novel approach sheds light on the safe application of IL-12-based T-cell therapy for large and heterogeneous solid tumors. View Publication

Early erythroid progenitors are transit-amplifying cells with high proliferative capacity committed to undergoing red cell differentiation. CD71/CD24low progenitors are less mature and have greater proliferative capacity than CD71/CD24high. We present protocols for isolation of CD71/CD24low progenitors from mouse fetal liver using both fluorescence-activated cell sorting (FACS) and immunomagnetic enrichment. CD71/CD24low progenitors isolated with both approaches show similar transcriptomes at single-cell resolution and exhibit characteristic proliferative responses to glucocorticoids. For complete details on the use and execution of this protocol,please refer to Li et al. (2019). View Publication

MicroRNAs (miRNAs/miRs) are small,endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling,we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8+ T cells. We observed that out of 362 measurable mature miRNAs,120 were differentially expressed by at least 2-fold in influenza-specific effector CD8+ CTLs compared with naive CD8+ T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139-mediated regulation of CTL effector responses,we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139-/- mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8+ T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8+ T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet,miR-139-/- CD8+ T cells are outcompeted by wild-type CD8+ T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes Using an in vitro model of T cell exhaustion,we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation,miR-139 expression is dispensable for influenza-specific CTL responses. View Publication

PURPOSE Glioblastoma (GBM) patients suffer from a dismal prognosis,with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM,prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here,we identify CD70 as a potential therapeutic target for recurrent GBM CSCs. EXPERIMENTAL DESIGN In the current study,we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells,and further define its function using established stem cell assays. We use CD70 knockdown studies,subsequent RNAseq pathway analysis,and in vivo xenotransplantation to validate CD70's role in GBM. Next,we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy,which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly,we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor,CD27,in immune infiltrates derived from freshly resected GBM tumor samples. RESULTS CD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations,notably putative M1 macrophages and CD4 T cells. CONCLUSION CD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME. View Publication

BACKGROUND Leucine rich repeat kinase 2 (LRRK2) and SNCA are genetically linked to late-onset Parkinson's disease (PD). Aggregated $\alpha$-synuclein pathologically defines PD. Recent studies identified elevated LRRK2 expression in pro-inflammatory CD16+ monocytes in idiopathic PD,as well as increased phosphorylation of the LRRK2 kinase substrate Rab10 in monocytes in some LRRK2 mutation carriers. Brain-engrafting pro-inflammatory monocytes have been implicated in dopaminergic neurodegeneration in PD models. Here we examine how $\alpha$-synuclein and LRRK2 interact in monocytes and subsequent neuroinflammatory responses. METHODS Human and mouse monocytes were differentiated to distinct transcriptional states resembling macrophages,dendritic cells,or microglia,and exposed to well-characterized human or mouse $\alpha$-synuclein fibrils. LRRK2 expression and LRRK2-dependent Rab10 phosphorylation were measured with monoclonal antibodies,and myeloid cell responses to $\alpha$-synuclein fibrils in R1441C-Lrrk2 knock-in mice or G2019S-Lrrk2 BAC mice were evaluated by flow cytometry. Chemotaxis assays were performed with monocyte-derived macrophages stimulated with $\alpha$-synuclein fibrils and microglia in Boyden chambers. RESULTS $\alpha$-synuclein fibrils robustly stimulate LRRK2 and Rab10 phosphorylation in human and mouse macrophages and dendritic-like cells. In these cells,$\alpha$-synuclein fibrils stimulate LRRK2 through JAK-STAT activation and intrinsic LRRK2 kinase activity in a feed-forward pathway that upregulates phosphorylated Rab10. In contrast,LRRK2 expression and Rab10 phosphorylation are both suppressed in microglia-like cells that are otherwise highly responsive to $\alpha$-synuclein fibrils. Corroborating these results,LRRK2 expression in the brain parenchyma occurs in pro-inflammatory monocytes infiltrating from the periphery,distinct from brain-resident microglia. Mice expressing pathogenic LRRK2 mutations G2019S or R1441C have increased numbers of infiltrating pro-inflammatory monocytes in acute response to $\alpha$-synuclein fibrils. In primary cultured macrophages,LRRK2 kinase inhibition dampens $\alpha$-synuclein fibril and microglia-stimulated chemotaxis. CONCLUSIONS Pathologic $\alpha$-synuclein activates LRRK2 expression and kinase activity in monocytes and induces their recruitment to the brain. These results predict that LRRK2 kinase inhibition may attenuate damaging pro-inflammatory monocyte responses in the brain. View Publication

Fibroblast growth factor receptor 1 (FGFR1) is overexpressed in multiple types of solid tumors,including head and neck squamous cell carcinoma (HNSCC). Being associated with poor prognosis,FGFR1 is a potential therapeutic target for aggressive tumors. T cell-based cancer immunotherapy has played a central role in novel cancer treatments. However,the potential of antitumor immunotherapy targeting FGFR1 has not been investigated. Here,we showed that FGFR-tyrosine kinase inhibitors (TKIs) augmented antitumor effects of immune checkpoint inhibitors in an HNSCC mouse model and upregulated tumoral MHC class I and MHC class II expression in vivo and in vitro. This upregulation was associated with the mitogen-activated protein kinase signaling pathway,which is a crucial pathway for cancer development through FGFR signaling. Moreover,we identified an FGFR1-derived peptide epitope (FGFR1305-319) that could elicit antigen-reactive and multiple HLA-restricted CD4+ T cell responses. These T cells showed direct cytotoxicity against tumor cells that expressed FGFR1. Notably,FGFR-TKIs augmented antitumor effects of FGFR1-reactive T cells against human HNSCC cells. These results indicate that the combination of FGFR-TKIs with immunotherapy,such as an FGFR1-targeting peptide vaccine or immune checkpoint inhibitor,could be a novel and robust immunologic approach for treating patients with FGFR1-expressing cancer cells. View Publication

BACKGROUND Monocytes are thought to be involved in venous thrombosis but the role of individual monocyte subpopulations on thrombus formation,clot inflammation,and degradation is an important unresolved issue. We investigate the role of inflammatory Ly6Chi monocytes in deep vein thrombosis and their potential therapeutic impact. METHODS Frequencies and compositions of blood monocytes were analyzed by flow cytometry in CCR2-/- (C-C chemokine receptor type 2) and wild-type mice of different ages and after treatment with the NR4A1 (nuclear receptor group 4 family A member 1,Nur77) agonist CnsB (cytosporone B). TF (tissue factor) sufficient and deficient Ly6Chi monocytes were adoptively transferred into aged CCR2-/- mice. Thrombus formation and size were followed by ultrasound over a 3-week period after surgical reduction of blood flow (stenosis) in the inferior vena cava. RESULTS Reduced numbers of peripheral monocytes in aged (>30 w) CCR2-/- mice are accompanied by reduced thrombus formation after inferior vena cava ligation. Reducing the number of inflammatory Ly6Chi monocytes in wild-type mice by CsnB treatment before ligation,similarly suspends clotting,while later treatment (d1 or d4) reduces thrombus growth and accelerates resolution. We describe how changes in inflammatory monocyte numbers affect the gradual differentiation of monocytes in thrombi and show that only tissue factor-competent Ly6Chi monocytes restore thrombosis in aged CCR2-/- mice. CONCLUSIONS We conclude that the number of inflammatory Ly6Chi monocytes controls deep vein thrombosis formation,growth,and resolution and can be therapeutically manipulated with a NR4A1 agonist at all disease stages. View Publication

Asthma and its heterogeneity change with age. Increased airspace neutrophil numbers contribute to severe steroid-resistant asthma exacerbation in the elderly,which correlates with the changes seen in adults with asthma. However,whether that resembles the same disease mechanism and pathophysiology in aged and adults is poorly understood. Here,we sought to address the underlying molecular mechanism of steroid-resistant airway inflammation development and response to corticosteroid (Dex) therapy in aged mice. To study the changes in inflammatory mechanism,we used a clinically relevant treatment model of house-dust mite (HDM)-induced allergic asthma and investigated lung adaptive immune response in adult (20-22 wk old) and aged (80-82 wk old) mice. Our result indicates an age-dependent increase in airway hyperresponsiveness (AHR),mixed granulomatous airway inflammation comprising eosinophils and neutrophils,and Th1/Th17 immune response with progressive decrease in frequencies and numbers of HDM-bearing dendritic cells (DC) accumulation in the draining lymph node (DLn) of aged mice as compared with adult mice. RNA-Seq experiments of the aged lung revealed short palate,lung,and nasal epithelial clone 1 (SPLUNC1) as one of the steroid-responsive genes,which progressively declined with age and further by HDM-induced inflammation. Moreover,we found increased glycolytic reprogramming,maturation/activation of DCs,the proliferation of OT-II cells,and Th2 cytokine secretion with recombinant SPLUNC1 (rSPLUNC1) treatment. Our results indicate a novel immunomodulatory role of SPLUNC1 regulating metabolic adaptation/maturation of DC. An age-dependent decline in the SPLUNC1 level may be involved in developing steroid-resistant airway inflammation and asthma heterogeneity. View Publication

Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells,suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells,but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration,we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells,we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1,which led to a high-affinity LFA-1 conformation,decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME,through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1,represent a new strategy to improve the efficiency of immunotherapy for cancer treatment. View Publication

Extracellular vesicles (EVs) have potential as minimally invasive biomarkers. However,the methods most commonly used for EV retrieval rely on ultracentrifugation,are time-consuming,and unrealistic to translate to standard-of-care. We sought a method suitable for EV separation from blood that could be used in patient care. Sera from breast cancer patients and age-matched controls (n = 27 patients; n = 36 controls) were analysed to compare 6 proposed EV separation methods. The EVs were then characterised on 8 parameters. The selected method was subsequently applied to independent cohorts of sera (n = 20 patients; n = 20 controls),as proof-of-principle,investigating EVs' gremlin-1 cargo. Three independent runs with each method were very reproducible,within each given method. All isolates contained EVs,although they varied in quantity and purity. Methods that require ultracentrifugation were not superior for low volumes of sera typically available in routine standard-of-care. A CD63/CD81/CD9-coated immunobead-based method was most suitable based on EV markers' detection and minimal albumin and lipoprotein contamination. Applying this method to independent sera cohorts,EVs and their gremlin-1 cargo were at significantly higher amounts for breast cancer patients compared to controls. In conclusion,CD63/CD81/CD9-coated immunobeads may enable clinical utility of blood-based EVs as biomarkers. View Publication

Although critical for host defense,innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during Coronavirus Disease 2019 (COVID-19) ARDS,compared to ARDS from other respiratory pathogens,is unclear. Moreover,mechanisms underlying the beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using single-cell RNA sequencing and plasma proteomics,we discovered that,compared to bacterial ARDS,COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin signaling. Dexamethasone during severe COVID-19 affected circulating neutrophils,altered IFNactive neutrophils,downregulated interferon-stimulated genes and activated IL-1R2+ neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils and preferential steroid-induced immature neutrophil expansion,potentially affecting outcomes. Our single-cell atlas (see 'Data availability' section) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19. View Publication