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Human neutrophilic granulocytes form the largest pool of innate immune cells for host defense against bacterial and fungal pathogens. The dynamic changes that accompany the metamorphosis from a proliferating myeloid progenitor cell in the bone marrow into a mature non-dividing polymorphonuclear blood cell have remained poorly defined. Using mass spectrometry-based quantitative proteomics combined with transcriptomic data,we report on the dynamic changes of five developmental stages in the bone marrow and blood. Integration of transcriptomes and proteome unveils highly dynamic and differential interactions between RNA and protein kinetics during human neutrophil development,which can be linked to functional maturation of typical end-stage blood neutrophil killing activities. View Publication

Epitope-specific CD4+ T cells can be labeled in complex cell mixtures from secondary lymphoid organs with fluorophore-labeled peptide:major histocompatibility complex class II (p:MHCII) tetramers and then detected by flow cytometry. Magnetic enrichment of tetramer-bound cells before flow cytometry increases the sensitivity of detection to the point where epitope-specific cells can be studied even when very rare at early and late times after the host has been exposed to the epitope. This method is very useful for studying polyclonal epitope-specific CD4+ T cells under physiological conditions. {\textcopyright} 2019 by John Wiley {\&} Sons,Inc. View Publication

Abdominal aortic aneurysm (AAA) is a prevalent life-threatening disease,where aortic wall degradation is mediated by accumulated immune cells. Although cytokines regulate inflammation within the aorta,their contribution to AAA via distant alterations,particularly in the control of hematopoietic stem cell (HSC) differentiation,remains poorly defined. Here we report a pathogenic role for the interleukin-27 receptor (IL-27R) in AAA,as genetic ablation of IL-27R protects mice from the disease development. Mitigation of AAA is associated with a blunted accumulation of myeloid cells in the aorta due to the attenuation of Angiotensin II (Ang II)-induced HSC expansion. IL-27R signaling is required to induce transcriptional programming to overcome HSC quiescence and increase differentiation and output of mature myeloid cells in response to stress stimuli to promote their accumulation in the diseased aorta. Overall,our studies illuminate how a prominent vascular disease can be distantly driven by a cytokine-dependent regulation of bone marrow precursors. View Publication

(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists,RO6871304,and RO6871085,as well as a CB2R inverse agonist,RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions,binding,cell signaling ({\ss}-arrestin and cAMP) and early absorption,distribution,metabolism,excretion,and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity,in comparison to the reported CB2R ligand,HU910,using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice,and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils,respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R {\textgreater} CB1R,with both ligands acting as full agonists in cAMP and {\ss}-arrestin assays (EC50s in low nM range). When tested in EIU,topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist,RO6851228. The CB2R agonist,RO6871304,decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-,and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085,as well as HU910,decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases. View Publication

Hodgkin Lymphoma (HL) is a malignancy that frequently affects young adults. Although,there are effective treatments not every patient responds,necessitating the development of novel therapeutic approaches,especially for relapsed and refractory cases. The two TNF receptor family members CD30 and CD137 are expressed on Hodgkin and Reed Sternberg (HRS) cells,the malignant cells in HL. We found that this co-expression is specific for HRS cells. Based on this discovery we developed a bispecific antibody that binds preferentially to the CD30,CD137-double positive HRS cells. The CD30,CD137 bispecific antibody gets internalized into HRS cells opening up the possibility to use it as a carrier for a toxin. This antibody also induces antibody-dependent,cell-mediated cytotoxicity in CD30,CD137-double positive HRS cells. The enhances specificity of the CD30,CD137 bispecific antibody to HRS cells makes it a promising candidate for development as a novel HL treatment. View Publication

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however,unipotent neutrophil progenitors are not well defined. Here,we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly,we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models,including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases,including cancer. View Publication

Neurologic melioidosis occurs in both human and animals; however,the mechanism by which the pathogen Burkholderia pseudomallei invades the central nervous system (CNS) remains unclear. B. pseudomallei-loaded Ly6C cells have been suggested as a putative portal; however,during melioidosis,lipopolysaccharide (LPS) can drive disruption of the blood-brain barrier (BBB). This study aims to test whether the Trojan horse-like mechanism occurs during endotoxemia. The expression levels of cerebral cytokines,chemokines and cell adhesion molecules; the activation of astrocytes,microglia and endothelial cells; and the increased vascular permeability and brain-infiltrating leukocytes were evaluated using B. pseudomallei,B. thailandensis,B. cenocepacia and B. multivorans LPS-induced brains. Accordingly,different degrees of BBB damage in those brains with endotoxemia were established. The B. multivorans LPS-induced brain exhibited the highest levels of disruptive BBB according to the above mediators/indicators. Into these distinct groups of endotoxemic mice,B. pseudomallei-loaded Ly6C cells or free B. pseudomallei were adoptively transferred at equal bacterial concentrations (103 CFU). The bacterial load and number of cases of meningeal neutrophil infiltration in the brains of animals treated with B. pseudomallei-loaded Ly6C cells were higher than those in brains induced by free B. pseudomallei in any of the endotoxemic groups. In particular,these results were reproducible in B. multivorans LPS-induced brains. We suggest that B. pseudomallei-loaded cells can act as a Trojan horse and are more effective than free B. pseudomallei in invading the CNS under septic or endotoxemic conditions even when there is a high degree of BBB disruption. View Publication

Human mucosal tissues and skin contain two distinct types of dendritic cell (DC) subsets,epidermal Langerhans cells (LCs) and dermal DCs,which can be distinguished by the expression of C-type lectin receptors,Langerin and DC-SIGN,respectively. Although peripheral blood monocytes differentiate into these distinct subsets,monocyte-derived LCs (moLCs) induced by coculture with GM-CSF,IL-4,and TGF-$\beta$1 coexpress both Langerin and DC-SIGN,suggesting that the environmental cues remain unclear. In this study,we show that LC differentiation is TGF-$\beta$1 dependent and that cofactors such as IL-4 and TNF-$\alpha$ promote TGF-$\beta$1-dependent LC differentiation into Langerin+DC-SIGN- moLCs but continuous exposure to IL-4 blocks differentiation. Steroids such as dexamethasone greatly enhanced TNF-$\alpha$-induced moLC differentiation and blocked DC-SIGN expression. Consistent with primary LCs,dexamethasone-treated moLCs express CD1a,whereas monocyte-derived DCs (moDCs) express CD1b,CD1c,and CD1d. moDCs but not moLCs produced inflammatory cytokines after stimulation with CD1b and CD1d ligands mycolic acid and $\alpha$-galactosylceramide,respectively. Strikingly,CD1a triggering with squalene on moLCs but not moDCs induced strong IL-22-producing CD4+ helper T cell responses. As IL-22 is an important cytokine in the maintenance of skin homeostasis,these data suggest that CD1a on LCs is involved in maintaining the immune barrier in the skin. View Publication

Effective humoral immunity requires class switch recombination (CSR) catalyzed by activation-induced cytidine deaminase (AID). In response to T cell-dependent (TD) Ags,CSR can be induced by CD40 signaling in B cells. TNFR-associated factors 2 and 3 (TRAF2/TRAF3) function as adaptors of the CD40 signaling pathway. B cell-intrinsic TRAF2 or TRAF3 (B-TRAF2 or B-TRAF3) knockout mice were previously reported to have indistinguishable phenotypes in gene expression,B cell survival and development,and enlarged peripheral lymphoid organs. However,it remains unknown whether deficiency of B-TRAF2 or B-TRAF3 differentially affects TD humoral immune responses and CD40-induced CSR. In this article,we show that B-TRAF2 is essential for optimal isotype switching induced by in vivo TD Ag immunization or by engaging CD40 in vitro. Our data clarify the controversial role of B-TRAF3 and confirm its dispensability in CD40-induced CSR. Mechanistically,CD40-induced AID expression was markedly impaired by B-TRAF2,but not B-TRAF3,deficiency. Moreover,B-TRAF2 deficiency causes defective activation of the NF-$\kappa$B1 complex in a CD40-autonomous manner,and restoring CD40-induced NF-$\kappa$B1 activation in TRAF2-deficient B cells rescues AID expression and CSR. We conclude that TRAF2 is essential but TRAF3 is dispensable for TD humoral immunity and CD40-induced CSR. Our studies provide significant biological bases for optimizing treatment of B cell-associated immune disorders by targeting CD40 signaling. View Publication

Indoleamine 2,3-dioxygenase 2 (Ido2) is a recently identified catalytic enzyme in the tryptophan-kynurenine pathway that is expressed primarily in monocytes and dendritic cells. To elucidate the biological role of Ido2 in immune function,we introduced lipopolysaccharide (LPS) endotoxin shock to Ido2 knockout (Ido2 KO) mice,which led to higher mortality than that in the wild type (WT) mice. LPS-treated Ido2 KO mice had increased production of inflammatory cytokines (including interleukin-6; IL-6) in serum and signal transducer and activator of transcription 3 (stat3) phosphorylation in the spleen. Moreover,the peritoneal macrophages of LPS-treated Ido2 KO mice produced more cytokines than did the WT mice. By contrast,the overexpression of Ido2 in the murine macrophage cell line (RAW) suppressed cytokine production and decreased stat3 expression. Finally,RAW cells overexpressing Ido2 did not alter nuclear factor $\kappa$B (NF-$\kappa$B) or stat1 expression,but IL-6 and stat3 expression decreased relative to the control cell line. These results reveal that Ido2 modulates IL-6/stat3 signalling and is induced by LPS,providing novel options for the treatment of immune disorders. View Publication

Despite showing success in treating melanoma and haematological malignancies,adoptive cell therapy (ACT) has generated only limited effects in solid tumors. This is,in part,due to a lack of specific antigen targets,poor trafficking/infiltration and immunosuppression in the tumor microenvironment. In this study,we combined ACT with oncolytic virus vaccines (OVV) to drive expansion and tumor infiltration of transferred antigen-specific T cells,and demonstrated that the combination is highly potent for the eradication of established solid tumors. Consistent with other successful immunotherapies,this approach elicited severe autoimmune consequence when the antigen targeted was a self-protein. However,modulation of IFN$\alpha$/$\beta$ signaling,either by functional blockade or rational choice of an OVV backbone,ameliorated autoimmune side effects without compromising antitumor efficacy. Our study uncovers a pathogenic role for IFN$\alpha$/$\beta$ in facilitating autoimmune toxicity during cancer immunotherapy and offers a safe and powerful combinatorial regimen with immediate translational applications. View Publication