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To achieve a functional cure for HIV,treatment regimens that eradicate latently HIV-infected cells must be established. For this,many groups have attempted to reactivate latently-infected cells to induce cytopathic effects and/or elicit cytotoxic T lymphocyte (CTL)/NK cell-mediated immune responses to kill these cells. We believe that not only the reactivation of latently-infected cells,but also the induction of strong CTL responses,would be required for this. Here,we used typical immune activators that target pattern recognition receptors (PRRs). For our experimental model,we identified eight SIV-infected cynomolgus monkeys that became natural controllers of viremia. Although plasma viral loads were undetectable,we could measure SIV-DNA by qPCR in peripheral blood mononuclear cells (PBMCs). Using these PBMCs,we screened 10 distinct PRR ligands to measure IFN-alpha and IFN-gamma production. Among these,STING ligands,cGAMP and c-di-AMP,and the TLR7/8 agonist R848 markedly increased cytokine levels. Both R848 and STING ligands could reactivate latently-infected cells in both cynomolgus monkeys and human PBMCs in vitro. Furthermore,c-di-AMP increased the frequency of SIV Gag-specific CD8+ T cells including polyfunctional CD8+ T cells,as compared to that in untreated control or R848-treated cells. Together,STING ligands might be candidates for HIV treatment. View Publication

Dysregulation of the JAK/STAT signaling pathway is associated with Multiple Sclerosis (MS) and its mouse model,Experimental Autoimmune Encephalomyelitis (EAE). Suppressors Of Cytokine Signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe,brain-targeted,atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3DeltaLysM),which is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE,however,their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3DeltaLysM mice show a hyper-activated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically,Socs3-deficient neutrophils exhibit enhanced STAT3 activation,a hyper-activated phenotype in response to G-CSF,and upon G-CSF priming,increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall,our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3DeltaLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress,which may explain the detrimental role of G-CSF in MS patients. View Publication

Subclinical hypothyroidism is associated with cardiovascular diseases,yet the underlying mechanism remains largely unknown. Herein,in a common population (n = 1,103),TSH level was found to be independently correlated with both carotid plaque prevalence and intima-media thickness. Consistently,TSH receptor ablation in ApoE-/- mice attenuated atherogenesis,accompanied by decreased vascular inflammation and macrophage burden in atherosclerotic plaques. These results were also observed in myeloid-specific Tshr-deficient ApoE-/- mice,which indicated macrophages to be a critical target of the proinflammatory and atherogenic effects of TSH. In vitro experiments further revealed that TSH activated MAPKs (ERK1/2,p38alpha,and JNK) and IkappaB/p65 pathways in macrophages and increased inflammatory cytokine production and their recruitment of monocytes. Thus,the present study has elucidated the new mechanisms by which TSH,as an independent risk factor of atherosclerosis,aggravates vascular inflammation and contributes to atherogenesis. View Publication

BACKGROUND The inhibition of Janus kinases (JAKs) and subsequent signal transducers and activators of transcription (STATs) by tofacitinib represents a new therapeutic strategy in inflammatory bowel diseases (IBD) as clinical trials have led to approval of tofacitinib for ulcerative colitis (UC) and hint at a possible efficacy for Crohn`s disease (CD). However,the impact of tofacitinib on cellular response of monocytes,which are key players in inflammatory responses,has not been investigated so far. We aimed to analyze JAK/STAT-inhibition by tofacitinib in monocytes of IBD patients and healthy controls. METHODS Primary monocytes of IBD patients with active disease and healthy controls (n = 18) were analyzed for cytokine expression and phenotype after granulocyte macrophage colony-stimulating factor (GM-CSF)/interferon (IFN)$\gamma$-stimulation and tofacitinib pretreatment (1-1000 nM) and capacity to induce Foxp3+-regulatory T cells (Tregs) in cocultures. In total,20 UC patients and 21 CD patients were included. Additionally,dose-dependent inhibition of JAK/STAT-phosphorylation was analyzed in controls. RESULTS Pro-inflammatory costimulation with GM-CSF/IFN$\gamma$ resulted in significant tumor necrosis factor (TNF$\alpha$) and interleukin (IL)-6 increase,whereas IL-10 expression decreased in monocytes. Tofacitinib modulated the responses of activated monocytes toward a regulatory phenotype through reduced TNF$\alpha$ and IL-6 secretion and enhanced Treg induction in cocultures. However,in monocytes from active IBD patients,higher tofacitinib dosages were needed for blockade of pro-inflammatory cytokines. Tofacitinib induced stronger regulatory phenotypes in monocytes of UC patients,including more effective inhibition of pro-inflammatory pathways and better restoration of anti-inflammatory mechanisms as compared with CD-derived monocytes. CONCLUSION Tofacitinib dose-dependently reprograms monocytes toward a more regulatory cell type. This beneficial effect possibly results from selective JAK/STAT-blockade by adequate tofacitinib dosage with inhibition of pro-inflammatory responses and permission of a balance-shift toward regulatory pathways. View Publication

The higher-order architecture observed in biological systems,like viruses,is very effective in nucleic acid transport. The replications of this system has been attempted with both synthetic and naturally occurring polymers with mixed results. Here we describe a peptide/siRNA quaternary complex that functions as an siRNA delivery system. The rational design of a peptide assembly is inspired by the viral capsids,but not derived from them. We selected the collagen peptide (COL) to provide the structural stability and the folding framework,and hybridize it with the cell penetrating peptide (CPP) that allows for effective penetration of biological barriers. The peptide/siRNA quaternary complex forms stoichiometric,10 nm nanoparticles,that show fast cellular uptake ({\textless}30 min),effective siRNA release,and gene silencing. The complex provides capsid-like protection for siRNA against nucleases without being immunostimulatory,or cytotoxic. Our data suggests that delivery vehicles based on synthetic quaternary structures that exhibit higher-order architecture may be effective in improving delivery and release of nucleic acid cargo. View Publication

Parasitic helminths cause significant damage as they migrate through host tissues to complete their life cycle. While chronic helminth infections are characterized by a well-described Type 2 immune response,the early,tissue-invasive stages are not well understood. Here we investigate the immune pathways activated during the early stages of Heligmosomoides polygyrus bakeri (Hpb),a natural parasitic roundworm of mice. In contrast to the Type 2 immune response present at later stages of infection,a robust Type 1 immune signature including IFNg production was dominant at the time of parasite invasion and granuloma formation. This early response was associated with an accumulation of activated Natural Killer (NK) cells,with no increase of other innate lymphoid cell populations. Parabiosis and confocal microscopy studies indicated that NK cells were recruited from circulation to the small intestine,where they surrounded parasitic larvae. NK cell recruitment required IFN$\gamma$ receptor signaling,but was independent of CXCR3 expression. The depletion of tissue-infiltrating NK cells altered neither worm burden nor parasite fitness,but increased vascular injury,suggesting a role for NK cells in mediating tissue protection. Together,these data identify an unexpected role for NK cells in promoting disease tolerance during the invasive stage of an enteric helminth infection. View Publication

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

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here,we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes,14 potent neutralizing antibodies were identified,with the most potent one,BD-368-2,exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2,respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally,the 3.8 {\AA} cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover,we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether,we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases. View Publication