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Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover,Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands,such as HIV and CpG respectively,turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions,and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection,but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here,we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α,TNF-α,IFN-γ and IL-12,and CCR5-interacting chemokines (MIP-1α and MIP-1β) in NK-pDCs co-cultures. At high HIV-1BaL concentrations,the addition of NK cells did not promote the release of these mediators,suggesting that once efficiently triggered by the virus,pDCs could not integrate new activating signals delivered by NK cells. However,high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly,we identified the alarmin HMGB1,released at pDC-NK cell synapse,as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover,HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1,HMGB1-specific antibodies,sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether,these findings identify HMGB1 as a trigger for IFN-α-mediated TRAIL expression at the surface of pDCs and NK cells,and they suggest a novel mechanism of innate control of HIV-1 infection. View Publication

The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury,although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation,respectively,attenuating the activity of the C3/C5 convertases and,consequently,avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study,we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly,FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture,resembling immature MoDCs,lower expression of the maturation marker CD83 and the costimulatory molecules CD40,CD80,and CD86,decreased production of key proinflammatory Th1-cytokines (IL-12,TNF-α,IFN-γ,IL-6,and IL-8),and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover,FH-treated MoDCs show low Ag uptake and,when challenged with LPS,display reduced CCR7 expression and chemotactic migration,impaired CD4(+) T cell alloproliferation,inhibition of IFN-γ secretion by the allostimulated T cells,and,conversely,induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus,this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity,transplantation,and autoimmunity. View Publication

Group 2 innate lymphoid cells (ILC2 cells) are important for type 2 immune responses and are activated by the epithelial cytokines interleukin 33 (IL-33),IL-25 and thymic stromal lymphopoietin (TSLP). Here we demonstrated that IL-1β was a critical activator of ILC2 cells,inducing proliferation and cytokine production and regulating the expression of epithelial cytokine receptors. IL-1β also governed ILC2 plasticity by inducing low expression of the transcription factor T-bet and the cytokine receptor chain IL-12Rβ2,which enabled the conversion of these cells into an ILC1 phenotype in response to IL-12. This transition was marked by an atypical chromatin landscape characterized by the simultaneous transcriptional accessibility of the locus encoding interferon-γ (IFN-γ) and the loci encoding IL-5 and IL-13. Finally,IL-1β potentiated ILC2 activation and plasticity in vivo,and IL-12 acted as the switch that determined an ILC2-versus-ILC1 response. Thus,we have identified a previously unknown role for IL-1β in facilitating ILC2 maturation and plasticity. View Publication

HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP,apolipoprotein B mRNA-editing,enzyme-catalytic,polypeptide-like 3 (APOBEC3) cytidine deaminases,and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase,which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA,and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr,found in all primate lentiviruses,and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx,hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions,but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here,we identify HLTF,a postreplication DNA repair helicase,as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81,which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus,separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast,we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery,suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. View Publication

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors,but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors,including the HIV co-receptors CD4 and CCR5,that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues,facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation,which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4(+) T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention. View Publication

Associations between chronic antigen stimulation,T cell dysfunction,and the expression of various inhibitory receptors are well characterized in several mouse and human systems. During chronic hepatitis B virus (HBV) infection (CHB),T cell responses are blunted with low frequencies of virus-specific T cells observed,making these parameters difficult to study. Here,using mass cytometry and a highly multiplexed combinatorial peptide-major histocompatibility complex (pMHC) tetramer strategy that allows for the detection of rare antigen-specific T cells,we simultaneously probed 484 unique HLA-A*1101-restricted epitopes spanning the entire HBV genome on T cells from patients at various stages of CHB. Numerous HBV-specific T cell populations were detected,validated,and profiled. T cells specific for two epitopes (HBVpol387 and HBVcore169) displayed differing and complex heterogeneities that were associated with the disease progression,and the expression of inhibitory receptors on these cells was not linearly related with their extent of T cell dysfunction. For HBVcore169-specific CD8+ T cells,we found cellular markers associated with long-term memory,polyfunctionality,and the presence of several previously unidentified public TCR clones that correlated with viral control. Using high-dimensional trajectory analysis of these cellular phenotypes,a pseudo-time metric was constructed that fit with the status of viral infection in corresponding patients. This was validated in a longitudinal cohort of patients undergoing antiviral therapy. Our study uncovers complex relationships of inhibitory receptors between the profiles of antigen-specific T cells and the status of CHB with implications for new strategies of therapeutic intervention. View Publication

The types and magnitude of Ag-specific immune responses can be determined by the functional plasticity of dendritic cells (DCs). However,how DCs display functional plasticity and control host immune responses have not been fully understood. In this study,we report that ligation of DC-asialoglycoprotein receptor (DC-ASGPR),a C-type lectin receptor (CLR) expressed on human DCs,resulted in rapid activation of Syk,followed by PLCgamma2 and PKCdelta engagements. However,different from other Syk-coupled CLRs,including Dectin-1,signaling cascade through DC-ASGPR did not trigger NF-kappaB activation. Instead,it selectively activated MAPK ERK1/2 and JNK. Rapid and prolonged phosphorylation of ERK1/2 led to sequential activation of p90RSK and CREB,which consequently bound to IL10 promoter and initiated cytokine expression. In addition,DC-ASGPR ligation activated Akt,which differentially regulated the activities of GSK-3alpha/beta and beta-catenin and further contributed to IL-10 expression. Our observations demonstrate that DC-ASGPR induces IL-10 expression via an intrinsic signaling pathway,which provides a molecular explanation for DC-ASGPR-mediated programing of DCs to control host immune responses. View Publication

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS),but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight,we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin beta7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore,phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFalpha secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFalpha production,while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms,but not markers of immune activation,decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation,but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms. View Publication

MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-kappaB activation. We discovered nanomolar,selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580,locking the protease in an inactive conformation. Interestingly,we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability,reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency,we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein,the most potent of the allosteric inhibitors rescued NF-kappaB and JNK signaling in patient lymphocytes. Following compound washout,MALT1 substrate cleavage was partly recovered. Thus,a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue,inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies. View Publication

During metazoan development,immune surveillance and cancer dissemination,cells migrate in complex three-dimensional microenvironments1-3. These spaces are crowded by cells and extracellular matrix,generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell4,5. Most mesenchymal and epithelial cells and some-but not all-cancer cells actively generate their migratory path using pericellular tissue proteolysis6. By contrast,amoeboid cells such as leukocytes use non-destructive strategies of locomotion7,raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes,and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus,which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore,cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted,migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration,our findings link the fundamental organization of cellular polarity to the strategy of locomotion. View Publication

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes,including the release of proteases,phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation,or NETosis,is a specific and highly efficient process,which is induced by a variety of stimuli leading to expulsion of DNA,proteases and antimicrobial peptides to the extracellular space. However,uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here,we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly,neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF),a Panx1 channel inhibitor,decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus,we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target. View Publication

Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however,most patients across cancer types still fail to respond. Consequently,there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy,which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable,we designed an $\alpha$HER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models,desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice,an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus,antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy. View Publication