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RATIONALE: Excessive recruitment of polymorphonuclear leukocytes (PMNs) to the lung promotes acute lung injury (ALI). Chemokine receptors and adhesion molecules initiate leukocyte-endothelial interactions,but mediators of PMN migration through the alveolo-capillary membrane remain to be identified. p21-Activated kinase (PAK) is an effector of small GTPases and has been implicated in cell migration. OBJECTIVES: To test the role of PAK in ALI. METHODS: An inhibitory PAK peptide was used to determine the role of PAK in cytoskeletal actin polymerization,cell adhesion,and oxidative burst. PMN migration was investigated in vitro and in a murine model of lipopolysaccharide-induced lung injury. MEASUREMENTS AND MAIN RESULTS: PMN migration into lung interstitium and alveolar space was suppressed by an inhibitory PAK peptide. Neutrophils that had taken up the inhibitory PAK peptide were unable to enter the alveolar space. CXCL2/3,an important PMN chemoattractant in murine lung injury,induced PAK phosphorylation in PMNs. Blocking PAK function inhibited chemotaxis,chemokine-induced cytoskeletal actin polymerization,and adhesion-induced oxidative burst. CONCLUSIONS: We conclude that neutrophil PAK is a critical mediator of PMN migration and may be an attractive target in ALI. View Publication

Abnormal activation of B lymphocytes is a feature commonly seen in human immunodeficiency virus type 1 (HIV-1)-infected persons. However,the mechanism(s) responsible for this dysfunction is still poorly understood. Having recently shown that CD40L,the ligand for CD40,is inserted within emerging HIV-1 particles,we hypothesized that the contact between virus-anchored host CD40L and CD40 on the surface of B lymphocytes might result in the activation of this cell type. We report here that CD40L-bearing viruses,but not isogenic virions lacking host-derived CD40L,can induce immunoglobulin G and interleukin-6 production. Furthermore,such viral entities were found to induce B-cell homotypic adhesion. These effects were paralleled at the intracellular level by the nuclear translocation of the ubiquitous transcription factor NF-kappaB. The presence of host-derived CD40L within virions resulted in an increased virus attachment to B cells and a more-efficient B-cell-mediated transfer of HIV-1 to autologous CD4(+) T lymphocytes. All the above processes were independent of the virus-encoded envelope glycoproteins. Altogether,the data gathered from this series of investigations suggest that the incorporation of host-encoded CD40L in HIV-1 is likely to play a role in the B-cell abnormalities that are seen in infected individuals. View Publication

Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here,we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1),we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4(+) T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together,our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore,the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures. View Publication

NOD.B10-H2(b) and NOD/LtJ mice manifest,respectively,many features of primary and secondary Sjögren's syndrome (SjS),an autoimmune disease affecting primarily the salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). B lymphocytes play a central role in the onset of SjS with clinical manifestations dependent on the appearance of autoantibodies reactive to multiple components of acinar cells. Previous studies with NOD.IL4(-/-) and NOD.B10-H2(b).IL4(-/-) mice suggest that the Th2 cytokine,IL-4,plays a vital role in the development and onset of SjS-like disease in the NOD mouse model. To investigate the molecular mechanisms by which IL-4 controls SjS development,a Stat6 gene knockout mouse,NOD.B10-H2(b).C-Stat6(-/-),was constructed and its disease profile was defined and compared with that of NOD.B10-H2(b).C-Stat6(+/+) mice. As the NOD.B10-H2(b).C-Stat6(-/-) mice aged from 4 to 24 wk,they exhibited leukocyte infiltration of the exocrine glands,produced anti-nuclear autoantibodies,and showed loss and gain of saliva-associated proteolytic enzymes,similar to NOD.B10-H2(b).C-Stat6(+/+) mice. In contrast,NOD.B10-H2(b).C-Stat6(-/-) mice failed to develop glandular dysfunction,maintaining normal saliva flow rates. NOD.B10-H2(b).C-Stat6(-/-) mice were found to lack IgG1 isotype-specific anti-muscarinic acetylcholine type-3 receptor autoantibodies. Furthermore,the IgG fractions from NOD.B10-H2(b).C-Stat6(-/-) sera were unable to induce glandular dysfunction when injected into naive recipient C57BL/6 mice. NOD.B10-H2(b).C-Stat6(-/-) mice,like NOD.B10-H2(b).IL4(-/-) mice,are unable to synthesize IgG1 Abs,an observation that correlates with an inability to develop end-stage clinical SjS-like disease. These data imply a requirement for the IL-4/STAT6-pathway for onset of the clinical phase of SjS-like disease in the NOD mouse model. View Publication

Recent studies have shown that NK-dendritic cell (DC) interaction plays an important role in the induction of immune response against tumors and certain viruses. Although the effect of this interaction is bidirectional,the mechanism or molecules involved in this cross-talk have not been identified. In this study,we report that coculture with NK cells causes several fold increase in IL-12 production by Toxoplasma gondii lysate Ag-pulsed DC. This interaction also leads to stronger priming of Ag-specific CD8+ T cell response by these cells. In vitro blockade of NKG2D,a molecule present on human and murine NK cells,neutralizes the NK cell-induced up-regulation of DC response. Moreover,treatment of infected animals with Ab to NKG2D receptor compromises the development of Ag-specific CD8+ T cell immunity and reduces their ability to clear parasites. These studies emphasize the critical role played by NKG2D in the NK-DC interaction,which apparently is important for the generation of robust CD8+ T cell immunity against intracellular pathogens. To the best of our knowledge,this is the first work that describes in vivo importance of NKG2D during natural infection. View Publication

We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse,we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding,at the molecular level,how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here,we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally,we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization,as well as T cell migration in vitro. Altogether,our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility. View Publication

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5,an immune-regulatory transcription factor. We show that the protein kinases IKK$\alpha$,IKK$\beta$,IKK$\epsilon$,and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization,thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets,we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs,linking IRF5 with immune regulatory and proinflammatory gene expression. Thus,TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome. View Publication

The developmental pathways of broadly neutralizing antibodies (bNAbs) against HIV are of great importance for the design of immunogens that can elicit protective responses. Here we show the maturation features of the HIV-neutralizing anti-V1V2 VRC26 lineage by simultaneously sequencing the exon together with the downstream intron of VRC26 members. Using the mutational landscapes of both segments and the selection-free nature of the intron region,we identify multiple events of amino acid mutational convergence in the complementarity-determining region 3 (CDR3) of VRC26 members,and determine potential intermediates with diverse CDR3s to a late stage bNAb from 2 years prior to its isolation. Moreover,we functionally characterize the earliest neutralizing intermediates with critical CDR3 mutations,with some emerging only 14 weeks after initial lineage detection and containing only {\~{}}6{\%} V gene mutations. Our results thus underscore the utility of analyzing exons and introns simultaneously for studying antibody maturation and repertoire selection. View Publication

CRISPR pools are being widely employed to identify gene functions. However,current technology,which utilizes DNA as barcodes,permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities,we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate {\textgreater}100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies,we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR,we simultaneously analyzed multiple phenotypic markers,including phospho-signaling,on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes,the immunoproteasome component Psmb8 and a chaperone Rtp4,are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution. View Publication

PURPOSE Tumor-associated macrophages (TAMs) and the hyperactivation of phosphoinositide 3-kinase(PI3K)/AKT pathway are involved in the pathogenesis of Hodgkin lymphoma (HL) and affect disease outcome. Since the $\delta$ and $\gamma$ isoforms of PI3K are overexpressed in Hodgkin/Reed-Sternberg (HRS) cells and the tumor microenvironment (TME),we propose that the PI3K$\delta$/$\gamma$ inhibitor RP6530 might affect both HRS cells and TME,ultimately leading to an enhanced antitumor response. EXPERIMENTAL DESIGN HL cell lines (L-540,KM-H2 and L-428) and primary human macrophages were used to investigate the activity of RP6530 in vitro and in vivo in HL cell line xenografts. RESULTS In vitro,RP6530 besides killing and inhibiting the proliferation of HL cells,downregulated lactic acid metabolism,switching the activation of macrophages from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state. By RNA sequencing,we define tumor glycolysis as a specific PI3K$\delta$/$\gamma$-dependent pathway implicated in the metabolic reprogramming of cancer cells. We identify the metabolic regulator Pyruvate Kinase M2 (PKM2) as the main mediator of tumor-induced immunosuppressive phenotype of macrophages. Furthermore,we show in human tumor xenografts that RP6530 repolarizes TAMs into pro-inflammatory macrophages and inhibits tumor vasculature,leading to tumor regression. Interestingly,HL patients experiencing objective responses (CR and PR) in a phase 1 trial using RP6530 showed a significant inhibition of circulating MDSCs and an average mean reduction in serum TARC levels of 40{\%} (range,4-76{\%}). CONCLUSIONS Our results support PI3K$\delta$/$\gamma$ inhibition as a novel therapeutic strategy that targets both malignant cells and the TME to treat HL patients. View Publication

CD4+ T cells,especially T-helper (Th) cells (Th1,Th2 and Th17) and regulatory T cells (Treg) play pivotal role in the pathogenesis of multiple sclerosis (MS),a demyelinating autoimmune disease occurring in central nervous system (CNS). Astragaloside IV (ASI,CAS: 84687-43-4) is one of the saponins isolated from Astragalus membranceus,a traditional Chinese medicine with immunomodulatory effect. So far,whether ASI has curative effect on experimental autoimmune encephalomyelitis (EAE),an animal model of MS,and how it affects the subsets of CD4+ T cells,as well as the underlying mechanism have not been clearly elucidated. In the present study,ASI was found to ameliorate the progression and hamper the recurrence of EAE effectively in the treatment regimens. It significantly reduced the demyelination and inflammatory infiltration of CNS in EAE mice by suppressing the percentage of Th1 and Th17 cells,which was closely associated with the inhibition of JAK/STAT and NF-$\kappa$B signaling pathways. ASI also increased the percentage of Treg cells in spleen and CNS,which was accompanied by elevated Foxp3. However,in vitro experiments disclosed that ASI could regulate the differentiation of Th17 and Treg cells but not Th1 cells. In addition,it induced the apoptosis of MOG-stimulated CD4+ T cells probably through modulating STAT3/Bcl-2/Bax signaling pathways. Together,our findings suggested that ASI can modulate the differentiation of autoreactive CD4+ T cells and is a potential prodrug or drug for the treatment of MS and other similar autoimmune diseases. View Publication

CD24 expression on pro-B cells plays a role in B cell selection and development in the bone marrow. We previously detected higher CD24 expression and frequency within IgD+ na{\{i}}ve and memory B cells in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) compared with age-matched healthy controls (HC). Here we investigated the relationship between CD24 expression and B cell maturation. In vitro stimulation of isolated B cells in response to conventional agonists were used to follow the dynamics of CD24 positivity during proliferation and differentiation (or maturation). The relationship between CD24 expression to cycles of proliferation and metabolism in purified B cells from HC was also investigated using phospho-flow (phosphorylation of AMPK-pAMPK) 1proton nuclear magnetic resonance and Mitotracker Far-red (Mitochondrial mass-MM). In vitro in the absence of stimulation there was an increased percentage of CD24+ viable B cells in ME/CFS patients compared to HC (p {\textless} 0.05) following 5 days culture. Following stimulation with B cell agonists percentage of CD24+B cells in both na{\"{i}}ve and memory B cell populations decreased. P {\textless} 0.01). There was a negative relationship between percentage of CD24+B cells with MM (R2 = 0.76; p {\textless} 0.01) which was subsequently lost over sequential cycles of proliferation. There was a significant correlation between CD24 expression on B cells and the usage of glucose and secretion of lactate in vitro. Short term ligation of the B cell receptor with anti-IgM antibody significantly reduced the viability of CD24+ memory B cells compared to those cross-linked by anti-IgD or anti-IgG antibody. A clear difference was found between na{\""{i}}ve and memory B cells with respect to CD24 expression and pAMPK most notably a strong positive association in IgD+IgM+ memory B cells. In vitro findings confirmed dysregulation of CD24-expressing B cells from ME/CFS patients previously suggested by immunophenotype studies of B cells from peripheral blood. CD24-negative B cells underwent productive proliferation whereas CD24+ B cells were either unresponsive or susceptible to cell death upon BCR-engagement alone. We suggest that CD24 expression may reflect variations in energy metabolism on different B cell subsets.""" View Publication