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The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However,little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional,or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite,thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria. View Publication

To expand the breadth and extent of current multiple myeloma (MM)-specific immunotherapy,we have identified various antigens on CD138+ tumor cells from newly diagnosed MM patients (n = 616) and confirmed B-cell maturation antigen (BCMA) as a key myeloma-associated antigen. The aim of this study is to target the BCMA,which promotes MM cell growth and survival,by generating BCMA-specific memory CD8+ CTL that mediate effective and long-lasting immunity against MM. Here we report the identification of novel engineered peptides specific to BCMA,BCMA72-80 (YLMFLLRKI),and BCMA54-62 (YILWTCLGL),which display improved affinity/stability to HLA-A2 compared to their native peptides and induce highly functional BCMA-specific CTL with increased activation (CD38,CD69) and co-stimulatory (CD40L,OX40,GITR) molecule expression. Importantly,the heteroclitic BCMA72-80 specific CTL demonstrated poly-functional Th1-specific immune activities [IFN-gamma/IL-2/TNF-alpha production,proliferation,cytotoxicity] against MM,which were correlated with expansion of Tetramer+ and memory CD8+ CTL. Additionally,heteroclitic BCMA72-80 specific CTL treated with anti-OX40 (immune agonist) or anti-LAG-3 (checkpoint inhibitor) display increased immune function,mainly by central memory CTL. These results provide the framework for clinical application of heteroclitic BCMA72-80 peptide,alone and in combination with anti-LAG3 and/or anti-OX40 therapy,in vaccination and/or adoptive immunotherapeutic strategies to generate long-lasting anti-tumor immunity in patients with MM or other BCMA expressing tumors. View Publication

Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate lipid biosynthesis and adipogenesis by controlling the expression of several enzymes required for cholesterol,fatty acid,triacylglycerol and phospholipid synthesis. In vertebrates,SREBP activation is mainly controlled by a complex and well-characterized feedback mechanism mediated by cholesterol,a crucial bio-product of the SREBP-activated mevalonate pathway. In this work,we identified acto-myosin contractility and mechanical forces imposed by the extracellular matrix (ECM) as SREBP1 regulators. SREBP1 control by mechanical cues depends on geranylgeranyl pyrophosphate,another key bio-product of the mevalonate pathway,and impacts on stem cell fate in mouse and on fat storage in Drosophila. Mechanistically,we show that activation of AMP-activated protein kinase (AMPK) by ECM stiffening and geranylgeranylated RhoA-dependent acto-myosin contraction inhibits SREBP1 activation. Our results unveil an unpredicted and evolutionary conserved role of SREBP1 in rewiring cell metabolism in response to mechanical cues. View Publication

Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study,we identified EphB4-ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell-cell contact,resulting in bidirectional signaling. We found that EphB4-ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8+ and CD4+Foxp3- T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4-ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression,particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4-ephrin-B2 inhibition with RT. In vitro,EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall,our study is the first to implicate the role of EphB4-ephrin-B2 in tumor immune response. Moreover,our findings suggest that EphB4-ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy. SIGNIFICANCE: These findings present EphB4-ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC. View Publication

In life sciences,the material properties of suspended cells have attained significance close to that of fluorescent markers but with the advantage of label-free and unbiased sample characterization. Until recently,cell rheological measurements were either limited by acquisition throughput,excessive post processing,or low-throughput real-time analysis. Real-time deformability cytometry expanded the application of mechanical cell assays to fast on-the-fly phenotyping of large sample sizes,but has been restricted to single material parameters as the Young's modulus. Here,we introduce dynamic real-time deformability cytometry for comprehensive cell rheological measurements at up to 100 cells per second. Utilizing Fourier decomposition,our microfluidic method is able to disentangle cell response to complex hydrodynamic stress distributions and to determine viscoelastic parameters independent of cell shape. We demonstrate the application of our technology for peripheral blood cells in whole blood samples including the discrimination of B- and CD4+ T-lymphocytes by cell rheological properties. View Publication

Human immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized,especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here,we show,using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy,that urethral macrophages contain integrated HIV-1 DNA,RNA,proteins and intact virions in virus-containing compartment-like structures,whereas viral components remain undetectable in urethral T cells. Moreover,urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide,while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor,CD206 and interleukin-4 receptor,but not CD163. To our knowledge,these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication. View Publication

Helminthic infections modulate host immunity and may protect their hosts from developing immunological diseases like inflammatory bowel disease. Induction of regulatory T cells (Tregs) may be an important part of this protective process. Heligmosomoides polygyrus bakeri infection also promotes the production of the regulatory cytokines TGF-beta and IL-10 in the gut. In the intestines,TGF-beta helps induce regulatory T cells. This study used Foxp3/IL-10 double reporter mice to investigate the effect of TGF-beta on the differentiation of colon and mesenteric lymph node-derived murine Foxp3- IL-10- CD4+ T cells into their regulatory phenotypes. Foxp3- IL-10- CD4+ T cells from H. polygyrus bakeri-infected mice,as opposed to T cells from uninfected animals,cultured in vitro with TGF-beta and anti-CD3/CD28 mAb differentiated into Foxp3+ and/or IL-10+ T cells. The IL-10-producing T cells nearly all displayed CD25. Smad7 is a natural inhibitor of TGF-beta signaling. In contrast to gut T cells from uninfected mice,Foxp3- IL10- CD4+ T cells from H. polygyrus bakeri-infected mice displayed reduced Smad7 expression and responded to TGF-beta with Smad2/3 phosphorylation. The TGF-beta-induced Tregs that express IL-10 blocked colitis when transferred into the Rag/CD25- CD4+ T cell transfer model of inflammatory bowel disease. TGF-beta had a greatly diminished capacity to induce Tregs in H. polygyrus bakeri-infected transgenic mice with constitutively high T cell-specific Smad7 expression. Thus,infection with H. polygyrus bakeri causes down-modulation in Smad7 expression in intestinal CD4+ T cells,which allows the TGF-beta produced in response to the infection to induce the Tregs that prevent colitis. View Publication

CD4 T cells play a critical role in promoting the development of autoimmunity in type 1 diabetes. The diabetogenic CD4 T cell clone BDC-2.5,originally isolated from a NOD mouse,has been widely used to study the contribution of autoreactive CD4 T cells and relevant Ags to autoimmune diabetes. Recent work from our laboratory has shown that the Ag for BDC-2.5 T cells is a hybrid insulin peptide (2.5HIP) consisting of an insulin C-peptide fragment fused to a peptide from chromogranin A (ChgA) and that endogenous 2.5HIP-reactive T cells are major contributors to autoimmune pathology in NOD mice. The objective of this study was to determine if poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) loaded with the 2.5HIP Ag (2.5HIP-coupled PLG NPs) can tolerize BDC-2.5 T cells. Infusion of 2.5HIP-coupled PLG NPs was found to prevent diabetes in an adoptive transfer model by impairing the ability of BDC-2.5 T cells to produce proinflammatory cytokines through induction of anergy,leading to an increase in the ratio of Foxp3+ regulatory T cells to IFN-gamma+ effector T cells. To our knowledge,this work is the first to use a hybrid insulin peptide,or any neoepitope,to re-educate diabetogenic T cells and may have significant implications for the development of an Ag-specific therapy for type 1 diabetes patients. View Publication

Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca-1) cells to reconstitute aged BM and rejuvenate the aged heart,and examined the underlying molecular mechanisms. BM Sca-1+ or Sca-1- cells from young (2-3 months) or aged (18-19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca-1+,young Sca-1-,old Sca-1+,and old Sca-1- . Four months later,expression of rejuvenation-related genes (Bmi1,Cbx8,PNUTS,Sirt1,Sirt2,Sirt6) and proteins (CDK2,CDK4) was increased along with telomerase activity and telomerase-related protein (DNA-PKcs,TRF-2) expression,whereas expression of senescence-related genes (p16INK4a,P19ARF,p27Kip1 ) and proteins (p16INK4a,p27Kip1 ) was decreased in Sca-1+ chimeric hearts,especially in the young group. Host cardiac endothelial cells (GFP- CD31+ ) but not cardiomyocytes were the primary cell type rejuvenated by young Sca-1+ cells as shown by improved proliferation,migration,and tubular formation abilities. C-X-C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP+ ) cells isolated from young Sca-1+ chimeric hearts. Protein expression of Cxcr4,phospho-Akt,and phospho-FoxO3a in endothelial cells derived from the aged chimeric heart was increased,especially in the young Sca-1+ group. Reconstitution of aged BM with young Sca-1+ cells resulted in effective homing of functional stem cells in the aged heart. These young,regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells. 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