技术资料

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

Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk,a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability,clonogenic hematopoietic potential,and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells,together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel,the supportive cells self-assemble into a hypoxic stromal network,stimulating CD34+ CD38+ cell formation,while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks,in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly,the primary in vitro niche model supports HSPCs with no cytokine addition. Overall,the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches,in the context of normal hematopoiesis or blood-related diseases. View Publication

A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified,but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety,providing sustained drug release,and simultaneously delivering multiple drugs to target tissues and cells. Here,we formulated hybrid nanocarriers that incorporate physicochemically diverse LRAs and target lymphatic CD4+ T cells. We identified one LRA combination that displayed synergistic latency reversal and low cytotoxicity in a cell model of HIV and in CD4+ T cells from virologically suppressed patients. Furthermore,our targeted nanocarriers selectively activated CD4+ T cells in nonhuman primate peripheral blood mononuclear cells as well as in murine lymph nodes,and substantially reduced local toxicity. This nanocarrier platform may enable new solutions for delivering anti-HIV agents for an HIV cure. View Publication

The in vitro 3D culture of intestinal epithelium is a valuable resource in the study of its function. Organoid culture exploits stem cells' ability to regenerate and produce differentiated epithelium. Intestinal organoid models from rodent or human tissue are widely available whereas large animal models are not. Livestock enteric and zoonotic diseases elicit significant morbidity and mortality in animal and human populations. Therefore,livestock species-specific models may offer novel insights into host-pathogen interactions and disease responses. Bovine and porcine jejunum were obtained from an abattoir and their intestinal crypts isolated,suspended in Matrigel,cultured,cryopreserved and resuscitated. 'Rounding' of crypts occurred followed by budding and then enlargement of the organoids. Epithelial cells were characterised using immunofluorescent staining and confocal microscopy. Organoids were successfully infected with Toxoplasma gondii or Salmonella typhimurium. This 3D organoid model offers a long-term,renewable resource for investigating species-specific intestinal infections with a variety of pathogens. View Publication

Gastrointestinal (GI) parasites,hookworms in particular,have evolved to cause minimal harm to their hosts,allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed,hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs),exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here,we analyze EVs from the rodent parasite Nippostrongylus brasiliensis,which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids,indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins,including proteins frequently present in exosomes (like tetraspanin,enolase,14-3-3 protein,and heat shock proteins),and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species,many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties,we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6,IL-1$\beta$,IFN$\gamma$,and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast,high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system,such as IBD. View Publication

The risk for non-Hodgkin lymphoma (NHL) is markedly increased in persons living with human immunodeficiency virus (HIV) infection,and remains elevated in those on anti-retroviral therapy (cART). Both the loss of immunoregulation of Epstein-Barr virus (EBV) infected cells,as well as chronic B-cell activation,are believed to contribute to the genesis of AIDS-related NHL (AIDS-NHL). However,the mechanisms that lead to AIDS-NHL have not been completely defined. A subset of B cells that is characterized by the secretion of IL10,as well as the expression of the programmed cell death ligand-1 (PD-L1/CD274),was recently described. These PD-L1+ B cells can exert regulatory function,including the dampening of T-cell activation,by interacting with the program cell death protein (PD1) on target cells. The role of PD-L1+ B cells in the development of AIDS-NHL has not been explored. We assessed B cell PD-L1 expression on B cells preceding AIDS-NHL diagnosis in a nested case-control study of HIV+ subjects who went on to develop AIDS-NHL,as well as HIV+ subjects who did not,using multi-color flow cytometry. Archival frozen viable PBMC were obtained from the UCLA Multicenter AIDS Cohort Study (MACS). It was seen that the number of CD19+CD24++CD38++and CD19+PD-L1+cells was significantly elevated in cases 1-4 years prior to AIDS-NHL diagnosis,compared to controls,raising the possibility that these cells may play a role in the etiology of AIDS-NHL. Interestingly,most PD-L1+ expression on CD19+ cells was seen on CD19+CD24++CD38++ cells. In addition,we showed that HIV can directly induce PD-L1 expression on B cells through interaction of virion-associated CD40L with CD40 on B cells. 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