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In recent years,the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated,but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs,and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor,Gleevec (imatinib mesylate; Novartis International,Basel,Switzerland,http://www.novartis.com),to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5,sarcomeric proteins beta-MHC and MLC-2v,and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy,the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating,myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters. View Publication

In humans,recent clinical and experimental data from hematopoietic stem cell transplantation revealed that donor-derived alloreactive NK cells exert a beneficial graft versus leukemia effect. The existence of donor-derived alloreactive NK cells can be predicted on the basis of donor killer cell Ig-like receptor (KIR) gene profile and HLA class I typing of both donor and recipient. Moreover,the size of the alloreactive NK cell population can be directly assessed by the combined use of anti-KIR-specific mAb. In this study,in an attempt to improve the definition of alloreactive NK cell subsets,we assessed the KIR genotype and phenotype in a cohort of 44 donors. This approach allowed the identification of two different KIR2DL3 alleles (KIR2DL3*005 and the novel allele KIR2DL3*015) that did not react with the anti-KIR2DL3-specific ECM41 mAb. In contrast,both alleles were recognized at the cell surface by several mAb reacting with KIR2DL2/L3/S2. Notably,KIR2DL3*005 was also stained by the anti-KIR2DL1/S1-specific EB6B and 11PB6 mAb. Functional analysis revealed that,despite its particular mAb reactivity,the specificity of KIR2DL3*005 for HLA-C molecules did not differ from that of other KIR2DL2/L3 alleles. Finally,site-directed mutagenesis demonstrated that glutamine at position 35 is required for ECM41 staining,whereas glutamic acid 35 and arginine 50 are relevant for staining with EB6B or 11PB6 mAb. Our present data represent a substantial progress in the characterization of the NK cell repertoire and an improved phenotypic/functional definition of given KIR(+) subsets. View Publication

Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells and are key cells of the immune system,acquiring different phenotypes in accordance with their localization during the immune response. A subset of inflammatory DCs is derived from circulating monocytes (Mo) and has a key role in inflammation and infection. The pathways controlling Mo-DC differentiation are not fully understood. Our objective was to investigate the possible role of nicotinamide adenine dinucleotide phosphate reduced form oxidases (NOXs) in Mo-DC differentiation. In this study,we revealed that Mo-DC differentiation was inhibited by NOX inhibitors and reactive oxygen species scavengers. We show that the Mo-DC differentiation was dependent on p22phox,and not on gp91phox/NOX2,as shown by the reduced Mo-DC differentiation observed in chronic granulomatous disease patients lacking p22phox. Moreover,we revealed that NOX5 expression was strongly increased during Mo-DC differentiation,but not during Mo-macrophage differentiation. NOX5 was expressed in circulating myeloid DC,and at a lower level in plasmacytoid DC. Interestingly,NOX5 was localized at the outer membrane of the mitochondria and interacted with p22phox in Mo-DC. Selective inhibitors and small interfering RNAs for NOX5 indicated that NOX5 controlled Mo-DC differentiation by regulating the JAK/STAT/MAPK and NFκB pathways. These data demonstrate that the NOX5-p22phox complex drives Mo-DC differentiation,and thus could be critical for immunity and inflammation. View Publication

Improved understanding of expression of immune checkpoint receptors (ICR) on tumor-infiltrating lymphocytes (TIL) may facilitate more effective immunotherapy in head and neck cancer (HNC) patients. A higher frequency of PD-1+ TIL has been reported in human papillomavirus (HPV)+ HNC patients,despite the role of PD-1 in T-cell exhaustion. This discordance led us to hypothesize that the extent of PD-1 expression more accurately defines T-cell function and prognostic impact,because PD-1high T cells may be more exhausted than PD-1low T cells and may influence clinical outcome and response to anti-PD-1 immunotherapy. In this study,PD-1 expression was indeed upregulated on HNC patient TIL,and the frequency of these PD-1+ TIL was higher in HPV+ patients (P = 0.006),who nonetheless experienced significantly better clinical outcome. However,PD-1high CD8+ TILs were more frequent in HPV- patients and represented a more dysfunctional subset with compromised IFN-γ secretion. Moreover,HNC patients with higher frequencies of PD-1high CD8+ TIL showed significantly worse disease-free survival and higher hazard ratio for recurrence (P < 0.001),while higher fractions of PD-1low T cells associated with HPV positivity and better outcome. In a murine HPV+ HNC model,anti-PD-1 mAb therapy differentially modulated PD-1high/low populations,and tumor rejection associated with loss of dysfunctional PD-1high CD8+ T cells and a significant increase in PD-1low TIL. Thus,the extent of PD-1 expression on CD8+ TIL provides a potential biomarker for anti-PD-1-based immunotherapy. Cancer Res; 77(22); 6353-64. textcopyright2017 AACR. View Publication

The adaptive immune response involves T cell differentiation and migration to sites of inflammation. T cell trafficking is initiated by rolling on inflamed endothelium. Tethers and slings,discovered in neutrophils,facilitate cell rolling at high shear stress. Here,we demonstrate that the ability to form tethers and slings during rolling is highly inducible in T helper 1 (Th1),Th17,and regulatory T (Treg) cells but less in Th2 cells. In vivo,endogenous Treg cells rolled stably in cremaster venules at physiological shear stress. Quantitative dynamic footprinting nanoscopy of Th1,Th17,and Treg cells uncovered the formation of multiple tethers per cell. Human Th1 cells also showed tethers and slings. RNA sequencing (RNA-seq) revealed the induction of cell migration and cytoskeletal genes in sling-forming cells. We conclude that differentiated CD4 T cells stabilize rolling by inducible tether and sling formation. These phenotypic changes approximate the adhesion phenotype of neutrophils and support CD4 T cell access to sites of inflammation. View Publication

BACKGROUND: Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects,including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments,with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However,current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover,FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific,whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations,we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs,and maintained the capacity to suppress conventional T cell responses directed against tyrosinase,as well as bystander T cell responses. Using this methodology in a model tumor system,murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging. CONCLUSIONS/SIGNIFICANCE: These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy. View Publication

RATIONALE: Chitin is a ubiquitous polysaccharide in fungi,insects,allergens,and parasites that is released at sites of infection. Its role in the generation of tissue inflammation,however,is not fully understood. OBJECTIVES: We hypothesized that chitin is an important adjuvant for adaptive immunity. METHODS: Mice were injected with a solution of ovalbumin and chitin. MEASUREMENTS AND MAIN RESULTS: We used in vivo and ex vivo/in vitro approaches to characterize the ability of chitin fragments to foster adaptive immune responses against ovalbumin and compared these responses to those induced by aluminum hydroxide (alum). In vivo,ovalbumin challenge caused an eosinophil-rich pulmonary inflammatory response,Th2 cytokine elaboration,IgE induction,and mucus metaplasia in mice that had been sensitized with ovalbumin plus chitin or ovalbumin plus alum. Toll-like receptor-2,MyD88,and IL-17A played critical roles in the chitin-induced responses,and MyD88 and IL-17A played critical roles in the alum-induced responses. In vitro,CD4(+) T cells from mice sensitized with ovalbumin plus chitin were incubated with ovalbumin-stimulated bone marrow-derived dendritic cells. In these experiments,CD4(+) T-cell proliferation,IL-5,IL-13,IFN-γ,and IL-17A production were appreciated. Toll-like receptor-2,MyD88,and IL-17A played critical roles in these in vitro adjuvant properties of chitin. TLR-2 was required for cell proliferation,whereas IL-17 and TLR-2 were required for cytokine elaboration. IL-17A also inhibited the generation of adaptive Th1 responses. CONCLUSIONS: These studies demonstrate that chitin is a potent multifaceted adjuvant that induces adaptive Th2,Th1,and Th17 immune responses. They also demonstrate that the adjuvant properties of chitin are mediated by a pathway(s) that involves and is regulated by TLR-2,MyD88,and IL-17A. View Publication

The B cell-activating factor of the TNF family receptor (BAFF-R),encoded by the TNFRSF13C gene,is critically important for transitional B cell survival to maturity. Thus,ligation of BAFF-R by BAFF delivers a potent survival signal. Reports implicating the BAFF/BAFF-R signaling axis in the pathogenesis of autoimmune human diseases and B lineage malignancies have largely prompted studies focusing on BAFF expression; however,there is an equally critical need to better understand BAFF-R expression. Initial BAFF-R expression,although characterized in murine B cells,has not yet been reported in human B lymphopoiesis. In this study,we first demonstrate that BAFF-R expression is absent from early precursors and is acquired by bone marrow B cells newly expressing the BCR. We next focused on identifying the specific genomic region that controls BAFF-R expression in mature B cells (i.e.,the TNFRSF13C promoter). To accomplish this,we used in silico tools examining interspecies genomic conservation in conjunction with reporter constructs transfected into malignant B and plasma cell lines. DNase protection assays using nuclear extracts from BAFF-R-expressing cells suggested potential regulatory sites,which allowed the generation of EMSA probes that bound NFs specific to BAFF-R-expressing cells. With a more stringent analysis of interspecies homology,these assays identified a site at which a single nucleotide substitution could distinctly impact promoter activity. Finally,chromatin immunoprecipitation assays revealed the in vivo binding of the specific transcription factor c-Rel to the most proximal genomic region,and c-Rel small interfering RNA transfections in BAFF-R-expressing lines demonstrated a coincident knockdown of both c-Rel and BAFF-R mRNA. View Publication

Monomeric HIV envelope vaccines fail to elicit broadly neutralizing antibodies or to protect against infection. Neutralizing antibodies against HIV bind to native functionally active Env trimers on the virion surface. Gag-Env pseudovirions recapitulate the native trimer and could serve as an effective epitope presentation platform for study of the neutralizing antibody response in HIV-infected individuals. To address if pseudovirions can recapitulate native HIV virion epitope structures,we carefully characterized these particles,concentrating on the antigenic structure of the coreceptor binding site. By blue native gel shift assays,Gag-Env pseudovirions were shown to contain native trimers that were competent for binding to neutralizing monoclonal antibodies. In enzyme-linked immunosorbent assay,pseudovirions exhibited increased binding of known CD4-induced antibodies after addition of CD4. Using flow cytometric analysis,fluorescently labeled pseudovirions specifically identified a subset of antigen-specific B cells in HIV-infected subjects. Interestingly,the sequence of one of these novel human antibodies,identified during cloning of single HIV-specific B cells and designated 2C6,exhibited homology to mAb 47e,a known anti-CD4-induced coreceptor binding site antibody. The secreted monoclonal antibody 2C6 did not bind monomeric gp120,but specifically bound envelope on pseudovirions. A recombinant form of the antibody 2C6 acted as a CD4-induced epitope-specific antibody in neutralization assays,yet did not bind monomeric gp120. These findings imply specificity against a quaternary epitope presented on the pseudovirion envelope spike. These data demonstrate that Gag-Env pseudovirions recapitulate CD4 and coreceptor binding pocket antigenic structures and can facilitate identification of B-cell clones that secrete neutralizing antibodies. View Publication

Recently,it has been shown that certain combinations of TLR ligands act in synergy to induce the release of IL-12 by DCs. In this study,we sought to define the critical parameters underlying TLR synergy. Our data show that TLR ligands act synergistically if MyD88- and TRIF-dependent ligands are combined. TLR4 uses both of these adaptor molecules,thus activation via TLR4 proved to be a synergistic event on its own. TLR synergy did not affect all aspects of DC activation but enhanced primarily the release of certain cytokines,particularly IL-12,whereas the expression of costimulatory molecules remained unchanged. Consequently,synergistic activation of DC did not affect their ability to induce T cell proliferation but resulted in T(H)1-biased responses in vitro and in vivo. Furthermore,we examined the impact of TLR ligand combinations on primary DC in vitro but observed only modest effects with a combination of CpG + Poly (I:C). However,noticeable synergy in terms of IL-12 production by DCs was detectable in vivo after systemic administration of CpG + Poly (I:C). Finally,we show that synergy is partially dependent on IFNAR signaling but does not require the release of IFNs to the enviroment,suggesting an autocrine action of type I IFNs. View Publication

Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented,their ability to directly recognize pathogens is less well defined. We have recently reported FimH,a bacterial fimbrial protein,as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here,we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha] and cytotoxicity. Importantly,NK cells directly recognize FimH-expressing pathogens as FimH(+),but not FimH(-),bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling,as NK cells from both TLR4(-/-) and MyD88(-/-) mice as well as human NK-92 cells,which lack TLR4,were all unresponsive to FimH. In addition,TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4-MyD88 pathways to aid innate protection against cancer or microbial infections. View Publication

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses,the function of some,like virion protein 11/12 (VP11/12),have not been clearly defined. Previously,we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis,M. J. Wagner,R. C. Doepker,J. M. Maciejko,C. M. Crider,K. R. Jerome,and J. R. Smiley,J. Virol. 82:6098-6108,2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here,we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394),a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However,we were unable to detect an interaction between ICP0 and Lck in T lymphocytes,and we show that ICP0,US3,and UL13 are not strictly required for Lck activation. In contrast,VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus,VP11/12 likely modulates host cell signaling pathways for the benefit of the virus. View Publication