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The lack of natural killer (NK) cell-specific markers,as well as the overlap among several common surface antigens and functional properties,has obscured the delineation between NK cells and dendritic cells. Here,novel subsets of peripheral blood CD3/14/19(neg) NK cells and monocyte/dendritic cell (DC)-like cells were identified on the basis of CD7 and CD4 expression. Coexpression of CD7 and CD56 differentiates NK cells from CD56+ monocyte/DC-like cells,which lack CD7. In contrast to CD7+CD56+ NK cells,CD7(neg)CD56+ cells lack expression of NK cell-associated markers,but share commonalities in their expression of various monocyte/DC-associated markers. Using CD7,we observed approximately 60% of CD4+CD56+ cells were CD7(neg) cells,indicating the actual frequency of activated CD4+ NK cells is much lower in the blood than previously recognized. Functionally,only CD7+ NK cells secrete gamma interferon (IFNgamma) and degranulate after interleukin-12 (IL-12) plus IL-18 or K562 target cell stimulation. Furthermore,using CD7 to separate CD56+ NK cells and CD56+ myeloid cells,we demonstrate that unlike resting CD7+CD56+ NK cells,the CD7(neg)CD56+ myeloid cells stimulate a potent allogeneic response. Our data indicate that CD7 and CD56 coexpression discriminates NK cells from CD7(neg)CD56+ monocyte/DC-like cells,thereby improving our ability to study the intricacies of NK-cell subset phenotypes and functions in vivo. View Publication

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However,limited information is available regarding the immunologic features of iPSCs. In this study,expression of MHC and T cell co-stimulatory molecules in hiPSCs,and the effects on activation,proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation,hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ,TNF-α and IL-17,hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10,and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity,which may result from their induction of IL-10-secreting Treg. View Publication

We investigated roles for chemoattractants in dissemination of HIV-1 by examining the induction of T cell-active chemokines in HIV-1-infected human monocyte-derived macrophages and dendritic cells. Of the 12 chemokines analyzed,mRNAs for two,CXCL10 and CXCL11,ligands for the chemokine receptor CXCR3,were up-regulated in both cell types upon infection by HIV-1. Induction of these chemokine genes in infected cultures was dependent on both viral entry and reverse transcriptase activity,but not on the HIV-1 envelope glycoprotein. Conditioned medium from infected cells was chemotactic for freshly isolated human CD4+ T cells,and chemotaxis was abolished by pretreatment with an Ab against CXCR3. A lymph node from an HIV-1-infected individual expressed CXCL10 and CXCL11 mRNAs in the paracortex,including venules,as detected by in situ hybridization,whereas neither mRNA was detected after highly active antiretroviral therapy. Because CCR5 on CD4+ T cells is found predominantly on cells that also express CXCR3,these data implicate CXCL10 and CXCL11 in the recruitment of susceptible T cells to HIV-1-infected lymph nodes,macrophages,and dendritic cells. This recruitment might enhance the sequestration of T cells in infected lymphoid organs and the spread of infection between cells,contributing to the immunopathology of AIDS. View Publication

We provide evidence that tumor cells can induce apoptosis of NK cells by engaging the natural cytotoxicity receptors (NCR) NKp30,NKp44,and NKp46. Indeed,the binding between NCR on NK cells and their putative ligands on tumor target cells led to NK cell apoptosis,and this event was abolished by blocking NCR/NCR-ligand interaction by anti-NCR-specific mAbs. The engagement of NCR induced up-regulation of Fas ligand (FasL) mRNA,FasL protein synthesis,and release. In turn,FasL interacting with Fas at NK cell surface causes NK cell suicide,as apoptosis of NK cells was inhibited by blocking FasL/Fas interaction with specific mAbs. Interestingly,NK cell apoptosis,but not killing of tumor target cells,is inhibited by cyclosporin A,suggesting that apoptosis and cytolysis are regulated by different biochemical pathways. These findings indicate that NCR are not only triggering molecules essential for antitumor activity,but also surface receptors involved in NK cell suicide. View Publication

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins,as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells,by enzyme-linked immunosorbent assay,immunoprecipitation followed by Western blotting,and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system,and that all forms of the recombinant proteins have in vitro functional activity. Furthermore,we find that in addition to the homodimers of IL-17F and IL-17A,activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses. 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

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

Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here,we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7),a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2,a receptor that recognizes NAP-2,abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration. View Publication

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity,which links a given individual stimulus to a unique activated state. Here,we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells. View Publication

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies,but is absent on normal tissues,including hematopoietic progenitor cells,and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity,PRAME-specific cytotoxic T lymphocytes (CTLs),we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal,PRAME-specific CTL lines and elicited high-avidity CTLs,with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope,P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts,but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays,which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors,indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies. View Publication

We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition,anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling,leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6),a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells. View Publication

The human peripheral B-cell compartment displays a large population of immunoglobulin M-positive,immunoglobulin D-positive CD27(+) (IgM(+)IgD(+)CD27(+)) memory" B cells carrying a mutated immunoglobulin receptor. By means of phenotypic analysis� View Publication