Uzi Gileadi

NKT Cells Enhance CD4+ and CD8+ T Cell Responses to Soluble Antigen In Vivo through Direct Interaction with Dendritic Cells

Modification in the function of dendritic cells (DC), such as that achieved by microbial stimuli or T cell help, plays a critical role in determining the quality and size of adaptive responses to Ag. NKT cells bearing an invariant TCR (iNKT cells) restricted by nonpolymorphic CD1d molecules may constitute a readily available source of help for DC. We therefore examined T cell responses to i.v. injection of soluble Ag in the presence or the absence of iNKT cell stimulation with the CD1d-binding glycolipid α-galactosylceramide (α-GalCer). Considerably enhanced CD4+ and CD8+ T cell responses were observed when α-GalCer was administered at the same time as or close to OVA injection. This enhancement was dependent on the involvement of iNKT cells and CD1d molecules and required CD40 signaling. Studies in IFN-γR−/− mice indicated that IFN-γ was not required for the adjuvant effect of α-GalCer. Consistent with this result, enhanced T cell responses were observed using OCH, an analog of α-GalCer with a truncated sphingosine chain and a reduced capacity to induce IFN-γ. Splenic DC from α-GalCer-treated animals expressed high levels of costimulatory molecules, suggesting maturation in response to iNKT cell activation. Furthermore, studies with cultured DC indicated that potentiation of T cell responses required presentation of specific peptide and α-GalCer by the same DC, implying conditioning of DC by iNKT cells. The iNKT-enhanced T cell responses resisted challenge with OVA-expressing tumors, whereas responses induced in the absence of iNKT stimulation did not. Thus, iNKT cells exert a significant influence on the efficacy of immune responses to soluble Ag by modulating DC function.

Binding protein-dependent transport systems

Bacterial binding protein-dependent transport systems are the best characterized members of a superfamily of transporters which are structurally, functionally, and evolutionary related to each other. These transporters are not only found in bacteria but also in yeasts, plants, and animals including man, and include both import and export systems. Although any single system is relatively specific, different systems handle very different substrates which can be inorganic ions, amino acids, sugars, large polysaccharides, or even proteins. Some are of considerable medical importance, including Mdr, the protein responsible for multidrug resistance in human tumors, and the product of the cystic fibrosis locus. In this article we review the current state of knowledge on the structure and function of the protein components of these transporters, the mechanism by which transport is mediated, and the role of ATP in the transport process.

Competition Between CTL Narrows the Immune Response Induced by Prime-Boost Vaccination Protocols

Recombinant vaccines encoding strings of virus- or tumor-derived peptides and/or proteins are currently being designed for use against both cancer and infectious diseases. These vaccines aim to induce cytotoxic immune responses against several Ags simultaneously. We developed a novel tetramer-based technique, based on chimeric HLA A2/H-2Kb H chains, to directly monitor the CTL response to such vaccines in HLA-A2 transgenic mice. We found that priming and boosting with the same polyepitope construct induced immune responses that were dominated by CTL of a single specificity. When a mixture of viruses encoding single proteins was used to boost the polyepitope primed response, CTL of multiple specificities were simultaneously expanded to highly effective levels in vivo. In addition, we show that a preexisting response to one of the epitopes encoded within a polyepitope construct significantly impaired the ability of the vaccine to expand CTL of other specificities. Our findings define a novel vaccination strategy optimized for the induction of an effective polyvalent cytotoxic response.

Hepcidin regulation by innate immune and infectious stimuli.

Hepcidin controls the levels and distribution of iron, an element whose availability can influence the outcome of infections. We investigated hepcidin regulation by infection-associated cytokines, pathogen-derived molecules, and whole pathogens in vitro and in vivo. We found that IL-22, an effector cytokine implicated in responses to extracellular infections, caused IL-6-independent hepcidin up-regulation in human hepatoma cells, suggesting it might represent an additional inflammatory hepcidin agonist. Like IL-6, IL-22 caused phosphorylation of STAT3 and synergized with BMP6 potentiating hepcidin induction. In human leukocytes, IL-6 caused potent, transient hepcidin up-regulation that was augmented by TGF-β1. Pathogen-derived TLR agonists also stimulated hepcidin, most notably the TLR5 agonist flagellin in an IL-6-dependent manner. In contrast, leukocyte hepcidin induction by heat-killed Candida albicans hyphae was IL-6-independent, but partially TGF-β-dependent. In a murine acute systemic candidiasis model, C albicans strongly stimulated hepcidin, accompanied by a major reduction in transferrin saturation. Similarly, hepcidin was up-regulated with concomitant lowering of serum iron during acute murine Influenza A/PR/8/34 virus (H1N1) infection. This intracellular pathogen also stimulated hepcidin expression in leukocytes and hepatoma cells. Together, these results indicate that hepcidin induction represents a component of the innate immune response to acute infection, with the potential to affect disease pathogenesis.

Identification of NY-ESO-1 Peptide Analogues Capable of Improved Stimulation of Tumor-Reactive CTL

Expression of NY-ESO-1 in a high proportion of different human tumors makes this protein a very attractive vaccine target. NY-ESO-1 peptides, recognized by HLA-A2-restricted CTL, have recently been described. However, it remains unclear how efficiently tumors generate these epitopes, and whether peptide analogues can be used for optimal expansion and activation of NY-ESO-1-specific HLA-A2-restricted CTL. By generating unique CTL clones, we demonstrate that NY-ESO-1-positive tumor cells are efficiently killed by HLA-A2-restricted CTL specific for the peptide epitope NY-ESO-1 157–165. Presentation of this epitope is not affected by the presence or absence of the proteasome subunits low molecular proteins 2 and 7 and is not blocked by proteasome inhibitors, while it is impaired in the TAP-deficient cell line LBL 721.174. NY-ESO-1 157–165 peptide analogues were compared for their antigenicity and immunogenicity using PBL from melanoma patients. Three peptides, containing the carboxyl-terminal cysteine substituted for either valine, isoleucine, or leucine, were recognized at least 100 times more efficiently than the wild-type peptide by specific CTL. Peptide analogues were capable of stimulating the expansion of NY-ESO-1-specific CTL from PBL of melanoma patients much more efficiently than wild-type peptide. These findings define the processing requirements for the generation of the NY-ESO-1 157–165 epitope. Identification of highly antigenic NY-ESO-1 peptide analogues may be important for the development of vaccines capable of expanding NY-ESO-1-specific CTL in cancer patients.

Antigen Processing Defects in Cervical Carcinomas Limit the Presentation of a CTL Epitope from Human Papillomavirus 16 E6

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E629–38 that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201+ HPV16 E6+ cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E629–38 correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-γ, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E629–38 epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E629–38 epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.

Cutting Edge: Nonglycosidic CD1d Lipid Ligands Activate Human and Murine Invariant NKT Cells

Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than α-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/α-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.

Dendritic Cell Function Can Be Modulated through Cooperative Actions of TLR Ligands and Invariant NKT Cells

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand α-galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8+ T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and α-galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.

Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses

ABSTRACT Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2+ cells after intravenous injection and in CD11c+ dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8+ T-cell response in HLA-A2 transgenic mice and stimulated a CD4+ T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-Ab. As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.

Membrane topology of the integral membrane components, OppB and OppC, of the oligopeptide permease of Salmonella typhimurium.

The oligopeptide permease of Salmonella typhimurium is a periplasmic binding protein‐dependent transport system. Five gene products, OppABCDF, are required for the functioning of this transporter, two of which (OppB and OppC) are highly hydrophobic, integral membrane proteins and are responsible for mediating passage of peptides across the cytoplasmic membrane. OppB and OppC are each predicted, from their sequences, to span the membrane many times. In this paper we describe experimental evidence confirming these predictions using a combination of biochemical, immunological and genetic procedures. Each of these two proteins is shown to span the membrane six times, with the N‐ and C‐termini both being located at the cytoplasmic face of the membrane. Opp is apparently a typical member of the ABC (ATP‐binding cassette) superfamily of transporters. These findings, therefore, have general implications for the organization and function of other ABC transporters, including the human multidrug resistance protein and the product of the cystic fibrosis gene.