Kuo-Ching Sheng

Characterization of Mice Lacking the Tetraspanin Superfamily Member CD151

ABSTRACT The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially α3β1, α6β1, α7β1, and α6β4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of α3 and α6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo

Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose‐based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA‐specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA‐specific CD4+/CD8+ T cell and antibody responses. MDO also targeted lymph node DC to cross‐present OVA, leading to OTI CD8+ T cell proliferation. Moreover, upon challenge with B16‐OVA tumor cells, tumors in mice pre‐immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA‐immunized mice. This mannose‐based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.

Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells

Mannan, a polysaccharide isolated from yeast binds to C‐type lectins of the mannose receptor family, expressed by antigen‐presenting cells (APCs) including dendritic cells (DCs) and macrophages. As these receptors mediate endocytosis, they have been targeted with ligands to deliver antigens into APCs to initiate immune responses. Immunization with tumour antigen MUC1 conjugated to oxidized mannan (OM) or reduced mannan (RM) induced differential immune responses in mice, and only mice immunized with OM‐MUC1 elicited strong MUC1‐specific cytotoxic T lymphocyte responses and protected mice from a MUC1 tumour challenge. In this study, the adjuvant effect of mannan and its derivatives including OM and RM, in comparison to lipopolysaccharide, on DCs were investigated. Mannan, OM and RM were capable of stimulating mouse bone marrow‐derived DC in vitro, eliciting enhanced allogeneic T‐cell proliferation and enhancing OTI/OTII peptide‐specific T‐cell responses. Injection of mice with mannan, OM and RM induced a mature phenotype of lymph node and splenic DCs. Analysis by reverse transcription–polymerase chain reaction indicated that Manna, OM and RM also stimulated up‐regulation of inflammatory cytokines including interleukin‐1β and tumour necrosis factor‐α, and differential T helper 1 (Th1)/Th2 cytokines. Subsequent experiments demonstrated that activation of DCs was Toll‐like receptor‐4‐dependent. The data presented here, together with evidence reported previously on OM and RM in induction of immune responses in vivo, suggest that OM and RM exert a dual capacity to target antigen to APCs as well as mature DCs.

Tetraspanins CD37 and CD151 differentially regulate Ag presentation and T-cell co-stimulation by DC.

A major question in immunology is how DC can display limited amounts of individual peptide–MHC complexes and still induce cross‐linking of T‐cell receptors to initiate cellular responses. One suggested mechanism is that MHC exists at the cell surface in high avidity multimers, and tetraspanin proteins, known to laterally associate with both MHC classes I and II, promote MHC multimerisation. To validate this theory, we tested the ability of DC deficient in either one of two typical tetraspanin molecules: CD37 or CD151 to present peptide to Ag‐specific T cells. Surprisingly, although they exhibited no developmental or maturation defects, DC lacking either CD37 or CD151 expression were hyper‐stimulatory to T cells. We demonstrate that CD37 and CD151 control DC‐mediated T‐cell activation by two different mechanisms: CD151 regulates co‐stimulation whereas CD37 regulates peptide/MHC presentation. The implications of these results on the model suggesting that tetraspanins promote MHC multimerisation are discussed.

Dendritic Cells: Activation and Maturation - Applications for Cancer Immunotherapy

There is an increasing number of studies utilizing dendritic cell (DC) based therapies for cancer. With a powerful antigen-presentation capability, DCs have the potential to overcome tumor tolerance and induce anti-tumor immunity, when loaded with tumor antigens. In order to optimize this approach, methods have aimed to enhance immunopotency of therapeutic DCs. A thorough understanding of DC immunobiology would accelerate this process and provide advantageous procedures to increase anti-tumor responses. This review contains an analysis of recent advances on DC subsets, phenotypic characterization, localization, surface receptors and their ligands. The events of immune induction via DCs, involving initial recognition and uptake of antigens, migration, subsequent activation and maturation are revisited. Furthermore, the current methods used for DC-based cancer immunotherapy, including DCs pulsed with tumor antigens in forms of DNA, RNA, peptides, proteins and lysates, or DCs fused with tumor cells are summarized. Respective preclinical and clinical trials are in progress and hold promise for developing effective cancer vaccines.

A Complementary Role for the Tetraspanins CD37 and Tssc6 in Cellular Immunity

The cooperative nature of tetraspanin–tetraspanin interactions in membrane organization suggests functional overlap is likely to be important in tetraspanin biology. Previous functional studies of the tetraspanins CD37 and Tssc6 in the immune system found that both CD37 and Tssc6 regulate T cell proliferative responses in vitro. CD37−/− mice also displayed a hyper-stimulatory dendritic cell phenotype and dysregulated humoral responses. In this study, we characterize “double knockout” mice (CD37−/−Tssc6−/−) generated to investigate functional overlap between these tetraspanins. Strong evidence for a cooperative role for these two proteins was identified in cellular immunity, where both in vitro T cell proliferative responses and dendritic cell stimulation capacity are significantly exaggerated in CD37−/−Tssc6−/− mice when compared with single knockout counterparts. Despite these exaggerated cellular responses in vitro, CD37−/−Tssc6−/− mice are not more susceptible to autoimmune induction. However, in vivo responses to pathogens appear poor in CD37−/−Tssc6−/− mice, which showed a reduced ability to produce influenza-specific T cells and displayed a rapid onset hyper-parasitemia when infected with Plasmodium yoelii. Therefore, in the absence of both CD37 and Tssc6, immune function is further altered when compared with CD37−/− or Tssc6−/− mice, demonstrating a complementary role for these two molecules in cellular immunity.

Reactive Oxygen Species Level Defines Two Functionally Distinctive Stages of Inflammatory Dendritic Cell Development from Mouse Bone Marrow

Reactive oxygen species (ROS) have been implicated in various physiological activities. However, their role in dendritic cell (DC) activation and generation has not been investigated. Using the bone marrow-derived GM-CSF–induced ex vivo DC model, we characterize how induction of ROS correlates with inflammatory DC functionality and expansion. We describe that the functionality of GM-CSF–induced DCs is distinct in two developmental stages. Whereas division of DC-committed hematopoietic progenitor cells (HPCs) neared completion by day 6, the level of ROS soared after day 4. Day 3 ROSlo DCs were highly responsive to TLR stimuli such as LPS and zymosan by rapid upregulation of CD80, CD86, and MHC class II, in contrast to the low response of day 6 ROShi DCs. ROShi DCs could not initiate and sustain a significant level of NF-κB phosphorylation in response to LPS and zymosan, although demonstrating hyperactivation of p38 MAPK by LPS, in a fashion disparate to ROSlo DCs. ROSlo DCs stimulated a higher level of allogeneic and OVA-specific T cell proliferative responses, although ROShi DCs were much more proficient in processing OVA. In response to pathogenic stimuli, ROShi DCs also demonstrated rapid cellular adhesion and H2O2 release, indicating their role in immediate microbial targeting. Moreover, HPC expansion and DC generation were dependent on the surge of ROS in an NADPH oxidase-independent manner. These findings point to the potential role of cellular ROS in mediating functionality and development of DCs from HPCs during inflammation.

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells.

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24high (conventional CD8+ equivalent) and CD11bhigh (CD8− equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA+ plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-β, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.

Oxidized and reduced mannan mediated MUC1 DNA immunization induce effective anti-tumor responses

DNA immunization is an attractive form of vaccination, which has shown promising results only in small animal models. There is a need to develop efficient gene delivery systems. We previously demonstrated that oxidized (OM) and reduced mannan (RM) complexed to ovalbumin DNA via poly-l-lysine (PLL), were able to generate potent immune responses in mice. Herein, we further investigated the suitability of OMPLL and RMPLL as carriers for mucin 1 (MUC1) DNA vaccination for cancer immunotherapy. Studies presented here showed that immune responses in C57BL/6 mice induced by OMPLL-MUC1 DNA and RMPLL-MUC1 DNA immunization were more immunogenic compared to MUC1 DNA alone. Moreover, tumor protection was evident at a dose as low as 0.5 microg. In addition, strong T cell responses were induced in HLA-A2 transgenic and human MUC1 transgenic mice. These results demonstrate the potential of OM and RM as efficient non-viral gene delivery carriers for DNA vaccines for use in cancer immunotherapy.

A membrane penetrating multiple antigen peptide (MAP) incorporating ovalbumin CD8 epitope induces potent immune responses in mice

Cell penetrating peptides (CPP) represent a novel approach to facilitate cytoplasmic delivery of macromolecules. The DNA binding domain of Drosophila Antennapedia contains 60 amino acids and consists of 3 α-helices, with internalizing activity mapped to a 16-amino acid peptide penetratin (Antp) within the third α-helix. Here, we report on the use of penetratin to deliver a multiple antigen peptide (MAP) incorporating the immunodominant CD8 epitope of ovalbumin, SIINFEKL (MAPOVACD8). We demonstrate that penetratin linked to the MAPOVACD8 construct either by a disulfide (SS) or thioether (SC) linkage promotes the uptake, cross presentation and subsequent in vivo proliferation and generation of OVACD8 (SIINFEKL)-specific T cells. The MAPOVACD8 construct without penetratin is not presented by MHC class I molecules nor does it generate an in vivo IFN-γ response in C57BL/6 mice. Moreover, we clearly define the uptake and intracellular processing pathways of AntpMAPOVACD8 SS and SC revealing the majority of AntpMAPOVACD8 is taken up by DC via an endocytic, proteasome and tapasin independent mechanism. We also show that the uptake mechanism of AntpMAPOVACD8 is dose dependent and uptake or intracellular processing is not altered by the type of chemical linkage.