Haley W. Sinn

Cooperation between platelet-derived CD154 and CD4+ T cells for enhanced germinal center formation

It has been demonstrated previously that platelet‐derived CD154 communicates with the adaptive immune compartment, enhancing B and T cell responses in CD154−/− mice. The presence of platelets was also shown to be necessary for optimal production of immunoglobulin G (IgG) in normal C57BL/6 mice. These data led us to hypothesize that platelets perform a sentinel function, quickly relaying activating signals to the adaptive immune compartment. Here, we report that platelet‐derived CD154 increases serum IgG levels and germinal center formation under conditions where antigen‐specific CD4+ T cell numbers are limiting. We propose that in the physiologic setting where antigen‐specific B and T cells are rare, platelets function to enhance signals required for robust adaptive humoral immunity.

Localization of the novel Xin protein to the adherens junction complex in cardiac and skeletal muscle during development.

Previously, we demonstrated that chick embryos treated with antisense oligonucleotides against a striated muscle‐specific Xin exhibit abnormal cardiac morphogenesis (Wang et al. [1999] Development 126:1281–1294); therefore, we surmised a role for Xin in cardiac development. Herein, we examine the developmental expression of Xin through immunofluorescent staining of whole‐mount mouse embryos and frozen heart sections. Xin expression is first observed within the heart tube of embryonic day 8.0 (E8.0) mice, exhibiting a peripheral localization within the cardiomyocytes. Colocalization of Xin with both β‐catenin and N‐cadherin is observed throughout embryogenesis and into adulthood. Additionally, Xin is found associated with β‐catenin within the N‐cadherin complex in embryonic chick hearts by coimmunoprecipitation. Xin is detected earlier than vinculin in the developing heart and colocalizes with vinculin at the intercalated disc but not at the sarcolemma within embryonic and postnatal hearts. At E10.0, Xin is also detected in the developing somites and later in the myotendon junction of skeletal muscle but not within the costameric regions of muscle. In cultured C2C12 myotubes, the Xin protein is found in many speckled and filamentous structures, coincident with tropomyosin in the stress fibers. Additionally, Xin is enriched in the regions of cell–cell contacts. These data demonstrate that Xin is one of the components at the adherens junction of cardiac muscle, and its counterpart in skeletal muscle, the myotendon junction. Furthermore, temporal and spatial expressions of Xin in relation to intercalated disc proteins and thin filament proteins suggest roles for Xin in the formation of cell–cell contacts and possibly in myofibrillogenesis.

The Intercalated Disc Protein, mXinα, Is Capable of Interacting with β-Catenin and Bundling Actin Filaments

Targeted deletion of mXinα results in cardiac hypertrophy and cardiomyopathy with conduction defects (Gustafson-Wagner, E., Sinn, H. W., Chen, Y.-L., Wang, D.-Z., Reiter, R. S., Lin, J. L.-C., Yang, B., Williamson, R. A., Chen, J. N., Lin, C.-I., and Lin, J. J.-C. (2007) Am. J. Physiol. 293, H2680-H2692). To understand the underlying mechanisms leading to such cardiac defects, the functional domains of mXinα and its interacting proteins were investigated. Interaction studies using co-immunoprecipitation, pull-down, and yeast two-hybrid assays revealed that mXinα directly interacts with β-catenin. The β-catenin-binding site on mXinα was mapped to amino acids 535-636, which overlaps with the known actin-binding domains composed of the Xin repeats. The overlapping nature of these domains provides insight into the molecular mechanism for mXinα localization and function. Purified recombinant glutathione S-transferase- or His-tagged mXinα proteins are capable of binding and bundling actin filaments, as determined by co-sedimentation and electron microscopic studies. The binding to actin was saturated at an approximate stoichiometry of nine actin monomers to one mXinα. A stronger interaction was observed between mXinα C-terminal deletion and actin as compared with the interaction between full-length mXinα and actin. Furthermore, force expression of green fluorescent protein fused to an mXinα C-terminal deletion in cultured cells showed greater stress fiber localization compared with force-expressed GFP-mXinα. These results suggest a model whereby the C terminus of mXinα may prevent the full-length molecule from binding to actin, until the β-catenin-binding domain is occupied by β-catenin. The binding of mXinα to β-catenin at the adherens junction would then facilitate actin binding. In support of this model, we found that the actin binding and bundling activity of mXinα was enhanced in the presence of β-catenin.

Xin proteins and intercalated disc maturation, signaling and diseases

Intercalated discs (ICDs) are cardiac-specific structures responsible for mechanical and electrical communication among adjacent cardiomyocytes and are implicated in signal transduction. The striated muscle-specific Xin repeat-containing proteins localize to ICDs and play critical roles in ICD formation and cardiac function. Knocking down the Xin gene in chicken embryos collapses the wall of developing heart chambers and leads to abnormal cardiac morphogenesis. In mammals, a pair of paralogous genes, Xinalpha and Xinbeta exist. Ablation of the mouse Xinalpha (mXinalpha) does not affect heart development. Instead, mXinalpha-deficient mice show adult late-onset cardiac hypertrophy and cardiomyopathy with conduction defects. The mXinalpha-deficient hearts up-regulate mouse Xinbeta (mXinbeta, suggesting a partial compensatory role of mXinbeta. Complete loss of mXinbeta however, leads to failure of forming ICD, mis-localization of mXinalpha, and early postnatal lethality. In this review, we will briefly discuss recent advances in the anatomy and function of ICDs. We will then review what we know about Xin repeat-containing proteins and how this protein family promotes ICD maturation and stability for normal cardiac function.

Induction of protective immunity to RM-1 prostate cancer cells with ALVAC-IL-2/IL-12/TNF-α combination therapy

Human prostate cancers characteristically express low levels of major histocompatibility complex (MHC) Class I, which makes it challenging to induce protective antitumor responses involving T cells. Here we demonstrate that a whole cell tumor vaccine can induce protective T cell immunity to a low MHC Class I‐expressing mouse prostate cancer cell line, RM‐1. ALVAC recombinant canarypox viruses encoding interleukin‐2, interleukin‐12 and tumor necrosis factor‐α were used to create therapeutic vaccines in 2 different ways. The RM‐1 cells were pre‐infected in vitro with the viruses prior to injection (pre‐infection vaccine) or the RM‐1 cells were injected alone, followed by the viruses (separate injection vaccine). The vaccines were each tested subcutaneously or intradermally. The pre‐infection vaccine resulted in 100% clearance of primary tumors, whereas intradermal delivery of the separate injection vaccine cleared 40–60% of primary tumors. Despite the highly efficient primary tumor clearance by the pre‐infection vaccine, only the separate injection vaccine generated protection upon rechallenge. Tumor‐free survival induced by the separate injection vaccine required natural killer (NK) cells, CD4+, and CD8+ T cells. None of these cells alone were sufficient to induce tumor‐free survival to the primary challenge, demonstrating an important cooperativity between NK cells and T cells. Secondary clearance of tumors also required NK and CD8+ T cells, but not CD4+ T cells. We report for the first time the generation of T cell immunity to the RM‐1 prostate cancer cell line, demonstrating that it is possible to generate protective T cell immunity to a MHC I‐low expressing tumor.