Messenger RNA electroporation: an efficient tool in immunotherapy and stem cell research.

Over the last decades medicine has developed tremendously, but still many diseases are incurable. The last years, cellular (gene) therapy has become a hot topic in biomedical research for the potential treatment of cancer, AIDS and diseases involving cell loss or degeneration. Here, we will focus on two major areas within cellular therapy, cellular immunotherapy and stem cell therapy, that could benefit from the introduction of neo-expressed genes through mRNA electroporation for basic research as well as for clinical applications. For cellular immunotherapy, we will provide a state-of-the-art on loading antigen-presenting cells with antigens in the mRNA format for manipulation of T cell immunity. In the area of stem cell research, we will highlight current gene transfer methods into adult and embryonic stem cells and discuss the use of mRNA electroporation for controlling guided differentiation of stem cells into specialized cell lineages

Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl

Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP3R) via its BH4 domain, thereby suppressing IP3R Ca2+-flux properties and protecting against Ca2+-dependent apoptosis. Here, we directly compared IP3R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP3R nor inhibited IP3-induced Ca2+ release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP3R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP3R binding and inhibition. This difference in IP3R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP(3)Rs. In agreement with the IP3R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP3R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP(3)Rs and Ca2+-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca2+ signaling and apoptosis

Mycobacterium tuberculosis infection induces il12rb1 splicing to generate a novel IL-12Rβ1 isoform that enhances DC migration

RNA splicing is an increasingly recognized regulator of immunity. Here, we demonstrate that after Mycobacterium tuberculosis infection (mRNA) il12rb1 is spliced by dendritic cells (DCs) to form an alternative (mRNA) il12rb1Deltatm that encodes the protein IL-12Rbeta1DeltaTM. Compared with IL-12Rbeta1, IL-12Rbeta1DeltaTM contains an altered C-terminal sequence and lacks a transmembrane domain. Expression of IL-12Rbeta1DeltaTM occurs in CD11c(+) cells in the lungs during M. tuberculosis infection. Selective reconstitution of il12rb1(-/-) DCs with (mRNA) il12rb1 and/or (mRNA) il12rb1Deltatm demonstrates that IL-12Rbeta1DeltaTM augments IL-12Rbeta1-dependent DC migration and activation of M. tuberculosis-specific T cells. It cannot mediate these activities independently of IL12Rbeta1. We hypothesize that M. tuberculosis-exposed DCs express IL-12Rbeta1DeltaTM to enhance IL-12Rbeta1-dependent migration and promote M. tuberculosis-specific T cell activation. IL-12Rbeta1DeltaTM thus represents a novel positive-regulator of IL12Rbeta1-dependent DC function and of the immune response to M. tuberculosis.This work was supported by the Trudeau Institute and the National Institutes of Health (AI067723 to A. M. Cooper; AI49823 to D. L. Woodland [trainee: R. T. Robinson] and AI084397 to R. T. Robinson)

Editorial: Perinatal Derivatives and the Road to Clinical Translation, Volume I

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Sensitive detection of human papillomavirus type 16 E7-specific T cells by ELISPOT after multiple <it>in vitro </it>stimulations of CD8⁺T cells with peptide-pulsed autologous dendritic cells

<p>Abstract</p> <p>Background</p> <p>Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.</p> <p>Results</p> <p>In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E7_11–20peptide in order to induce an <it>in vitro </it>CD8⁺T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8⁺T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-γ ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-γ-secreting CD8⁺T cells in 5/5 healthy donors.</p> <p>Conclusion</p> <p>We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response <it>in vitro</it>. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses <it>in vitro</it>.</p

Multimodal <it>in vivo</it> imaging reveals limited allograft survival, intrapulmonary cell trapping and minimal evidence for ischemia-directed BMSC homing

Abstract Background Despite positive reports on the efficacy of stem cell therapy for the treatment of cardiovascular disease, the nature of stem cell homing to ischemic tissues remains elusive. Results We used a mouse model of peripheral tissue ischemia to study the survival and homing capacity of dual reporter gene (eGFP/Luciferase) expressing bone marrow-derived stromal cells (BMSC). Cell homing and survival were studied in the presence and absence of ciclosporin A (CsA) immunosuppression using bioluminescence imaging (BLI) together with confocal endomicroscopy. Different injection strategies were applied: central venous (CV), intra-arterial (IA) and intramuscular (IM). BLI and confocal endomicroscopy evidenced complete rejection of the IM injected allogeneic BMSC transplant within 5 to 10 days. Immunosuppression with CsA could only marginally prolong graft survival. IM injected BMSC did not migrate to the site of the arterial ligation. CV injection of BMSC resulted in massive pulmonary infarction, leading to respiratory failure and death. Intrapulmonary cell trapping was evidenced by confocal endomicroscopy, BLI and fluorescence microscopy. IA injection of BMSC proved to be a feasible and safe strategy to bypass the lung circulation. During the follow-up period, neither BLI nor confocal endomicroscopy revealed any convincing ischemia-directed homing of BMSC. Conclusions BLI and confocal endomicroscopy are complementary imaging techniques for studying the in vivo biology of dual reporter gene-expressing BMSC. Allogeneic BMSC survival is limited in an immunocompetent host and cannot be preserved by CsA immunosuppression alone. We did not find substantial evidence for ischemia-directed BMSC homing and caution against CV injection of BMSC, which can lead to massive pulmonary infarction.</p