RNA Interference as a Tool to Reduce the Risk of Rejection in Cell-Based Therapies

Remarkable progress in the experimental and clinical applications of cell-based therapies has identified stem cells and their derived products as potential candidates for regenerative therapies for many disorders. The use of autologous stem cells as source for regenerative therapeutic products is strongly limited by their low availability. Therefore, the future applications of in vitro pharmed therapeutic cell products will most likely occur in an allogeneic manner. However, the high variability of the human leukocyte antigen (HLA) represents a major obstacle to the application of off-the-shelf products. We have developed a strategy to decrease the immunogenicity of in vitro generated cell products by silencing HLA expression using RNAi. HLA expression was permanently silenced in CD34+ hematopoietic stem and progenitor cells and induced the pluripotent stem cells to generate HLA-universal cells sources, which were then used for the differentiation of low immunogenic cell products. In this chapter, we will provide an overview about an RNAi-based strategy to reduce the immunogenicity of cell-based therapies, and in particular in the generation of HLA-universal platelets and tissues

Dynamic Interaction between Immune Escape Mechanism and HLA-Ib Regulation

HLA molecules scan the intracellular proteome and present self- or non-self-peptides to immune effector cells. HLA-Ia (HLA-A, HLA-B and HLA-C) are the most polymorphic genes, resulting in various numbers of allelic variants expressed on the surface of almost all nucleated cells. In contrast to HLA-Ia molecules that activate the immune system during pathogenic invasion, the marginal polymorphic HLA-Ib molecules (HLA-E, HLA-F and HLA-G) are upregulated during pathogenic episodes and mediate immune tolerance. A fine tuning between downregulation of HLA-Ia and upregulation of HLA-Ib can be observed through immunological episodes that require to remain unrecognized by immune effector cells. While HLA-Ia molecules collaborate by presenting a wide range of peptides, every HLA-Ib molecule is highly specialized in its protective immune function and seems to be restricted in the presentation of peptides. Additionally, Ia molecules are expressed ubiquitously while the expression of HLA-Ib molecules is strictly restricted to certain tissues and occurs instantly on demand of the cells/tissue that attempt to be hidden from the immune system. The more knowledge becomes available for the function of HLA-Ib molecules; the question emerges if the molecular typing of HLA-Ib molecules would be reasonable to take a decision post treatment for personalized cellular therapies

Peptide Presentation Is the Key to Immunotherapeutical Success

Positive and negative selection in the thymus relies on T-cell receptor recognition of peptides presented by HLA molecules and determines the repertoire of T cells. Immune competent T-lymphocytes target cells display nonself or pathogenic peptides in complex with their cognate HLA molecule. A peptide passes several selection processes before being presented in the peptide binding groove of an HLA molecule; here the sequence of the HLA molecule’s heavy chain determines the mode of peptide recruitment. During inflammatory processes, the presentable peptide repertoire is obviously altered compared to the healthy state, while the peptide loading pathway undergoes modifications as well. The presented peptides dictate the fate of the HLA expressing cell through their (1) sequence, (2) topology, (3) origin (self/nonself). Therefore, the knowledge about peptide competition and presentation in the context of alloreactivity, infection or pathogenic invasion is of enormous significance. Since in adoptive cellular therapies transferred cells should exclusively target peptide-HLA complexes they are primed for, one of the most crucial questions remains at what stage of viral infection viral peptides are presented preferentially over self-peptides. The systematic analyzation of peptide profiles under healthy or pathogenic conditions is the key to immunological success in terms of personalized therapeutics

Heat shock protein 70/peptide complexes: potent mediators for the generation of antiviral T cells particularly with regard to low precursor frequencies

<p>Abstract</p> <p>Background</p> <p>Heat shock protein 70 (HSP70) has gained major attention as an adjuvant capable of inducing antigen-specific CD8⁺and CD4⁺T-cell responses. The ability of HSP70/peptide complexes to elicit cytotoxic T-cell (CTL) responses by cross-presentation of exogenous antigens via HLA class I molecules is of central interest in immunotherapy. We examined the role of HSP70/CMVpp65_495-503-peptide complex (HSP70/CMV-PC) in HLA class I-restricted cross-presentation for <it>ex vivo </it>expansion of CMV-specific CTLs.</p> <p>Methods</p> <p>CMV-specific T cells generated from PBMCs of HLA-A*02:01/CMV-seropositive donors were stimulated for 21 days with HSP70/CMV-PC and analyzed in functional assays. As a control PBMCs were cultured in the presence of CMVpp65_495-503peptide or HSP70. Increase of CMV-specific CTLs was visualized by pentameric HLA-A*02:01/CMVpp65_495-503complex.</p> <p>Results</p> <p>About 90% of HSP70/CMV-PC generated T cells were CMV-specific and exhibited significantly higher IFN-γ secretion, cytotoxic activity, and an increased heme oxygenase 1 (HO-1) gene expression as compared to about 69% of those stimulated with CMVpp65_495-503peptide. We decided to classify the HLA-A*02:01/CMV-seropositive donors as weak, medium, and strong responder according to the frequency of generated A2/CMV-pentamer-positive CD8⁺T cells. HSP70/CMV-PC significantly induces strong antiviral T-cell responses especially in those donors with low memory precursor frequencies. Blockage of CD91 with α2-macroglobulin markedly reduced proliferation of antiviral T cells suggesting a major role of this receptor in the uptake of HSP70/CMV-PC.</p> <p>Conclusion</p> <p>This study clearly demonstrates that HSP70/CMV-PC is a potent mediator to induce stronger T-cell responses compared to antiviral peptides. This simple and efficient technique may help to generate significant quantities of antiviral CTLs by cross-presentation. Thus, we propose HSP70 for chaperoning peptides to reach an efficient level of cross-presentation. HSP70/peptide complexes may be particularly useful to generate stronger T-cell responses in cases of low precursor frequencies and may help to improve the efficiency of antigen-specific T-cell therapy for minor antigens.</p

Personalized adoptive immunotherapy for patients with EBVassociated tumors and complications:Evaluation of novel naturally processed and presented EBV-derived T-cell epitopes

Morbidity and mortality of immunocompromised patients are increased by primary infection with or reactivation of Epstein-Barr virus (EBV), possibly triggering EBV+post-transplant lymphoproliferative disease (PTLD). Adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) promises a non-toxic immunotherapy to effectively prevent or treat these complications. To improve immunotherapy and immunomonitoring this study aimed at identifying and evaluating naturally processed and presented HLA-A*03:01-restricted EBV-CTL epitopes as immunodominant targets. More than 15000 peptides were sequenced from EBV-immortalized B cells transduced with soluble HLA-A*03:01, sorted using different epitope prediction tools and eleven candidates were preselected. T2 and Flex-T peptide-binding and dissociation assays confirmed the stability of peptide-MHC complexes. Their immunogenicity and clinical relevance were evaluated by assessing the frequencies and functionality of EBV-CTLs in healthy donors (n> 10) and EBV+PTLD-patients (n= 5) by multimer staining, Eli- and FluoroSpot assays. All eleven peptides elicited EBV-CTL responses in the donors. Their clinical applicability was determined by small-scale T-cell enrichment using Cytokine Secretion Assay and immunophenotyping. Mixtures of these peptides when added to the EBV Consensus pool revealed enhanced stimulation and enrichment efficacy. These EBV-specific epitopes broadening the repertoire of known targets will improve manufacturing of clinically applicable EBV-CTLs and monitoring of EBV-specific T-cell responses in patients

Discovery of immunodominant T-cell epitopes reveals penton protein as a second immunodominant target in human adenovirus infection

Additional file 2: Figure S2. Analysis of HAdV-specific T-cell responses against the novel immunodominant T-cell epitope in healthy donors

Repeated freezing procedures preserve structural and functional properties of amniotic membrane for application in ophthalmology

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing– thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-ß1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing. © 2020 by the authors. Licensee MDPI, Basel, Switzerland