No abstract

Cross-presenting CD8-+ conventional dendritic cells (cDCs) are important for the eradication of cancers and viral infections due to their ability to induce cytotoxic T lymphocyte (CTL) responses. Cross presenting DCs are a very rare subset and in the past, studies of these cells have been limited by their scarcity of specific cell surface markers. Therefore, methods for the detection and isolation of these cells were commonly based on a multitude of immunophenotypic criteria, such as the expression of CD11c and CD8- and the absence of CD3, CD4, SIRP-- and CD11b. Recently, it was demonstrated that crosspresenting cDCs in lymphoid and non-lymphoid tissues specifically express XCR1, which correlates with the ability to take up and cross-present exogenous antigens. Combining our recombinant REAfinity™ Anti-XCR1 mouse mAb with MACS® Technology based on UltraPure MicroBeads, we developed a new method for the fast and easy isolation of cross-presenting DCs. Using this method XCR1+ DCs can be routinely enriched with high recovery and purity without the need for time-consuming laborious flow sorting. This will facilitate the study of cross-presenting DC subset, which will ultimately help to develop new therapeutic strategies employing DCs in the future.

The value and safety of dendritic cell (DC) vaccines in cancer immunotherapy have been proven in several clinical studies using monocyte-derived dendritic cells (Mo-DCs). However, the parallel development of several different maturation and antigen-loading protocols might have led to cellular products with different therapeutic efficacy. Moreover a low immunological capability of the patients might cause the generation of functionally impaired DCs. Functionality of Mo-DCs can be improved by mRNA electroporation, an effective method for introducing either antigens for MHC class I-mediated presentation (cross presentation) or other genes that might modulate the functional properties of Mo-DCs in a desired way. Yet, a standardized and reproducible GMP-compliant manufacturing of such cellular product requires instrumentation with specific characteristics not always provided by conventional technologies. In order to address this need, we have developed a platform for automated manufacture of cellular products (CliniMACS Prodigy and CliniMACS Electroporator), enabling a reproducible and scalable generation of Mo-DCs and automated electroporation in a closed system that facilitates compliance with GMP requirements. To validate the functionality of our system we first optimized transfection rate using GFP-mRNA. Then, using mRNA encoding immune relevant antigens we generated Mo-DCs able to stimulate autologous, antigen specific, cytotoxic T cells. Moreover, we successfully induce expression of genes that are responsible for main DC functions. In summary, we developed an automated, large-scale, closed process that enables efficient transfection of Mo-DCs for antigen delivery and functional tuning