Construction of an optimal immunogen using mRNA transfected dendritic cells. Kira Minkis

ISBN: 9780549582458

Published:

276 pages


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Construction of an optimal immunogen using mRNA transfected dendritic cells.  by  Kira Minkis

Construction of an optimal immunogen using mRNA transfected dendritic cells. by Kira Minkis
| | PDF, EPUB, FB2, DjVu, talking book, mp3, ZIP | 276 pages | ISBN: 9780549582458 | 8.47 Mb

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that initiate and regulate innate and adaptive immune responses. These properties underlie the use of DCs as adjuvants in therapeutic anti-tumor vaccines. We have focused on... MoreDendritic cells (DCs) are professional antigen presenting cells (APCs) that initiate and regulate innate and adaptive immune responses.

These properties underlie the use of DCs as adjuvants in therapeutic anti-tumor vaccines. We have focused on improving DC based vaccines for melanoma using an approach whereby mRNA, encoding antigens (ags) of interest can be directly routed to DC. By making modifications that simultaneously enhance CD4+ T cell activation and DC function, we were able to differentiate DCs into more potent immunogens capable of presenting tumor associated antigens (TAAs), and maximizing CD8+ and CD4+ Th1 responses.

A melanoma-associated antigen, MelanA/Mart-1, was used as a model antigen. DCs were EP with in vitro transcribed mRNA encoding the MART-1 coding sequence conjugated to the lysosomal targeting sequence, LAMP-1 (lysoMART-1) in order to maximize delivery of antigen for processing onto MHC class II.

As a comparison, we used cytoplasmic MART-1 (cytoMART-1) whereby antigen is preferentially expressed in the cytoplasm.-Both cyto- and lysoMART-1 transfected DCs directly activated MART-1 specific CD8+ T cell clones, with lysoMART-1 EP DCs being the more potent stimulators.

LysoMART-1 EP DCs also enhanced the quality and quantity of MART-1 specific T cells primed from healthy donors and subjects with melanoma, respectively.-IL-12p70 mRNA transfected DCs produced significant quantities of IL-12p70. The secreted IL-12p70 enhanced natural killer cell activation. The cytokine profiles of CD4+ allo-specific T cell lines that expanded in response to transfected DC was altered, increasing the ratio of IFN-g to IL-5 produced in response to allogeneic targets.

Furthermore, the antigen specificity and TH1-skewing of CD8+ T cell lines that were induced from PBMC of normal donors and of melanoma patients by autologous MART-transfected DC was enhanced when the APCs were simultaneously transfected with IL-12.-Thus, EP with lysoMART-1 and IL-12p70 mRNA can enhance anti-tumor T cell responses both quantitatively, in terms of magnitude, and qualitatively, in terms of phenotype. Our results demonstrate the potential usefulness of mRNA transfection to introduce TAA and immunomodulatory cytokines into DC to induce innate and adaptive immune responses.



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