Development and validation of a Murine model for dendritic cell-based immunotherapy

Gillions, Imogen Maree (2012). Development and validation of a Murine model for dendritic cell-based immunotherapy MPhil Thesis, School of Medicine, The University of Queensland.

       
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Author Gillions, Imogen Maree
Thesis Title Development and validation of a Murine model for dendritic cell-based immunotherapy
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2012
Thesis type MPhil Thesis
Supervisor Jose Alejandro Lopez
Chris Schmidt
Andreas Suhrbier
Total pages 94
Total colour pages 11
Total black and white pages 83
Language eng
Subjects 1107 Immunology
Formatted abstract
Dendritic cell (DC)-based therapy, as a part of immunotherapy for cancer patients, can result in complete regression of metastatic disease. However, the mechanisms by which DC stimulate effective anti-tumour T-cell responses in existing disease are unknown. Current experimental models use short-term cultured spleen- or lymph node-derived DC to study their ability to cross present antigens and examine their roles in stimulating immune responses in vitro and in vivo. Cultured and fresh DC are heterogeneous and are cumbersome to expand to large populations. One approach to overcome these shortcomings is through the use of cell lines that could be easily manipulated for the investigation of multiple variables and combinations thereof. Due to the lack of human DC cell lines a murine model that reflects the conditions of clinical trials is a plausible alternative. This project examines the partially differentiated DC-like cell line DC2114, cloned from the splenic cells of transgenic mice with a C57BL/6 background. These transgenic mice express the SV-40 large-T antigen driven by CD11c promoter with a GFP tag. DC2114 has been used here to investigate the conditions required for optimal antigen uptake and cross-presentation. In addition to the reduction in animal culling and cytokine use, the use of cell lines is advantageous as they are a homogeneous cell population. These cells can be easily manipulated in a number of ways, including modification of gene expression, independent of effects on ontogeny - a potential shortfall of transgenic mice.

We show that DC2114 express the costimulatory molecules CD40, CD80 and CD86 on its surface as well as major histocompatibility complex (MHC) class II. DC2114 upregulate these costimulatory molecules and secrete the proinflammatory cytokine IL-12p70 in response to LPS, CpG, and Poly(I:C) stimulation. DC2114 stimulated the proliferation of mismatched responder lymphocytes from BALB/c mice, at a level comparable to that of bone-marrow derived DC. DC2114 efficiently present the H2-Kb-restricted ovalbumin peptide to naive TCR transgenic OT-I mice and were capable of cross-presenting ovalbumin (OVA) protein at low concentrations. DC2114 are also capable of presenting OVA antigen from Cos-7 cells that were transfected to express the protein in the cytoplasm, and which are not capable of presenting antigens directly (due to MHC mismatch). This cross-presentation to OT-I and presentation to OT-II T cells of whole cell associated antigen was further enhanced by oxidisation of the tumour antigen with HOCl.

These results provide the foundation for optimisation of the conditions required by DC to uptake, and subsequently cross-present, tumour derived antigens. In particular, they are key for further experiments leading to optimising the cross-presentation of whole tumour cells as an antigen source, shown in clinical trials to be twice as effective as defined antigen sources.
Keyword Dendritic cell
Cross-presentation
Maturation
Toll-like receptor
Tumour
Oxidation
DC2114

 
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Created: Wed, 05 Sep 2012, 10:11:58 EST by Imogen Gillions on behalf of Scholarly Communication and Digitisation Service