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Induction of antigen-specific cytotoxic T lymphocytes (CTLs) by dendritic cell vaccination: Loading of dendritic cells with whole tumour cell mRNA
Dendritic cells (DCs) are phenotypically distinct and extremely potent antigen-presenting cells that are critical for initiation of specific immune responses (1). DCs take up and process antigen as peptides within the major histocompatibility complex (MHC) and present these to T cells. They also deliver additional co-stimulatory signals facilitating activation and expansion of antigen-specific T cells. Because of their unique ability to induce primary immune responses, DCs are attractive targets for cancer immunotherapy.
We study induction of cytotoxic T lymphocytes (CTLs) after immunizations with DCs loaded with an antigen of interest. These antigens can be delivered to DCs in a number of forms: MHC-restricted peptides, proteins, DNA or mRNA for a single antigen species, or in the form whole tumor cell mRNA. The use of whole tumor cell mRNA may have the advantage of presenting a number of different antigens to the immune system.
Aim of the project:
We have previously shown that monocyte-derived DCs can be transfected with single species mRNA using electroporation (2). The aim of this study is to transfect DCs with whole tumor cell mRNA isolated from the prostate cancer cell line LNCaP, and to study the ability of these antigen-loaded DCs to stimulate CTL responses.
1) Isolate mRNA from the prostate cancer cell line LNCaP
2) Transfect DCs with this mRNA using electroporation
3) Examine the ability of these antigen-loaded DCs to stimulate CTL responses.
· Dendritic cell culture
· Isolation of mRNA using RNeasy kit
· Electroporation of dendritic cells
· Analysis of CTL responses by ELISPOT
Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998 Mar 19;392(6673):245-52.
Lundqvist A, Noffz G, Pavlenko M, Saeboe-Larssen S, Fong T, Maitland N, Pisa P. Nonviral and viral gene transfer into different subsets of human dendritic cells yield comparable efficiency of transfection. J Immunother. 2002 Nov-Dec;25(6): 445-54.
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