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Preparation of Activated Anti-Tumor Cells by Dendritic Cell Peptide Presentation
UMF Student Research Grant Application
As the role of the host immune system in tumor immunosurveillance is elucidated, increasing attention is being given to targeted immunotherapy as a promising cancer treatment. It is known that tumors express antigens which can induce host immune responses under experimental conditions. Yet tumor-reactive T cells are rarely found in vivo. The development of cytotoxic T cells depends on the action of activated antigen presenting cells, such as dendritic cells. Antigenic tumor peptides must be presented within the context of MHC class I molecules with appropriate co-stimulatory molecules. It has been hypothesized that the lack of T cell response in cancer patients may result from a deficiency of functional dendritic cells. (1) In animal models the reinfusion of dendritic cells which have been treated with tumor antigens has been shown to induce specific immunity, leading in some cases to a decrease in tumor mass. (2)
The hypothesis to be evaluated with this project is: Can anti-tumor cytotoxic T lymphocytes be generated by dendritic cell presentation of breast cancer peptides? The first goal will be to determine whether immature human dendritic cells derived from bone marrow precursors can be primed with tumor proteins to become mature cells which present processed peptides to autologous T cells. Secondly, the development of T cell cytotoxic activity against the breast cancer targets and IFN? production will be measured to determine whether the dendritic cells can successfully activate specific immunity in the T cells. The work described will be done exclusively by the applicant, with assistance from personnel in the [mentor’s] laboratory.
- Prepare a membrane lysate from an HLA-A2 breast cancer cell line, MCF-7.
- Purify immature dendritic cells from the bone marrow of an HLA-A2 donor.
- Purify T cells from the peripheral blood of the same donor.
- Verify the activated status of the dendritic cells after they have been pulsed with the membrane preparation.
- Grow autologous T cells in coculture with the activated dendritic cells.
- Determine whether the T cells become activated to proliferate and produce IFN?.
- Determine whether the T cells develop cytotoxic activity against the breast cancer target cells.
- Breast cancer cells will be lysed to form a preparation of tumor cell membranes.
- Dendritic cells will be grown from bone marrow derived progenitors. CD34 cells will be isolated from bone marrow by immunofluorescence. The stem cells will be cultured with GM-CSF, IL-4, and TNF to induce the differentiation of dendritic cells.
- T cells will be purified from peripheral blood.
- The dendritic cells will be primed by pulsing them with the breast cancer cell membrane preparation. (4)
- The transformation of dendritic cells from immature antigen-capturing cells to mature antigen presenting cells will be assessed by phenotyping with monoclonal antibody staining for CD54, CD40, CD80, CD83, CD86 and MHC molecules. The expression of these cell surface antigens increases with maturation. (5)
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- Banchereau J, Steinman R. (1998) Nature, 392, 245-252.