Optimizing Dendritic Cell-Tumor Fusion Vaccine for Acute Myeloid Leukemia Öffentlichkeit
Chang, Hsiao-Man (Spring 2020)
Published
Abstract
Conventional treatment for acute myeloid leukemia (AML) is with non-cancer selective chemotherapy or allogenic bone marrow transplantations. Developing a treatment option to specifically target AML without damaging healthy cells is a promising strategy for improving patient prognosis. A common approach within immunotherapy is cancer vaccine synthesis which utilizes the individual’s own antigen-presenting cells to enhance anti-tumor T cell activity.
Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system. The DC-AML fusion heterokaryon entity exposes DCs to tumor-associated antigens that are expressed on the tumor cells, allowing DCs to endogenously process and present tumor-associated antigens via major histocompatibility complex (MHC) classes, which can then elicit T cell responses.
The organization of this study included four parts: harvesting bone marrow from FLT3L injected mice, harvesting bone-marrow derived dendritic cells, fusing dendritic cells with murine AML cell line, C1498, and testing the efficacy of the fused vaccine in a mixed lymphocyte reaction (MLR) assay. Previous studies in our lab has shown that FMS-like tyrosine kinase 3 ligand (FLT3L) injections in mice bone marrow donors increased the content of pDCs in their graft. We showed that mouse receiving FLT3L injections had increased number of nucleated cells in the bone marrow. We also showed that FLT3L had a proliferative effect on dendritic cell expansion in vitro when added to adherent and nonadherent bone marrow cultures, but this increase in dendritic cells was not observed when only adherent cells were cultured with FLT3L. In addition, dendritic cells in FLT3L-supplemented cultures had increased CD80 and B220 expression levels, but decreased CD11c expression, which is consistent with pDC phenotype.
Results from the fusion percentages fusion of vaccine reveal that an extra two-time serum-free RPMI wash prior to fusion with polyethylene glycol improves fusion percentage. Upon testing for the efficacy of the vaccine in MLR assays, we observed that T cells primed with irradiated C1498 had enhanced cytotoxic activities compared to non-specific T cells.
Table of Contents
Table of Contents
I. Introduction
1. Overview of Acute Myeloid Leukemia…………………………………………...1
2. Personalized Vaccine……………………………………………………………...2
3. Dendritic Cell Maturation Overview ………………………………………….….3
4. Effect of FLT3L on Dendritic Cells……………………………………………....4
5. Effect of VIP Antagonist on Dendritic Cells……………………...……………....5
II. Hypotheses Tested ……………………………………………………………………6
III. Experimental Approach
1. Mice ………………………………………………………………………………7
2. FLT3L Induced Murine Bone Marrow Harvest ………………………………….7
3. Cytokine-induced Dendritic Cell Culture Following Bone Marrow Harvest…….8
4. Dendritic Cell-Tumor Vaccine Fusion with C1498 ……………………………...9
5. Immunostaining and Flow Cytometry Characterization of Fused Vaccine.…….10
6. T Cell Isolation ………………………………………………………………….11
7. MLR T cell Killing and Proliferation Assays……………………………………11
IV. Results
1. FLT3L Injection to Mice Increased the Numbers of Nucleated Cells in Bone Marrow…………………………………………………………………………...13
2. FLT3L Increased Number of Dendritic Cells Cultured from Non-Adherent and Adherent Bone Marrow Expansion In Vitro……………………………….…….14
3. FLT3L Increased B220 and CD80 and Decreased CD11c Markers on Mouse DC in Culture Consistent with pDC Differentiation…………………………………15
4. Culturing Adherent Bone Marrow Cells Yield Higher Numbers of Dendritic Cells than Culturing Nonadherent and Adherent Cells………………………………...16
5. Nonadherent Bone Marrow Cells Collected after 8 Hours and 16 Hours Exhibit Small Phenotypic Difference…………………………………………………….17
6. The Expression of Dendritic Cell Markers on Adherent and Nonadherent Cells Increased Between Day 5 and Day 10 of Culture……………………………….18
7. Washing with Serum-Free RPMI Media Improves the Fused-Cell Percentage…19
8. Antigen Specific Killing of C1498 is More Potent than Non-Specific Killing….20
V. Figures ………………………………………………………………………………22
VI. Discussion and Future Directions……………………………………………………41
VII. Works Cited………………………………………………………………………….48
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