Monitoring the Anti-Tumor Activity of Expanded CD8+ NKT Cells After Allogeneic Bone Marrow Transplantation Using Bioluminescent Imaging
Presenter: Matthias Edinger
Presenter's Affiliation: Stanford University School of Medicine
Type of Session: Plenary
Biolumiscent imaging is an imaging technique that allows the visualization of labeled cells within a living animal. Cells labeled with the Luc gene emit low levels of light that can be detected and quantified with low light CCD cameras. Only living cells contribute to the detected signal. This technique is used in this study to monitor the kinetics of tumor growth and regression and the anti-tumor activity of an expanded cell population of CD8+ NKT cells, previously described as cytokine-induced killer cells (CIK).
Materials and Methods
The tumor cell lines BCL1 and A20 were simultaneously transfected with the bioluminescent gene Luc and green flourescent protein (GFP) or yellow fluorescent protein (YFP). Luc allowed the imaging of cells in vivo, while GFP and YFP were used to re-isolate tumor cells using flow cytometry. To verify that CCD-detected signal represented tumor burden, mice were photographed, then the cells were separated and quantified.
NKT cells were generated by stimulating splenocytes with IFN-ã followed by anti-CD-3 antibody and IL-2. The resulting cells were CD3+ and CD8+, with 30-50% expressing NK-1.1.
Animals with Luc-labeled BCL1 were radiated and transplanted with either bone marrow, bone marrow + splenocytes, or bone marrow + NKT cells. This experiment was performed with both syngeneic transplant and allogeneic transplant.
In order to image the trafficking of NKT cells, a separate group of tumor-bearing mice were injected with Luc-labeled NKT cells.
Animals injected with 2.5 x 106 NKT cells showed a long term survival of 85%, while cells injected wtih 10 x 106 NKT cells showed a 30% survival rate.
Control animals injected with splenocytes had all died from acute graft-versus-host disease (GVHD) at day 40 (2.5 x 106 injected splenocytes) and day 14 (10 x 106 injected splenocytes).
Animals transplanted with allogeneic transplant only demonstrated a drastic increase in bioluminescent imaging, likely from a relapse of the Luc-labeled BCL1 lymphoma cells, and died.
Tumor-bearing mice injected with Luc-labeled NKT cells showed an increase in signal in the tumor by 3 days. This increased signal remained at the tumor site until the tumor was eradicated.
Bioluminescent imaging accurately visualizes and quantifies labeled cells in live animals, as demonstrated by the flow cytometry studies of cells co-transfected with Luc and GFP/YFP.
Biolumiscent imaging accurately and non-invasively allowed the imaging of tumor burden in BCL1 lymphoma models.
Bioluminescent imaging demonstated the trafficking and activity of labeled NKT cells.
This study provides information about the trafficking and anti-tumor effect of NKT cells, and demonstrates the usefulness of an exciting new imaging modality using bioluminescence. More studies are needed to further evaluate this exciting new technology.
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