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Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer
Brian A Rabinovich1, Yang Ye, Tamara Etto
1M. D. Anderson Cancer Center, 7455 Fannin Street, Houston, TX 77054, USA. brabinov@mdanderson.org
Abstract:
Antigen specific T cell migration to sites of infection or cancer is critical for an effective immune response. In mouse models of cancer, the number of lymphocytes reaching the tumor is typically only a few hundred, yet technology capable of imaging these cells using bioluminescence has yet to be achieved. A combination of codon optimization, removal of cryptic splice sites and retroviral modification was used to engineer an enhanced firefly luciferase (ffLuc) vector. Compared with ffLuc, T cells expressing our construct generated >100 times more light, permitting detection of as few as three cells implanted s.c. while maintaining long term coexpression of a reporter gene (Thy1.1). Expression of enhanced ffLuc in mouse T cells permitted the tracking of <3 x 10(4) adoptively transferred T cells infiltrating sites of vaccination and preestablished tumors. Penetration of light through deep tissues, including the liver and spleen, was also observed. Finally, we were able to enumerate infiltrating mouse lymphocytes constituting <0.3% of total tumor cellularity, representing a significant improvement over standard methods of quantitation including flow cytometry.
Insights
Researchers developed an enhanced firefly luciferase (ffLuc) vector for improved bioluminescence imaging of T cells. This new tool allows sensitive detection and tracking of small numbers of T cells in vivo, crucial for cancer immunotherapy research.
Area of Science:
- Immunology
- Biotechnology
- Molecular Biology
Background:
- Antigen-specific T cell migration is vital for immune responses against cancer and infections.
- Current imaging technologies struggle to detect the low numbers of T cells found in tumors.
- Sensitive detection methods are needed to track T cell infiltration and efficacy in vivo.
Purpose of the Study:
- To engineer an enhanced firefly luciferase (ffLuc) vector for sensitive bioluminescence imaging of T cells.
- To enable the tracking of adoptively transferred T cells in mouse models of cancer and vaccination.
- To improve the quantitation of infiltrating lymphocytes in tumors.
Main Methods:
- Codon optimization, removal of cryptic splice sites, and retroviral modification were used to create the enhanced ffLuc vector.
- T cells were engineered to express the enhanced ffLuc construct, allowing for increased light output.
- Bioluminescence imaging was performed on mouse models to track T cell migration and infiltration.
Main Results:
- Engineered T cells expressing the enhanced ffLuc vector produced >100 times more light than standard ffLuc.
- The enhanced ffLuc system enabled detection of as few as three implanted T cells.
- Tracking of <3 x 10(4) adoptively transferred T cells infiltrating tumors and vaccination sites was achieved.
- Light penetration through deep tissues like the liver and spleen was observed.
- Infiltrating lymphocytes constituting <0.3% of tumor cellularity were enumerated, surpassing standard methods.
Conclusions:
- The enhanced ffLuc vector significantly improves bioluminescence imaging sensitivity for T cells.
- This technology facilitates the tracking and quantitation of T cell migration in vivo.
- The findings offer a powerful tool for evaluating T cell-based immunotherapies and understanding immune responses.
