Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer

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 ؋ 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.bioluminescence ͉ immunology ͉ molecular biology

Disulfiram when Combined with Copper Enhances the Therapeutic Effects of Temozolomide for the Treatment of Glioblastoma

“…Stable BTIC lines expressing enhanced firefly luciferase (effLuc) and eGFP were generated using a self-inactivating lentiviral vector system as described previously (28,29). BT73R and BT206R are temozolomide-resistant BTIC lines generated from parental BT73 and BT206.…”

Optical Imaging for Stem Cell Differentiation to Neuronal Lineage

“…To overcome this, a wide variety of signal amplification approaches has been proposed to enhance in vivo detection limits. Rabinovich and colleagues [23] developed an improved optical reporter vector system containing an engineered luciferase enzyme obtained through codon optimization techniques, such as insertion of Woodchuck hepatitis virus in the viral backbone to enhance exportation of mRNA to the cytoplasm and addition of a (G 3 S) 2 linker located at the 3′ portion of the internal ribosomal entry site sequence to avoid abnormal RNA folding. In this result, an increase of more than 100-fold in the luciferase activity was found in T cells infected with an enhanced firefly luciferase retrovirus (v-effLuc), compared with general firefly luciferase retroviral vector (v-ffLuc)-infected T cells.…”

In Vivo Cell Tracking with Bioluminescence Imaging

“…BLI could track migration to sites of inflammation and measure the life span of adoptively transferred T cells for tumor immunotherapy [79]. Rabinovich et al [80] developed an enhanced version of FLuc that can be transduced into primary mouse T cells and could provide sufficient sensitivity in tracking <10,000 T cells within living mice. Edinger et al [81] demonstrated homing of cytokine induced killer cells derived from splenocytes, which were retrovirally transduced with both green fluorescent protein and FLuc, to sites of tumor growth followed by tumor eradication.…”