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A novel system for simultaneous in vivo tracking and biological assessment of leukemia cells and leukemia-reactive cytotoxic T-cells

A novel system for simultaneous in vivo tracking and biological assessment of leukemia cells and leukemia-reactive cytotoxic T-cells

Blood 102(11): 388a, November 16

Adoptive immunotherapy, using ex vivo generated and expanded CTLs can be effective in treating minimal residual disease. To determine the mechanisms by which adopive immunotherapy could reduce lethality to C1498 (acute myeloid leukemia (AML)) cells, a novel technique was developed to track, both leukemic blasts and adoptively transferred CTLs independently and simultaneously in vivo using whole body and confocal imaging techniques. Bone marrow-derived dendritic cells (DCs) were pulsed with AML lysates. Syngeneic splenocytes obtained from enhanced green fluorescent protein (eGFP) transgenic mice were co-cultured with DCs to prime AML reactive T-cells which were expanded by exposure to anti-CD3/CD28 mAb-coated magnetic microspheres. To track AML cells stable transfectants of C1498 expressing the red fluorescent protein DSRed2 were generated. Syngeneic mice were injected intravenously with a 100% lethal dose of C1498 (4X105/mouse). One day later ex vivo generated CTLs were injected intravenously. Migration, homing, and survival of adoptively transferred T-cells and AML cells were assessed. AML cells were widely metastatic and could be trafficked by whole-body imaging techniques. Three factors related to CTLs correlated with disease-free survival (DFS): 1) CTL dose: 5X106 versus 40X106 CTLs/mouse (p=0.04) with the higher dose resulting in up to a 50% DFS. The site of treatment failure was mainly the CNS where no CTLs could be identified. 2) Duration of ex vivo expansion (9 days versus 16 days, p=0.01). Short-term expanded CTLs could be found at high frequency in lymphoid organs for longer than 4 weeks after transfer whereas long term expanded CTLs were cleared from the system after 2 weeks. 3) L-selectin expression (L-selectin high fraction versus L-selectin low fraction, p=0.05) of CTLs fractionated prior to infusion. L-selectin low CTLs were only found in lymphoid organs at very low frequency and cleared rapidly from the organism, whereas the L-selectin high fraction homed readily to the lymph nodes, could be identified longer in non lymphoid organs and rescued up to 70% of mice. In sites of AML deposits, CTLs could be seen juxtaposed to AML cells which had a fragmented appearance. In summary, we have developed a new system in which CTLs and tumor cells can be simultaneously visualized in vivo and isolated based upon fluorescent properties. Such studies have provided insight as to the limitations of adoptive CTL transfer and should lead to new strategies to overcome tumor recurrence.

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Accession: 034312399

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