There is no clinically validated chimeric antigen receptor T-cell imaging platform to date. The sodium-iodide symporter represents a sensitive and clinically relevant reporter to image Car T-cells by PET scan. TLP PET imaging of NIS CAR T-cell allows investigators to non-invasively track infused cells in vivo and has a potential to predict efficacy and toxicity of CAR T-cell therapy.
Efficient imaging of CAR T-cells enables the evaluation of T-cell trafficking and expansion and allows the development of strategies to overcome the limitations. The method described in this protocol are simple and can be applied to other CAR constructs beyond the ones shown in this study. Start with the isolation of peripheral blood mononuclear cells or PBMCs from the blood sample using the standard density gradient technique.
To do so, gently add 15 milliliters of density gradient medium to a 50 milliliter density gradient separation tube avoiding bubble formation. To avoid cell trapping, dilute the blood sample with PBS containing 2%FBS at a one-to-one volume ratio and gently transfer the diluted blood on top of the density gradient medium without breaking the interface between the two. Then centrifuge the separation tube at 1, 200 times G for 10 minutes at room temperature.
Collect the supernatant into a fresh 50 milliliter conical tube, wash the cells with PBS containing 2%FBS by filling the tube up to 50 milliliter mark, and centrifuging the tube at 300 times G for eight minutes at room temperature. Aspirate the supernatant before resuspending the pelleted cells in 50 milliliters of PBS containing 2%FBS. Resuspend the pelleted PBMCs in PBS containing 2%FBS to a concentration of 50 times 10 to the sixth cells per milliliter.
Repeat the wash in a fresh 50 milliliter tube. Count the number of cells and then centrifuge the cell suspension at 300 times G for eight minutes at room temperature. For T-cell isolation, transfer isolated PBMCs to a 14 milliliter polystyrene round bottom tube.
Then use a negative selection magnetic bead kit to perform T-cell isolation by placing the PBMCs in negative selection antibody cocktail in a fully automated cell separator. After T-cell isolation, count the cells and culture the cells in T-cell expansion medium or TCM at a concentration of two times 10 to the sixth cells per milliliter. Next, mix the vial containing the anti-CD3/CD28 beads by swirling and pipette out the required volume of beads into a sterile 1.5 milliliter microcentrifuge tube.
For washing, resuspend the beads in one milliliter of TCM before placing tube on a magnet for one minute and aspirate the supernatant. After removing the tube from the magnet, resuspend the washed beads in one milliliter of TCM and repeat the wash twice. Resuspend the washed beads in one milliliter of TCM to transfer them to the T-cells culture, then dilute the T-cell bead suspension with TCM to a concentration of 1.0 times 10 to the sixth cells per milliliter before transferring it to a tissue culture treated six-well plate.
Incubate the well plate at 37 degrees Celsius and 5%carbon dioxide. To progress with the transduction of lentiviruses, thaw the frozen lentiviruses in coating chimeric antigen receptors or sodium-iodide symporter at four degrees Celsius. After 24 to 48 hours of T-cell stimulation, mix the T-cells to break up the clusters and add the freshly thawed lentivirus to the cells at a multiplicity of infection of five.
Incubate the transduced T-cells at 37 degrees Celsius and 5%carbon dioxide. On days three, four, and five of incubation, count the transduced T-cells and adjust the cell concentration to 1.0 times 10 to the sixth cells per milliliter by adding fresh pre-warmed TCM. For NIS transduced T-cells carrying the puromycin resistance gene, treat the cells with one microgram per milliliter of puromycin dihydrochloride on days three, four, and five.
On day six, break up the T-cell clusters and remove the anti-CD3/CD28 beads from the transduced T-cells by placing the plate on a magnet for one minute. Then place the collected cells back in the culture at a concentration of 1.0 times 10 to the sixth cells per milliliter. Wash an aliquot of the T-cell culture containing 50, 000 cells with flow buffer and resuspend the cells with 50 microliters of flow buffer.
For detecting CAR expression, stain the T-cells with one microliter of goat anti-mouse antibody. To exclude the dead cells, add 0.3 microliters of LIVE/DEAD Aqua. Incubate the stained cells in the dark at room temperature.
After 15 minutes, wash the cells with 150 microliters of flow buffer at 650 times G for three minutes at four degrees Celsius. To fix and permeabilize the stained cells, incubate the cells with 100 microliters of fixation medium for 20 minutes at four degrees Celsius. Then wash the cells twice with 100 microliters of a buffer containing a cell permeabilizing agent such as saponin and centrifuge.
After washing, resuspend the permeabilized cells in 50 microliters of a permeabilizing buffer. Then add 0.3 nanograms of anti-human ETNL NIS antibody and incubate at four degrees Celsius. After one hour of incubation, wash the cells by adding 150 microliters of flow buffer and centrifuge as demonstrated.
Then incubate the cells with 50 microliters of flow buffer containing 2.5 microliters of anti-rabbit secondary antibody for 30 minutes at four degrees Celsius. After washing the T-cells as demonstrated, resuspend the cells in 200 microliters of flow buffer to perform flow cytometry. Then determine the transduction efficiency of the cells.
On day eight, count and spin down the T-cells at 300 times G for eight minutes at four degrees Celsius before resuspending the cell pellet with a freezing medium at a concentration of 10 times 10 to the sixth cells per milliliter. Transfer one milliliter of the cell suspension to each labeled cryo vial to store at minus 80 degrees Celsius freezer for 48 hours. After 48 hours, transfer the T-cells to liquid nitrogen.
Prepare the mice injected with NIS positive BCMA CAR T-cells for the PET/CT imaging by weighing the mice and removing metal ear tags. Then inject 9.25 megabecquerel of freshly prepared F18 tetrafluoroborate into the mouse intravenously via tail vein injection. After 45 minutes of uptake period, proceed to acquire static PET images of an anesthetized mouse for 15 minutes, followed by CT image acquisition for five minutes with 360 degrees rotation and 180 projections.
In this representative analysis, co-transduction of two viruses into T-cells generated NIS positive BCMA CAR T-cells where over 90%of the cells were NIS positive. The analysis of T-cell growth kinetics from zero to eight days revealed that incorporation of BCMA CAR and NIS did not significantly impact T-cell expansion when compared to the untransduced cells. In the flow cytometric analysis of OPM-2, cells transduced with lentivirus represented the GFP expression upon treatment with puromycin.
The mice receiving BCMA positive luciferase positive OPM-2 cells were subjected to bioluminescence imaging to confirm the engraftment of OPM-2 cells. PET imaging of the F18 tetrafluoroborate administered mouse revealed NIS positive BCMA CAR T-cell distribution in the various organs and bone marrow. Additionally, the bone marrow cells harvested from the mouse displayed the engraftment of OPM-2 cells and delivery of the NIS positive BCMA CAR T-cells to the bone marrow.
For high-quality imaging of infused CAR T-cells, it is important to follow the steps of the NIS CAR T-cell generation protocol and to confirm the presence of dual NIS and CAR positive fractions.
