This method allows users to easily track T cells in vivo to evaluate the kinetics of homing and to monitor T cell persistence. The main advantage of this technique is that it can be repeated at multiple time points on the same animal without requiring sacrifice in flow cytometry or immunohistochemistry. This technique could be modified for tracking other types of immune cells as well.
To culture 293T cells, prepare one liter of DMEM by adding 110 milliliters of heat-inactivated fetal bovine serum and 11 milliliters of penicillin streptomycin. Thaw five times 10 to the sixth 293T cells and transfer them into a T175 flask containing DMEM. Split the cells one to 10 every three days for six days before the cells become more than 90%confluent.
For transfection, transfer 1.5 milliliters of media to two 50 milliliter tubes using a pipette. To one tube, add 10 micrograms of VSVG plasmid, 20 micrograms of gag pol, and 20 micrograms of click beetle red TDtomato or CBRTDR luciferase plasmid and mix. Add 100 microliters of DNA in vitro transfection reagent to another tube and mix.
Incubate both tubes at room temperature for five minutes. Transfer the media containing the DNA in vitro transfection reagent dropwise to the tube containing the DNA plasmids and mix gently with a pipette. Incubate at room temperature for 20 minutes.
During incubation, detach the 293T cells by adding five to 10 milliliters of 0.05%trypsin. Once the cells have detached, add 10 milliliters of media and centrifuge at 800 G for five minutes. Resuspend the cells in 1.5 milliliters of media.
Add the media containing the DNA in vitro transfection reagent and the DNA plasmids to the 293T cells and incubate at 37 degrees Celsius for 30 minutes. After incubation, transfer the suspension to a T175 flask. Add 18 milliliters of media and incubate at 37 degrees Celsius overnight.
The next day, carefully aspirate the media with a glass pipette without detaching the cells and replace it with 18 milliliters of fresh media. Again, incubate overnight at 37 degrees Celsius. The following day, remove the viral supernatant using a pipette put on ice and centrifuge at 800 G for five minutes to pellet the cells and debris.
Filter the viral supernatant using a 0.22 micron filter and immediately place it on ice. For in vitro activation of human T cells, add 10 milliliters of PBS containing 0.1%bovine serum albumin or BSA and two millimolar EDTA to a sterile 15 milliliter tube. Then to wash the beads, add beads into a tube and place the tube in a magnetic rack.
After two minutes, remove the PBS from the tube. After preparing DMEM, add washed beads and interleukin-2 or IL-2 to a final concentration of 30 international units per milliliter. Count the T cells using a hemo cytometer and Trypan blue stain.
After counting, add desired number of T cells to the media and gently invert the tube to mix. Plate 2.5 milliliters of media containing two million T cells, beads, and IL-2 in each well of a 24-well tissue culture plate. Culture at 37 degrees Celsius in humidified 5%carbon dioxide.
Examine the T cells under a 20X objective every day for three days to determine when to transduce with luciferase. Next, to prepare a RetroNectin plate, add two milliliters of 20 micrograms per milliliter RetroNectin in PBS to a well of an untreated six-well plate and incubate for two hours at room temperature. Aspirate the RetroNectin without scratching the bottom of the plate and add three milliliters of 2%BSA in PBS per well in the six-well plate.
Incubate for 30 minutes at room temperature. Next, for spinoculation, aspirate the BSA and wash the wells once with three milliliters of PBS. Continue or replace with fresh PBS and leave the plate covered at four degrees Celsius overnight.
The next day, add two milliliters of CBRTDR luciferase viral supernatant per well and centrifuge at 1, 240 G for 90 minutes at 32 degrees Celsius. For transduction, after counting the T cells, aspirate the viral supernatant and plate two million T cells per well in six milliliters of DMEM containing 30 international units per milliliter of human IL-2. Examine the T cells for two days.
When the cells are nearly confluent, gently wash them off the bottom of the plate using a pipette and transfer each well to a separate T75 flask. Add nine milliliters of media for a total of 15 milliliter volume per flask. The next day, add 15 milliliters of media and confirm successful transduction by performing flow cytometry.
After anesthetizing the mouse, using a 26 gauge needle, inject 20 million T cells in 100 microliters of media retroorbitally. Administer 1, 000 units of recombinant IL-2 subcutaneously to support T cell survival in vivo. Then administer the bispecific antibody retroorbitally or intraperitoneally.
For in vivo imaging, administer 100 microliters of three milligrams of D-luciferin by retroorbital injection with a 26 gauge needle in an anesthetized mouse. To capture images, place the mouse in the light tight chamber of the imager such that the flank bearing the xenograft is facing up toward the camera. Using the acquisition control panel, select luminescent, photograph, and overlay.
Set the exposure time to auto, the binning to medium, and f-stop to one and acquire images. After the first image, set the exposure time to match that automatically calculated for the first image so that subsequent images may be compared directly. Take another set of images with the mouse supine to evaluate T cell presence in the lungs.
Armed T cells trafficked to the tumor faster than unarmed T cells, leaving the lungs two days sooner. Quantitation of T cell infiltration into tumors over time was measured by bioluminescence and expressed as total flocks or radiance per pixel integrated over the tumor contour. T cell trafficking could be monitored for 28 days post-injection of the luciferase-transduced T cells, allowing for tracking T cell homing and persistence throughout treatment.
T cell trafficking into HER2 positive human breast cancer patient-derived xenografts in DKO mice is shown. Treatment with the HER2 bispecific antibody with an unsilenced FC had no effect on tumor growth compared to a controlled bispecific antibody. In contrast, treatment with HER2 bispecific antibody containing N297A mutation alone or in combination with a K322A mutation was able to halt tumor growth completely.
For groups with the silencing mutations, the T cell luminescence signal reached a higher peak on day three post-injection and persisted at a higher level compared to the controlled bispecific antibody and unsilenced HER2 bispecific antibody. Mice bearing neuroblastoma patient-derived xenografts demonstrated depletion of Ly6C positive macrophages significantly increased T cell homing to tumors, resulting in decreased tumor growth and prolonged survival. The effect was more pronounced in osteosarcoma patient-derived xenografts.
Among bispecific antibodies targeting the tumor antigen STEAP1, the IGGL SCFV format resulted in significantly more T cell infiltration on day six after the first dose of T cells which persisted for the duration of treatment. Such observations were not predicted by in vitro cytotoxicity assays. After tracking T cells in vivo, users can still perform immunohistochemistry to determine more specifically where the T cells are located within the tumor or other normal tissues.
