Murine Lymphocyte Labeling by ⁶⁴Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

The overall goal of this procedure is to stably label murine lymphocytes with a copper-modified radioactive monoclonal antibody to track lymphocyte temporal distribution and homing patterns in vivo by PET/CT in mouse models of inflammation or cancer. This method can help elucidate the sites of action and the key underlying biological principles of cell-based therapies, such as stem cell therapies in regenerative medicine or cancer immunotherapies. The main advantage of this technique is that it permits the imaging of cell migration over 48 hours with a high contrast and minimal detrimental effects on the cells.

This method can be easily transferred to any cell type of interest with specific membrane-bound receptors and corresponding monoclonal antibodies. Generally, individuals new to this method may struggle with the chelator conjugation and radiolabeling processes, resulting in a lower immunoreactive fraction and cell-specific activity. Begin by disinfecting a DO11.10 mouse with 70%ethanol.

Then fix the limbs with adhesive tape, and make a medial incision along the abdomen of the animal. Laterally pull both sides of the incision, separate the skin from the peritoneum, and locate the cervical, axial, brachial, and inguinal lymph nodes. Using blunt forceps, transfer the lymph nodes into a container of 1%FCS in PBS.

Then open the peritoneum, separate the spleen from the pancreas and connective tissue, and store the spleen with the lymph nodes. When all of the tissues have been collected, transfer the samples onto a 40-micron filter in a 15-milliliter conical tube, and use a syringe plunger to macerate the tissues. Then rinse the filter with 10 milliliters of FCS in PBS, and collect the single-cell suspension by centrifugation.

After lysing the red blood cells, resuspend the white blood cell pellet in fresh PBS plus FCS and CD4-positive MicroBeads according to the manufacturer's instructions. After 20 minutes at four degrees Celsius, wash the cells in up to 50 milliliters of fresh PBS plus FCS buffer, and resuspend the cells in the appropriate volume of PBS plus FCS for magnetic column elution of the CD4-negative cell populations. Load the cells onto a magnetic cell separation column, and collect the flow-through in a 15-milliliter conical tube.

At the end of the magnetic cell separation, use the plunger to flush the CD4-positive cell population into a new conical tube, and adjust the isolated CD4-positive T cell suspension concentration to one times 10 to the six cells per milliliter in fresh complete medium for four degrees Celsius storage. Collect the CD4-negative cells by centrifugation. Resuspend the pellet in anti-CD4, anti-CD8, mouse anti-rat monoclonal antibodies, and rabbit complement, and incubate for 45 minutes at 37 degrees Celsius.

After collecting the cells by centrifugation, resuspend the pellet in three milliliters of fresh complete medium, and irradiate the negatively selected antigen-presenting cells at 30 grays. Adjust the antigen-presenting cell concentration to five times 10 to the six cells per milliliter in fresh complete medium, and add 100 microliters of CD4-positive T cells and 100 microliters of antigen-presenting cells into the appropriate experimental wells of a 96-well flat bottom plate. Then add the appropriate stimuli to each well, and transfer the plate to a cell culture incubator.

For chicken ovalbumin-specific TH1 cell radiolabeling, first use a dose calibrator to draw 37 megabecquerels of the T cell-specific radioactive antibody of interest into a syringe without dead volume. Next, dispense the antibody into a reaction cup, and measure and subtract the remaining amount of reactivity in the syringe from the amount that was drawn. Then add one milliliter of saline to the cup to generate a 37-megabecquerel-per-milliliter solution.

Next, add two times 10 to the six OVA-TH1 cells in 0.5 milliliters of complete medium to each well of a 48-well plate, followed by 20 microliters of the radiolabeled antibody solution. After 30 minutes in a radiation-safe, 37-degrees Celsius, and 7.5%carbon dioxide cell culture incubator, pool the radiolabeled cells in a 50-milliliter conical tube for two washes in 10 milliliters of 37 degrees Celsius PBS. To measure the radiolabeled antibody uptake and efflux, immediately after the second wash, transfer one times 10 to the six monoclonal antibody-labeled OVA-TH1 cells in one milliliter of complete medium into each of 10 gamma counting tubes.

Pellet the cells by centrifugation, and transfer the supernatants into 10 new gamma counting tubes. Next, wash the cells in one milliliter of complete medium to remove any unbound radiolabeled monoclonal antibody, and transfer the supernatants into 10 new gamma counting tubes. Then add one milliliter of fresh complete medium to the cells, and measure the initial uptake values in a gamma counter.

For in vivo PET/CT imaging, immediately after radiolabeling, adjust the OVA-TH1 cells to a five times 10 to the seven cells per milliliter concentration in PBS, and draw 200 microliters of cells into a one-milliliter syringe equipped with a 30-gauge needle. Inject the cells intraperitoneally into an ova delayed-type hypersensitivity reaction diseased animal between the fourth and fifth nipple. Then, to facilitate co-registration of the PET and CT images during the image analysis, fix glass capillaries containing radiolabeled antibody solution under a small animal bed.

When the appropriate level of sedation is reached, apply ointment to the animal's eyes and use cotton swabs and surgical tape to immobilize the mouse on the bed. Mouse the mouse bed to the PET scanner, and center the field of view with a focus on the lungs to acquire a 20-minute static PET scan with an energy window of 350 to 650 kiloelectron volts. Transfer the mouse bed to the CT scanner.

Via scout view, center the field of view on the lungs. Acquire a planar CT image via 360 projections during a 360-degree rotation in the step-and-shoot mode with an exposition time of 350 milliseconds and a binning factor of four. The applied radioactive dose of 0.7 megabecquerels reduces the TH1 cell viability by only 8%while higher doses demonstrate an even more pronounced effect.

Compared to radiolabeling with copper-PTSM however, no significant advantage for the radiolabeled antibody is observed. T cell-specific radioactive antibody-labeled TH1 cells demonstrate no loss in functionality as evidences by interferon gamma secretion, a decreased efflux of radioactivity, and a reduced induction of apoptosis compared to copper-PTSM-labeled TH1 cells. Here, representative high-resolution static PET and anatomical CT images after adoptive transfer of the monoclonal antibody radiolabeled cells are shown, with the images fused in the coronal, axial, and sagittal view with the help of glass capillaries placed as just demonstrated.

Volumes of interest were drawn on the pulmonary and perithymic lymph nodes on the decay-corrected and normalized PET images to calculate the percentage of injected dose per cubic centimeter. OVA-TH1 cell migration tracking to the pulmonary and perithymic lymph nodes as ova presentation sites in an airway delayed-type hypersensitivity reaction reveals that the in vivo PET signals derived from the monoclonal antibody radiolabeled OVA-TH1 cells are considerably higher than the signals from the copper-PTSM-labeled cells. Further, the OVA-TH1 cell uptake values were significantly increased in the delayed-type hypersensitivity reaction diseased animals compared to their untreated control littermates.

Once mastered, this technique can be used to study the specific roles and sites of action of immune cells in animal models of human disease. While attempting this procedure, it's important to remember that each cell type might react differently to radioactivity and to adjust the amount of radioactivity used for the labeling accordingly. After watching this video, you should have a good understanding of how to isolate and culture antigen-specific T cells, label these cells with a radioactive antibody, and image their distribution and homing kinetics with PET/CT.

Don't forget that working with radioactivity can be extremely hazardous and that precautions such as using proper lead shielding, minimizing exposure times, and maximizing the distance to the radioactive source should always be taken while performing this procedure.