Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Our lab designs artificial immune receptors, like chimeric antigen receptors, and studies how they affect regulatory T cell biology. By inventing new DNA sequences and using humanized mouse models of disease, we aim to create engineered immune cell therapies for autoimmune disease, transplant rejection, cancer, and aging. When designing chimeric antigen receptors for regulatory T cells, it's crucial to consider their strength.

High-affinity CAR Tregs behave more like effector T cells, producing increased inflammatory cytokines and demonstrating greater killing activity. Our ongoing research indicates that reducing CAR affinity leads to improved cytokine profiles and function in CAR Tregs. CAR Treg field is nascent and lacks standardization.

Our protocol introduces a robust, universal approach for generating and testing CAR Tregs. This enhances reproducibility and speeds up innovation while also guiding labs new to CAR Treg research, especially given the challenges of Treg scarcity and specialized requirements. We study the mechanisms regulatory T cells use to maintain immune balance and promote tissue healing.

Our research includes understanding CAR Treg signaling and function, as well as exploring new disease contexts where CAR Treg therapy may offer unique benefits compared to current strategies. To begin, transfer the contents of the leukopak into a 50-milliliter conical tube. Add an equal volume of DPBS with 2%fetal bovine serum and gently mix with a pipette.

Spin the tube at 300g for 10 minutes at room temperature. Once the supernatant is aspirated, reconstitute the cell pellet in two milliliters of DPBS with 2%fetal bovine serum. Then, add eight milliliters of ammonium chloride solution to the cell suspension and mix by gentle inversion.

With the break off, spin the washed cells at 150g for 10 minutes at room temperature. After aspirating the supernatant, resuspend the cell pellet in 30 milliliters of DPBS with 2%fetal bovine serum. Then, spin down 10 to the power of eight to 10 to the power of nine PBMCs at 500g for five minutes at room temperature.

And resuspend in cell separation buffer at a concentration of five times 10 to the power of seven cells per milliliter. For fluorescence-assisted cell sorting of regulatory T cells, spin CD4 positive cells at 500g for five minutes. Then, reconstitute cells in 200 microliters of DPBS.

For each one million cells, add one microliter of anti-human CD4 FITC, one microliter of anti-human CD25 APC, and one microliter of anti-human CD127 PE.After gently vortexing the tube, place it in a four-degree Celsius refrigerator for 30 minutes. Once the cells are washed with 10 milliliters of DPBS with 2%fetal bovine serum, spin them at 500g for five minutes and gently resuspend the stained cells at 1.5 times 10 to the power of seven cells per milliliter in DPBS with 2%fetal bovine serum. Next, pass the stained cell suspension through a 40-micrometer filter cap into fluorescence-assisted cell sorting tubes.

Prepare 15-milliliter collection tubes containing three milliliters of RPMI10 medium and place them on ice. Finally, sort CD4 positive, CD25 high, CD127 negative regulatory T cells, and CD4 positive, CD25 low, CD127 positive conventional T cells using fluorescence-assisted cell sorting. To begin, take regulatory T cells isolated from human blood 48 hours after activation and resuspend them.

After counting the cells, spin at 500g for five minutes at room temperature. Resuspend regulatory T cells in RPMI10 at 1.25 times 10 to the power of six cells per milliliter with 1, 000 international units per milliliter of interleukin-2. Now, add each lentivirus aliquot to 2.5 times 10 to the power of five regulatory T cells in 200 microliters in a microcentrifuge tube.

Spinoculate at 1, 000g for one hour at 32 degrees Celsius. Move each 200-microliter reaction to a 24-well plate. Incubate the plate with the transduced regulatory T cells in a tissue culture incubator overnight.

Top up each well to two milliliters with RPMI10 medium with the final interleukin-2 concentration being 1, 000 international units per milliliter. Assess gene modification efficiency using flow cytometry, as shown here. To begin, take resuspended regulatory T cells with anti-CD3, CD28 beads in a 15-milliliter conical tube 48 hours after activation.

Incubate the cell suspension in a magnet for three minutes. While in the magnet, transfer the cells in the medium via pipette to a new tube. After bead removal, allow the debeaded regulatory T cells to rest in RPMI10 for two hours.

Then, spin regulatory T cells at 500g for five minutes. Once the supernatant is decanted, resuspend the cells in pre-warmed reduced serum medium at four times 10 to the power of six cells per milliliter. Aliquot cells in 100 microliters in low-protein binding 1.5-milliliter centrifuge tubes.

Add chimeric antigen receptor adeno-associated virus at a multiplicity of infection of 20, 000 to each sample and resuspend. Then, incubate the reaction tubes in the tissue culture incubator for one hour. During the incubation, prepare CRISPR-Cas9 ribonucleoprotein complexes by adding 8.3 microliters of Cas9 protein to 2.5 microliters of single guide RNA, targeting the track gene locus.

After mixing the components thoroughly, incubate the ribonucleoprotein mixture for 15 minutes at 37 degrees Celsius in the tissue culture incubator. Next, fill a fresh electroporation tube with three milliliters of high-osmolarity electroporation buffer. Insert the filled electroporation tube into the pipette station of the electroporation system until a click is heard.

Set electroporation conditions to 2, 200 volts, 20 milliseconds, one pulse in the electroporation system. When the one-hour incubation with the adeno-associated virus is complete, spin the cells with the virus at 300g for five minutes at room temperature. Once the supernatant is aspirated, resuspend the cell pellet in 100 microliters of the cell resuspension buffer provided by the electroporation system per sample.

Then, add 10.8 microliters of ribonucleoprotein complex per sample and mix well with a pipette without creating bubbles. Now, insert a 100-microliter electroporation tip by pushing the pipette to its second stop to open the clamp. Position the pipette's top head into the electroporation tip until the clamp securely engages with the piston's mounting stem.

Gradually release the button while maintaining downward pressure on the pipette to ensure the tip fits snugly without any gaps. Then, press the pipette to the first stop and immerse the electroporation tip into the cell ribonucleoprotein mixture. Gently pull up the sample into the pipette without any bubbles.

Insert the pipette with the mounted electroporation tip containing the sample vertically into the E tube until a click sound is heard. After confirming the optimal settings for human regulatory T cells, press Start on the touchscreen to electroporate the cells. Wait for the touchscreen to display complete.

Gently remove the pipette and immediately transfer the sample into the prepared six-well plate containing 2.5 milliliters of pre-warmed antibiotic-free RPMI10 medium with interleukin-2 per well. After gently rocking the plate in linear motions, place it in the tissue culture incubator. The following day, 16 to 18 hours later, replace the media with antibiotic-containing media.

Count the electroporated regulatory T cells and culture at 10 to the power of six cells per milliliter with 1, 000 international units per milliliter of interleukin-2.