This protocol outlines the production of immunotherapeutic rhesus T-cells that express two proteins:an antiviral chimeric antigen receptor and a chemokine receptor CXCR5 that target cells to lymphoid follicles. This relatively rapid nine-day procedure produces T-cells with less differentiated central memory phenotype with the potential for long-term persistent after infusion into animals. Adapting this method to human cells will allow production of immunotherapeutic T-cells with the potential to induce a durable remission to HIV without administration of antiretroviral drugs.
This method can be used broadly to produce rhesus T-cells expressing other proteins of interest. And with minor modifications, it can be used to generate transduced T-cells in other species including humans. The success of the transduction is dependent upon healthy stimulated PBMCs.
Care must be taken in collection, transport, and storage. Prior to transduction, it's important to visually assess stimulation of the cells. Start by thawing primary rhesus PBMCs.
Incubate the cells in a 37 degrees Celsius water bath with gentle agitation until only a small amount of ice remains. Gently pipette the cells into a conical tube. Then rinse the vial with one milliliter of basic medium and slowly add it to the cells.
Slowly add an additional nine milliliters of warm basic medium to the cells. Place the tube in the aerosol-resistant canister and centrifuge it at 600 times g for five minutes at 22 degrees Celsius. Aspirate the supernatant and resuspend the pellet in a small amount of growth medium.
To count the cells, add 10 microliters of cells to 10 microliters of trypan blue. Load the chamber slide and insert it into the counter. Push the capture button to count the cells.
Calculate the total number of cells by multiplying cell concentration by volume and dilute them to two million cells per milliliter in growth medium. Add anti-CD28 for a final concentration of five micrograms per milliliter. Then pipette three to six million cells per well into anti-CD3 coated plates.
Incubate the plates for two days at 37 degrees Celsius and 5%carbon dioxide. Warm a centrifuge to 32 degrees Celsius by running it at 2, 000 times g for about 30 minutes. Meanwhile, warm both serum-free and growth media in a 37 degrees Celsius water bath and thaw the virus with gentle swirling in a 37 degrees Celsius water bath.
Then place it on ice. Dilute the virus to a predetermined optimal multiplicity of infection in serum-free medium. Add two milliliters of the diluted retrovirus to each well of the fibronectin-coated plate using media alone for the negative control cells.
Place the plates in the prewarmed centrifuge in microplate carriers and centrifuge them at 2, 000 times g for two hours. If using immediately, aspirate the virus preparation from the wells and add two milliliters of growth medium. Check the stimulated PBMCs under an inverted microscope.
The cells should be round, bright, and they should refract light. They should also have a clumped appearance indicating stimulation. Collect the target cells and transfer them to a 50 milliliter conical tube.
Rinse each well once with one milliliter of growth medium and add that to the tube. Then count the cells as previously demonstrated. Pellet the cells by centrifuging them at 600 times g for five minutes at 32 degrees Celsius.
Aspirate the media from the pellet and resuspend the cells in growth medium at a concentration of 1.5 million cells per milliliter. Add one milliliter of the cell suspension to each virus-coated well and add MAC transduce cells to fibronectin-coated wells that did not receive virus. Centrifuge the plates at 1, 000 times g for 10 minutes at 32 degrees Celsius and incubate them at 37 degrees Celsius and 5%carbon dioxide for 48 hours.
Work with rhesus cells and gamma retroviruses requires the use of personal protective equipment such as lab coats and gloves and work must be carried out in a biosafety cabinet. All pipettes and solutions must be decontaminated. Pipette the contents of each well up and down with a five milliliter pipette to resuspend the cells and transfer them to a 50 milliliter conical tube.
Add one milliliter of growth medium to each well and pipette up and down to remove adherent cells. Then count the cells as previously described. Centrifuge the cells at 600 times g for 10 minutes at 25 degrees Celsius.
Then aspirate the media and resuspend the cells in expansion media to a concentration of one million cells per milliliter. Seed five milliliters of cells into each gas permeable well of a six-well plate and carefully layer an additional 25 milliliters of expansion media per well. Then incubate the cells undisturbed at 37 degrees Celsius and 5%carbon dioxide for four days.
To collect the cells from the wells, remove and discard 20 milliliters of the media taking care not to disturb the cells. Pipette the remaining media up and down to dislodge the cells. Use a sterile transfer pipette to rinse each well with three milliliters of media and collect the residual cells.
Then count them to check for cell number and viability. This protocol was used for transduction and expansion of cells from six different animals. Gas permeable wells were seeded at a starting density of five million cells and after four days of growth, a median density of 55.6 million cells per well was achieved.
The viability of the cells monitored by trypan blue exclusion was maintained at 83 to 95%throughout the protocol. Flow cytometry was used to observe co-expression of the transduced genes. On day five, a median of 42.8%of the cells were transduced with both CD4-MBL CAR and CXCR5.
While on day nine, median expression was 47.6%with a single round of transduction. Flow cytometry was also used to monitor memory phenotype. Prior to transduction and expansion, naive central memory and effector memory populations were identified.
The naive population was lost with culturing and the cells were primarily central memory cells by day five and day nine. Success in the transduction is dependent upon the use of high-quality reagents, highly viable cells and titrated virus as well as consistently following the timing of the protocol. The cells can be used for functional assays including migration and viral suppression assays as well as for infusion into animals for preclinical studies to test the efficacy of the immunotherapy.
We have moved forward with preclinical testing of CAR and CXCR5 T-cells in animals and hope to soon test this immunotherapy in HIV-infected individuals.
