High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

This protocol allows you to generate a large number of functional antigen-specific T cells which are a desired safe approach in treating autoimmune diseases. The main advantage of this technique is that it provides you with a highly efficient transduction process and a large number of functional T cells. This technique can be extended to treat type 1 diabetes and other autoimmune diseases in which antigens are known.

Furthermore, CAR T cells may be a promising treatment for certain types of cancer. This method can be used to generate immune cells expressing other CARs. It is important to have good accepted technique, read the entire protocol and plan the whole experiment in advance.

This two week long protocol includes mini-steps and success is dependent on proper performance of each step. Visual demonstrations of this method is critical for learners to follow correctly. Prepare Phoenix cells for transfection as described in the manuscript.

It is good practice to mark the side wall where medium will be added and removed to minimize blowing off cells. On day zero aspirate the supernatant from the cells and wash them with five milliliters of PBS. Carefully add seven milliliters of reduced serum medium dropwise to the side wall of the plate and transfer the cells back to the incubator.

Add 1.5 milliliters of reduced serum medium to two 14 millimeter round bottom polypropylene tubes. Add 40 micrometers of transfection reagent to one of the tubes and 15 micrograms of antibody redirected CAR plasmid along with five micrograms of packaging plasmid to the other. Incubate the tubes at room temperature for five minutes and then add transfection reagent to the plasmid tube.

Mix by gently pipetting the solution up and down three times and incubate it at room temperature for at least 20 minutes. Add three milliliters of the mixture to the Phoenix cells and place them in a tissue culture incubator. After four to five hours add one milliliter of FCS to the cells and culture them overnight at 37 degrees Celsius.

On the next day, remove the supernatant from the cells and add four milliliters of fresh prewarmed culture medium. Harvest the virus on day two by collecting the virus containing medium from the Phoenix cells and filtering it to remove residual cell debris. Add recombinant human IL-2 stock to the virus for a final concentration of 200 international units per milliliter and use it immediately for transduction.

Add four milliliters of fresh medium to the Phoenix cells and place them in the incubator overnight. Repeat virus collection on the next day for a second transduction and discard the cells. One day prior to seeding murine CD8 T cells, add one milliliter of a mixture of anti-mouse CD3 and CD28 antibodies to each well of a 24 well plate and incubate the plate overnight at four degree Celsius.

On day zero, harvest mouse spleens and put them on a cell strainer soaking in 10 milliliters of PBS in a cell culture dish on ice. In a cell culture hood, cut each spleen into three to five pieces and use a sterile plunger of a syringe to press the tissue through the wire mesh. Gently remove red blood cells by resuspending splenocytes in one to four diluted red cell lysis buffer and incubating them at room temperature for five minutes then dilute 10 microliters of the cell suspension with Trypan Blue for cell counting.

And pellet the rest of the cells by centrifuging at 350 times g for seven minutes. Use a mouse CD8 T cell isolation kit to enrich CD8 T cells and suspend the cell pellets in 400 microliters of buffer with 100 microliters of biotin antibody cocktail per one times 10 to the eighth cells. Mix the cell suspension well and incubate it for five minutes at four degrees Celsius to allow for antibody binding.

Then add 300 microliters of labeling buffer and 200 microliters of Anti-Biotin MicroBeads per one times 10 to the eighth cells. Mix well and incubate for another 10 minutes at four degrees Celsius. Meanwhile, set up a separation column on the separator and wash it with three milliliters of labeling buffer.

Put a 40 micrometer cell strainer on the top of a column and pass one milliliter of the bead and cell mixture through the strainer into the separation column. Collect the flow through into a prechilled 15 milliliter tube and wash the column according to the manufacturer’s directions, collecting effluent into the same tube. Count the cells and collect them by centrifugation at 350 times g for five minutes.

Wash them with two milliliters of T cell medium and centrifuge again. Then resuspend them in prewarmed T cell medium at a concentration of 250, 000 to 500, 000 cells per milliliter. Wash the previously prepared CD3 and CD28 antibody coated plates with one milliliter of sterile PBS three times and add two milliliters of the cell suspension to each well.

Use a swirling motion to dispense cells evenly. As a control, plate the same number of CD8 T cells into a single non-coated well of the plate and incubate the cells at 37 degrees Celsius in a 10%carbon dioxide gas tank incubator for 48 hours. On day one, prepare human fibronectin fragment coated plates by adding 0.5 milliliters of fibronectin to the wells of a 24-well plate and incubating it over night at four degrees Celsius.

On the next day, remove the fibronectin solution and add one milliliter of 2%BSA and PBS per well. Incubate the plate at room temperature for 30 minutes and wash the treated wells with one milliliter of sterile PBS. The plate is ready to use after removing wash solution.

Count the activated CD8 T cells using Trypan Blue. Collect them by centrifugation. And resuspend them at five million viable cells per milliliter for transduction.

Add 100 microliters of the activated CD8 cell suspension to each well of the fibronectin coated plate. Then add 1.5 to two milliliters of the previously prepared virus containing medium and mix using a swirling motion to evenly dispensed the cells. Seal the plate in a Ziploc bag and centrifuge it at 2, 000 times g for 90 minutes at 37 degrees Celsius.

Remove the plate from the centrifuge and take it to a biological safety cabinet. Carefully remove the plastic bag and ensure that the outside of the plate is not contaminated with medium. Transfer the plate to a 37 degrees Celsius carbon dioxide incubator.

After four hours, remove one milliliter of medium from each well, replace it with one milliliter of prewarmed complete T cell medium and put the plate back in the incubator. The critical aspects of this protocol are a high titer virus, healthy activated T cells, and the appropriate transduction method. Cells were co-stained with PE size seven conjugated anti-CD8, AF647 conjugated anti-CD3, and BV 421 conjugated anti-CD28 for flow cytometric analysis.

Approximately, 70%of the CD8 T cells co-expressed GFP which indicates CAR expression. They also co-expressed CD28 and CD3. Importantly, all of the test GFP positive cells also co-stained with I-Ag7 insulin BR3 tetramers which indicates expression of CAR on cell surface but not with the control tetramer.

Furthermore, the sorted test and control CAR T cells each secreted high levels of interferon gamma only after coculture with target cells expressing their cognate ligands which confirms that the transduced cells have a CD8 effector T cell phenotype directed toward the target of the parent antibodies. Transduction of virus producer cells, isolation of primary CD8 T cells, and activation of these T cells are the most important steps of this experiment. Having healthy activated T cells and transduction of these T cells with appropriate reagents ensures favorable results.

This method can be used to transduce other T cells but it must be customized for the specific T cell type. This protocol paves the way for the prevention of type 1 diabetes with antigen-specific chimeric antigen receptor T cell therapy.