Expansion of Human Peripheral Blood γδ T Cells using Zoledronate

This video demonstrates the large scale expansion of human peripheral blood gamma delta T cells using zoledronate. First whole blood is subject to density gradient centrifugation to isolate peripheral blood mononuclear cells or P BMCs. The P BMCs are stimulated with zoledronate to cause monocytes to become antigen presenting cells, and also with IL two to stimulate gamma delta T cells.

The cells are incubated for up to 14 days in order to proliferate gamma delta T cells. In the culture analysis by flow cytometry demonstrates that the majority of harvested cells display CD three positive V gamma nine positive phenotype. The main advantage of this technique over existing method is that ate is already licensed for clinical applications at Zometa.

Therefore, a reliable reagent is easily available for the clinical grade gamma delta T-cell preparation, demonstrating the procedure will be makoto condo and amichi izumi cell culture. Technicians from my laboratory Begin this procedure by isolating peripheral blood mononuclear cell from whole blood collected in a BD vacutainer CPT cell preparation tube containing sodium heparin anticoagulant, and a fial high peak density fluid, plus a polyester gel barrier, which separates the two liquids. Centrifuge the sample at room temperature in a horizontal swinging bucket rotor for 20 minutes at 1800 times G.With the break off after centrifugation, the contents of the tube will be separated into five layers.

The top yellow layer consists of plasma. Below that, a whitish layer contains peripheral blood mononuclear cells and platelets, which sit on top of the lighter yellow density solution. A polyester gel layer in white separates this from the granular sites, which form a grayish layer that sit on top of the red blood cells at the bottom of the tube.

Using a two milliliter pipette, carefully collect a fraction of the plasma layer, leaving five to 10 millimeters of plasma above the interface and transfer it into a 15 milliliter conical tube. Place the tube at four degrees Celsius to use for culture. Next, use a two milliliter pipette to harvest the PBMC enriched fraction at the interface and transfer it to a 15 milliliter conical tube.

To wash the PBMC, add 10 milliliters of PBS and invert the tube five times. Then centrifuge the tube for five minutes at 400 times G.Repeat the washing steps twice. Then after reusing the cell palette in five milliliters of PBS, use a hemo cytometer to determine the cell number.

Typically, 1.3 times 10 to the six cells are recovered from one milliliter of whole blood. A fraction of pbmc containing one times 10 to the six cells is separated and kept on ice. For analysis by flow cytometry.

To expand the gamma delta T cells in the PBMC sample centrifuge, the cell suspensions in 15 milliliter conical tubes for five minutes at 400 times G at room temperature. Following the spin, discard the supernatants and resus suspend the cells in cell culture medium, supplemented with 1000 IU per milliliter IL two and five micromolar zoledronate at a density of one times 10 to the six cells per milliliter. Zoledronate inhibits faril pyrophosphate synthase in monocytes leading to the accumulation of isopentenyl pyrophosphate and the shift of monocytes to antigen presenting cells.

Next pipette. One milliliter of medium containing one times 10 to the six cells into each well of a 24 well plate cells should be at 0.5 times 10 to the six centimeter squared. So for larger cultures, scale accordingly to each well of a 24 well plate.

Add either the 100 microliters autologous plasma collected earlier, pooled human AB serum or FBS. Place the plates in a humidified 37 degrees Celsius 5%carbon dioxide every two to three days. Replace the medium with fresh serum supplemented medium containing human 1000 IU per milliliter of IL two.

When the cells reach a density of 0.5 to two times 10 to the six cells per milliliter, split them one to two into new wells or flasks. Observe the cells over the course of several days. Shown here are the cells on day zero.

The smaller cells are lymphocytes. The larger cells are monocytes. About 24 hours later, monocytes adhere to the bottom of the well.

After two days, some lymphocytes attach to monocytes by three days, gamma delta T cells begin to form clusters By five days, large clusters of cells can be seen. In contrast, no clusters or aggregates will be observed when gamma delta T cell growth is not adequate. When cluster formation is delayed.

As shown here, the growth of other cell types such as CD four positive or CD eight positive alpha beta T cells or NK cells dominate the growth of gamma delta T cells. On day 12 to 14 extensively pipette the cells with 10 milliliter pipette and harvest the cells into 50 milliliter conical tubes. The cells can now be analyzed by flow cytometry to determine the frequency, phenotype, and functions of gamma delta T cells.

To perform phenotypic analysis of pbmc or expanded gamma delta T cells begin by transferring 200 microliters samples containing two times 10 to the fifth cells to each of nine fluorescent activated cell sorting tubes. Add two milliliters of cold PBS and centrifuge for five minutes at 400 times G then resuspend the pellets into 50 microliters. Fax buffer, add five microliters of each of the required antibodies to the samples.

Incubate the samples on ice in the dark for 20 minutes. Following the incubation, add two milliliters, fax buffer to each sample, and then vortex centrifuge samples for five minutes at 400 times G at four degrees celsius. After the spin, carefully decant the supernatant, re suspend the cells in 300 microliters, fax, buffer, and vortex.

Then analyze the samples on a flow cytometer to evaluate the function of gamma delta T cells. Their capacity to produce interferon gamma is analyzed by intracellular interferon gamma staining. Resus suspend gamma delta T cells at two times 10 to the six cells per milliliter in RPM I 10 containing felden A at 20 micrograms per milliliter.

Felden A inhibits secretion of interferon gamma resulting in the accumulation of interferon gamma in the cell. Next, transfer 100 microliters of gamma delta T-cell suspension to each well of a 96 well plate containing 100 microliters of RPMI 10 only, or RPMI 10 with 20 nanograms per milliliter of four. Ball 12 ate 13 acetate plus two micrograms per milliliter of ion mycin or Z duddy cells.

Mix by pipetting up and down several times. Incubate for four hours in a 37 degree Celsius 5%carbon dioxide incubator. Then centrifuge the plate for five minutes at 400 times G at four degrees Celsius after resus, suspending the pellets in 200 microliters of cold PBS.

Transfer the samples to fax tubes. Then add four milliliters of cold PBS and centrifuge the tubes for five minutes at 400 times G at four degrees Celsius following the spin resuspend. The pellets in 50 microliters of fax buffer with five microliters.

FE conjugated anti TCRV gamma nine and 2.5 microliters PE SCI five conjugated anti CD three monoclonal antibody incubate protected from light for 15 minutes at room temperature. After the incubation, add 100 microliters of intra prep reagent one and incubate for 15 minutes. At room temperature, add four milliliters of PBS to each tube and centrifuge for five minutes at 400 times G at room temperature after centrifuging resus, suspend the pellet in 100 microliters of intra prep reagent two incubate for five minutes.

At room temperature, add five microliters of PE conjugated anti interferon gamma monoclonal antibody to the test tube. Incubate protected from light for 15 minutes. At room temperature, add four milliliters of PBS to each tube and centrifuge for five minutes at 400 times G.At room temperature, remove the supernatant by aspiration and resuspend the cell pellet.

In 0.5 milliliters of fax buffer. Analyze the cells by flow cytometer gate on CD three positive TCR v gamma nine positive cells and examine the expression of interferon gamma. Typical surface phenotype of gamma delta T cells is demonstrated as shown here.

Gamma delta T cells were identified by their expression of CD three and TCRV gamma nine on day zero. The percentage of CD three positive tcr RV gamma nine positive gamma delta T cells in PBMC was 1.6%The dominant populations of gamma delta T cells were CD 27 positive, CD 45, RA positive naive, or CD 27 positive, CD 45 RA negative central memory phenotypes. The expressions of CD 27 and CD 45 RA are used to determine their naive or memory phenotype.

CD 27 positive. CD 45. RA positive is naive.

CD 27 positive. CD 45 RA negative is central memory CD 27 negative. CD 45.

RA negative is effector memory. CD 27 negative. CD 45.

RA positive is terminally differentiated phenotype. The activation status of the cells was analyzed by NK G 2D or CD 69 as shown here. Gamma delta T cells express NK G 2D, but do not express CD 69.

After 14 days of culture, the frequency of gamma delta T cells increased to more than 93.8%of the cultured cells. In successful gamma delta T-cell cultures, they displayed CD 27 negative CD 45 RA negative effector memory phenotype. The cultured gamma delta T cells upregulated nkg 2D and CD 69 expression.

The intracellular interferon gamma staining demonstrated that gamma delta T cells produced interferon gamma in response to pma CIN treatment or Z dwy cells that accumulated IPP. After zoledronate treatment, 97%and 19.6%of gamma delta T cells produced interferon gamma in response to PMA CIN and Z ddy cell stimulation respectively. These results indicate that zoledronate can efficiently stimulate and expand functional gamma delta T cells.

The kinetics of gamma delta T-cell culture expanded by zoledronate and IL two, absolute number of cultured cells exponentially increased up to 12 to 14 days. Enrichment of gamma delta T cells was achieved relatively early. Almost 80%of cultured cells were gamma delta T cells By day seven, an absolute number of gamma delta T cells at the indicated time points demonstrate that approximately 2.2 times 10 to the eight gamma delta T cells can be obtained from one times 10 to the sixth PPMC containing 1.6 times 10 to the fourth gamma delta T cells After its development.

This technique paves the way for researcher in the field of cancer immunotherapy to explore clinical application in many different types of cancer. This culture method has been used in phase one clinical trials, evaluating the safety and feasibility of the donate expanded gamma delta T-cell transfer therapy in patient with cancer.