The overall goal of this procedure is to evaluate the NK cell cytotoxic response to HIV one infected cells. This is accomplished by first isolating and infecting CD four positive T cells from venous blood. Then natural killer cells are isolated from the same donor.
Next uninfected CD four positive T cells are isolated away from the infected cells. Finally, the infected cells and the NK cells are used as targets and effectors respectively in one of two assays to assess NK cells cytotoxicity. Ultimately, results can be obtained that show that NK cells can respond to HIV V one infected cells through upregulation of CD 1 0 7, a expression as determined by flow cytometric analysis or through chromium 51 release as detected by a gamma counter.
This method can answer key questions in the HIV one field, such as do NK cells respond to HIV V one infected cells. What receptor ligand interactions are important in NK T-cell interactions And do HIV one viral proteins modulate the NK cell response to H HIV V one infected cells After stimulating CD four positive T cells that have been isolated from human peripheral blood and stimulated by anti CD three, anti CD 28 coupled beads for 72 hours. Count the resulting T cell population by hemo cytometer and then split the culture into two groups at approximately two times 10 to the six T cells to a 15 milliliter conical tube for use as the uninfected control group and place the remainder of the CD four positive T-cell suspension into a 50 milliliter conical tube for the infection group.
Centrifuge both groups at 300 G at 22 degrees Celsius for 10 minutes and then aspirate the snat Resus. Suspend the CD four positive T cells from the 50 milliliter tube directly in culture fluids containing virons and add poly brain to a final concentration of 10 milligrams per milliliter. Then spin infect the cells at 1200 times G for two hours at 20 to 25 degrees Celsius.
Here the cells are being infected with DHIV three at a multiplicity of five. Upon completion of the spin infection, remove the supernatant and resuspend the infected cells at a density of three times 10 to the six per milliliter. In RPMI complete supplemented with 100 units per milliliter IL two.
Then culture, the uninfected cells and the newly infected cells for 72 hours in a six well plate. If a replication incompetent virus was used to infect the cells or for five to seven days if a replication competent virus was used, collect the supernat containing the infected CD four positive T-cell cultures and centrifuge the cells at 300 G for 10 minutes at 22 degrees Celsius with the break on aspirate the supernat and Reese. Suspend the pellet in two milliliters of PBS with 2%FBS and two millimolar EDTA and count the cells on a hemo cytometer using a CD four positive positive isolation kit.
Remove the CD four positive uninfected T cells. Follow the manufacturer's instructions for the isolation of the CD four positive T cells with the exception that A one anti CD four B per cell ratio is used. Then remove the dead cells from the purified virus harboring cell population using a dead cell removal kit.
According to the manufacturer's instructions, collect the living T-cell population and centrifuge the purified cells at 300 G for 10 minutes. With the break on, then remove the snat resus, suspend the pellet. In one milliliter of RPMI complete and count the cells Peripheral blood NK cells are first isolated from the same donor as were the CD four positive T cells.
After overnight culture count the NK cells with a hemo cytometer centrifuge them at 300 G for 10 minutes with the break on and then resus suspend the cells In RPMI complete adjusting the final concentration to 10 to the six cells per milliliter. Next, collect and centrifuge the purified, infected and control uninfected T-cell suspensions at 300 G for 10 minutes. With the break on, remove the SANE and resuspend the pellets.
In RPMI complete at two times 10 to the six cells per milliliter for a positive control about two times 10 to the six of NK lysis susceptible. K 5 62 cells are used in this video centrifuge the K 5 62 cells at 300 G for 10 minutes with the break on then remove the snat and resuspend The pellet. In RPMI complete at two times 10 to the six cells per milliliter.
After all the cell populations have been washed and resuspended at their experimental concentrations place NK cells together with either K 5 62 cells, uninfected T cells, or HIV infected T cells. In individual wells of a 96 well V bottom plate, only one replicate per target cell type will be used to measure nonspecific NK cell degranulation. Add 100 microliters of NK effector cells and 100 microliters of medium only into one.
Well add fluorochrome conjugated anti CD 1 0 7 A fits the antibody to each well in a 10 microliter volume and centrifuge the plate at 20 G for two minutes with the break on at 22 degrees Celsius. Then incubate the plate at 37 degrees Celsius and 5%carbon dioxide for four hours after incubation. Centrifuge the plate at 400 G for five minutes with the break on at 22 degrees Celsius and then remove the SANE to using a two milliliter aspirating pipette fitted with a P 200 tip.
Next, add 100 microliters of flow buffer to each well and then stain the cells with anti CD 3 14 20 Pacific blue antibodies and anti CD 56 A PC antibody on ice for 20 minutes. After the cells have been stained, add 200 microliters of flow buffer to each well and centrifuge the plate at 400 G for five minutes. With the break on at 22 degrees Celsius, carefully aspirate the flow buffer from each well.
Using a two milliliter aspirating pipette fitted with a 200 microliter micro pipette tip, making sure not to disturb the pellet, wash the cells again and then resus, suspend the pellets in 200 microliters of 1%paraform aldehyde in calcium and magnesium free PBS. Finally, transfer the cell suspensions to flow tubes and bring the total volume in the tubes up to 300 microliters by adding approximately 100 microliters of 1%paraform aldehyde in calcium and magnesium free PBS to each one. Then read two times 10 to the fourth NK cell events by flow cytometry using facts diva software and analyze the data using FlowJo software label purified HIV V one infected and uninfected T-cell targets with 125 micro curies chromium 51 in saline per 10 to the six cells for two hours at 37 degrees Celsius and 5%carbon dioxide in a 15 milliliter conical tube as a positive control, also labeled 10 to the six K 5 62 cells with 100 micro curies chromium 51 for two hours.
Under the same culture conditions. Use RPMI complete to bring the final volume of staining solution for each cell population to 500 microliters. The volume of chromium 51 added to the target cells is calculated using the universal radioactive decay calculator found on the PerkinElmer website while the target cells are labeling.
Remove previously isolated NK cells from culture and count them centrifuge the NK cells at 300 G for 10 minutes with the break on and resuspend the pellets in RPMI complete split the resulting cell suspensions into three 15 milliliter conical tubes. Adjust the cell density to 10 to the fifth per milliliter in the first tube, 2.5 times 10 to the fifth per milliliter in the second tube, and five times 10 to the fifth per milliliter. In the third tube, the NK cells will be used as effector cells at one to one, 2.5 to one and five to one ratio respectively in the chromium 51 release assay.
Remove the now labeled target cells from the incubator and add 4.5 milliliters of RPMI complete to each tube. Centrifuge the tubes at 300 G for 10 minutes. Then carefully pour the supernatant into a container for radioactive liquid waste without disturbing the pellet.
Wash the target cells two more times to remove all unabsorbed chromium 51. The supernatant from the third wash may be discarded in a container for non-radioactive liquid waste. Now re resuspend the targets in RPMI complete to the final cell density of 10 to the fifth per milliliter shown as an example of a typical setup for a chromium 51 release assay.
Note that each group is done in triplicate aliquot 100 microliters of labeled targets into each of the indicated wells of a 96 V bottom well plate for a final cell number of 10 to the fourth target cells per well. Next, add 100 microliters of the NK suspensions to the target cells in the appropriately indicated effector target cell ratios. Then add 100 microliters of targets to the corresponding wells in which the effector cells are absent for the maximum and spontaneous release groups.
Supplement the spontaneous release group with an additional 100 microliters of RPMI complete centrifuge the plate at 20 G for two minutes with the break on and then incubate the plate at 37 degrees Celsius and 5%carbon dioxide for four hours. After the four hour incubation, add five microliters of a one to 10 dilution of human red blood cells in RPMI complete to each well accept the maximum release wells, then add 100 microliters of 10%sodium ESAL sulfate to the maximum release wells and centrifuge the plate at 400 G for five minutes. To palate the cells label a gamma counter tube for each of the experimental and control wells.
On the 96 well plate harvest 100 microliters of cell-free super name from each well and place the super name from each well into the pre level tubes. Measure the amount of chromium 51 in each tube in a PerkinElmer 24 70 automatic gamma counter. The following scatter plots demonstrate a typical flow cytometric gating strategy for analyzing the results of a CD 1 0 7.
A degranulation assay. First single cells are gated, excluding clumps or doublets on a plot of FSCA versus FSCH. Next, the single cell gate plotted as FSCA versus SSC is gated on the lymphocyte population indicated again by the pink coval.
The lymphocyte gate is then plotted as CD 3 14 20 versus CD 56 gating on the CD 56 positive and CD 3 14 20 negative population as indicated by the vertical pink oval. Here the NK GA is plotted as CD 1 0 7 A versus CD 56 to visualize NK cells that have degranulated in the following two figures, representative results from assessing the cytotoxic response of natural killer cells against HIV one infected cells are shown here. NK cells were evaluated for their ability to degranulate without targets and in response to K 5 62 cells, primary CD four positive T cells and HIV infected primary T cells as assessed by the percentage of NK cells that express surface CD 1 0 7 A.The numbers in each quadrant represent the percentage of total NK cells in this figure.
NK cells were evaluated for their ability to lye K 5 62 cells uninfected CD four positive T cells, and HIV one infected T cells in acromium 51 release assay at effector cell target cell ratios of 2.5 to one five to one, and 7.5 to one After its development. This technique helped pave the way for researchers in the area of HIV V one biology to explore how natural killer cells respond to HIV one infected cells in HIV infected patients.
