The overall goal of this assay, is to enumerate and characterize Simian Immunodeficiency Virus-specific CD8 positive T-cell populations generated by vaccination or primary infection. This method can help answer key questions in the T-cell immunology field, such as what is the frequency of antigen-specific CD8 T-cells induced either by vaccination or infection. The main advantage of this technique is that it can quantify antigen-specific CD8 T-cells in biological samples obtained directly ex vivo without the need for in vitro re-stimulation.
The implications of this technique extend toward the evaluation of incision specific CD8 T-cell in the rhesus macaque, and for studying CDB T-cell responses in multiple disease settings. Generally, individuals new to this method will struggle because of the incomplete knowledge available for rhesus macaque MHC genetics, and because the flow cytometric staining requires precise antibody application. Begin by re-suspending the rhesus PBMC in R10 medium.
Add a 1.6 times 10 to the seventh cells per milliliter concentration. Aliquot 50 microliters of cells to the appropriate number of flow cytometry tubes, and add 50 microliters of protein kinase inhibitor to each tube. Vortex each tube and incubate the samples at 37 degrees Celsius for 30 minutes.
The correct amount of titrated peptide MHC tetramer must be diluted in enough master mix to stain all of the experimental tubes. Therefore, it is important to prepare a 15%excess of the master mix, to account for pippetting errors. While the cells are incubating, pellet the protein aggregates in the tetramer solution by centrifugation, to minimize the background staining.
Next, dilute the tetramers in enough stain buffer, so that 25 microliters of the tetramer master mix can be added to each flow cytometry tube. Then vortex the sample tubes and incubate the PBMC tetramer solutions in the dark, at room temperature for 45 minutes. At the end of the incubation, add 50 microliters of staining master mix to the appropriate sample tubes.
And mix the tubes for vortexing. After 25 minutes in the dark at room temperature, wash the cells with wash buffer, pellet the cells by centrifugation, and careful decant the supernatants without disturbing the pellets. Vortex the cells in the leftover buffer in each tube, and fix the samples with 250 microliters of 2%paraformaldehyde per tube.
Immediately vortex and incubate the tubes in the dark, at 4 degrees Celsius. After 20 minutes, wash the cells in fresh wash buffer. Pellet the cells by centrifugation, and decant the supernatant without disturbing the pellet.
Re-suspend the cells in 500 microliters of permeabilization buffer, vortex, and incubate at room temperature for 10 minutes in the dark. Then wash and pellet the cells. Then decant the supernatants as described previously.
Proceed to adding 50 microliters of intracellular stain master mix to the appropriate tubes. Vortex each sample, and incubate at room temperature in the dark for 30 minutes. After the incubation is done, wash and pellet the cells.
The samples are now ready to be acquired in a flow cytometer. Mamu-B017.01 tetramers typically yield a dim staining, which can be substantially improved by pre-treatment of the cells, with a protein kinase inhibitor. To avoid background staining, MHC class one matched cells that do or do not contain the antigen-specific CD8 positive T-cell population of interest, should be labelled with the relevant tetramer and multiple dilutions, to determine the optimum amount that results in the best separation between tetramer positive and negative CD8 positive T-cells.
In these graphs, the gating strategy used to analyze the magnitude and memory phenotype of vaccine-induced Gag CM9 specific CD8 positive T-cells responses in a Mamu-A 01 positive rhesus macaque, are shown. Note that the tetramer positive CD8 positive T-cell population, is well-separated with minimal background staining. And with the memory subsets clearly delineated by their CD28, CCR7 and Granzyme B expression within the tetramer gate.
Once mastered, this technique can be completed in about three hours if it is performed properly. While attempting this procedure, it is important to remember to add the correct reagents to each antibody or tetramer master mix, and to work with the experimental tubes each time as demonstrated. After its development, this technique paved the way for researchers in the field of immunology to explore the specificity and phenotype of antigen-specific CD8 T-cells.
After watching this video, you should have a good understanding of how to set up an MHC class one tetramer staining in the assay of the rhesus macaque model of human HIV-1 AIDS research. Don't forget that working with biological specimens from rhesus macaques requires special precautions such as wearing the appropriate personal protective equipment during the sample processing.
