The overall goal of the following experiment is to identify rare epitope specific T cells from the endogenous T cell repertoire of mice. This is achieved by first staining harvested lymphoid cells with peptide MHC tetramer reagents, which will fluorescently label the relevant epitope specific T cells. These tetramer labeled cells are then stained with magnetic microbeads conjugated to antibodies specific for the fluorescent tag on the tetramer.
Next, the magnetically labeled cells are passed through a magnetized column in order to enrich the sample for tetramer bound epitope specific T cells. Finally, the cells are stained for the desired experimental cell markers and multi parameter flow cytometry is performed to detect and quantify the rare tetramer enriched epitope specific T-cell populations. The main advantage of using this technique over other existing methods like the TCR Transgenic T-cell adoptive transfer system, is that studies are performed on real polyclonal populations of epitope specific T-cells that develop naturally within the immune system.
Moreover, the high sensitivity of detection afforded by this technique enables studies of these populations when they're present at extremely low frequencies. This method can help answer many key questions in the field of immunology, such as how many naive T cells may recognize a given antigen from a pathogenic microbe, and how the clonal composition of an epitope specific T cell population changes throughout the course of an immune response. Before starting the dissection, add one milliliter of complete EHAA medium to a 60 millimeter culture dish containing a small square of 100 micron nylon mesh, and place the dish on ice.
Then remove the spleen from a euthanized mouse and as many easily accessible lymph nodes as possible. Place the tissues on top of the nylon mesh in the culture dish. Use the flat top of a closed 1.5 millimeter micro fuge tube to gently mash the lymphoid tissues on the nylon mesh.
To liberate the lymphocytes, add another milliliter of media to the dish and then pipette the solution up and down to work the cells into a suspension. Now, place a new nylon mesh on top of a fresh 15 milliliter polypropylene centrifuge tube, and then transfer the cells through the new mesh. Rinse the dish and mesh with another milliliter of cold media and pull the volumes through the new piece of mesh into the 15 milliliter tube.
Repeat the rinse one more time. After adding cold sorter buffer to a final volume of 15 milliliters, centrifuge the tube for five minutes at 300 times G and four degrees Celsius. Then carefully aspirate the snat resus.
Suspend the cell pellet in FC block to a final volume equal to approximately twice that of the pellet itself. If a large degree of cell clumping has occurred. Carefully remove the cell clump with a pipette tip to stain For antigen specific T cells.
Add fluorescently tagged peptide MHC tetramer to the resuspended cell pellet at the optimized concentration. Then mix the cell suspension and incubated at the optimized time and temperature for tetramer binding. Next, add cold sorter buffer to bring the volume up to 15 milliliters and centrifuge keeping the cells at four degrees Celsius.
From now on carefully aspirate the supernatant. Then resuspend the cell pellet in sorter buffer to a final volume of 200 microliters. Now add 50 microliters of mil tenny antibody conjugated microbeads that will specifically bind to the fluorescent tag of the tetramer mix, and then incubate the tetramer bound cells and beads at four degrees Celsius.
After 20 minutes, wash the cells in sorter buffer while the cells are in the centrifuge. Place a milani LS magnetic column on a quadro max magnet and position a new 15 milliliter polypropylene centrifuge tube. Directly underneath the column, add three milliliters of sorter buffer to the top of the column, allowing it to drain into the 15 milliliter tube.
Then place a 100 micron nylon mesh square on top of the column. When the cells have finished spinning, carefully aspirate the supernatant and resuspend the pellet in three milliliters of sorter buffer, transfer the cell suspension through the nylon mesh onto the top of the column. When the cell suspension has completely drained into the column, rinse the original tube with another three milliliters of sorter buffer and transfer through the mesh onto the column.
Pooling the wash in the collection tube, discard the nylon mesh. Then when the buffer has completely drained into the tube, wash the column two more times with three milliliters of sorter buffer each time. Then remove the column from the magnet and place the column over a new 15 milliliter polypropylene centrifuge tube.
Now add another five milliliters of sorter buffer to the column. Immediately insert the plunger into the top of the column, and in one continuous motion, push the plunger all the way down, forcing the buffer out of the column into the tube. After spinning down both the tube containing the eluded bound fraction and the tube containing the flow through unbound fraction, carefully aspirate the supernatant from the bound fraction and resuspend the cell pellet in sorter buffer to a final volume of exactly 95 microliters, aspirate and resuspend the unbound fraction to a final volume of two milliliters before staining the cell fractions further, add 200 microliters of counting beads into a five milliliter fax tube and transfer five microliters of cells into this tube.
Set these tubes aside at four degrees Celsius for analysis. Later now, combine a master mix of antibodies to stain surface markers on the cells according to the table. For the bound fraction, add a dose of antibody cocktail directly to the cells for the unbound fraction.
Transfer 90 microliters of the cells to a five milliliter fax tube and add a dose of antibody cocktail to the transferred sample for each fluorochrome to be used. Mix 50 microliters of the leftover unbound fraction of cells in a five milliliter fax tube with one microliter of anti CD four antibody conjugated to the appropriate fluorochrome. Set aside an unstained control as well next vortex, and then incubate all of the samples at four degrees Celsius for 30 minutes.
Then after washing each tube in five milliliters of sorter buffer, carefully aspirate the supernatant from the bound fraction samples and resuspend the cells in 200 microliters of sorter buffer. Transfer the cells into 1.2 milliliter, fax micro tubes, and then rinse their previous tubes with another 200 microliters of sorter buffer, pooling the rinses into the corresponding micro tubes. If clumps are apparent, pass the cells through a 50 micron filter for the unbound fraction and compensation controls, decant the supernatant and resuspend the pellets in one milliliter of sorter buffer.
Analyze the stained samples using a sequence of successive inclusion gates for identifying CD four positive or CD eight positive T cells as illustrated here for the bound fraction samples. Collect as many cells as possible up to a maximum of 2 million total events for the unbound fraction samples. Collect 1 million total events.
Finally, using the same machine settings, collect 10, 000 events from the counting bead samples. This first figure DEPICTS representative flow cytometry plots of peptide MHC two tetramer enriched spleen and lymph node samples from naive mice for bound and unbound cell fractions. A succession of gates was set to select lymphoid scatter positive side scatter with low dump negative CD three positive events of these CD four positive or CD eight positive events were gated for the analysis of epitope specific T cells or background staining.
Aliquots of unstained cells from the bound and unbound fractions were mixed with fluorescent counting beads and analyzed separately. These density plots depict representative data for mice previously immunized with the relevant peptide plus complete adjuvant. The same serial gating strategy as in the previous experiment, was used to remove any autofluorescence and other unwanted events.
From the analysis of the CD four positive T-cell populations, the CD eight positive T-cell population served as a useful internal negative control for peptide MHC two tetramer staining of CD four positive T cells. The absolute numbers of epitope specific T cells in a sample are calculated by multiplying the total number of all the cells in the bound fraction of the enriched sample as determined from the bead count analysis with the proportion of these cells that are tetramer positive as determined from the cell staining analysis for the representative naive mouse data shown in the first series of density plots, the total cell count was 4, 411, and the bead count was 5, 589. The bead stock concentration was two times 10 to the fifth per milliliter.
The bead volume was 0.2 milliliters and the cell volume was 0.005 milliliters. Thus, these data were used to generate the cell concentration of 6.31 times 10 to the sixth per milliliter. The cell concentration was then multiplied by the total sample volume to generate the total cell number.
This data in turn was multiplied by the percent of the tetramer positive events from the cell analysis to determine that the number of epitope specific CD four positive T cells from this naive mouse was 98. For the representative immunized mouse data in the second series of density plots, the total number of epitope specific CD four positive T cells was 1.53 times 10 to the fourth. The efficiency of the enrichment declines as the number of epitope specific T cells increases.
So tetramer positive cells may be seen in the unbound fraction of samples containing very high frequencies of epitope specific T cells. In such cases, the number of epitope specific T cells present in the unbound fraction can be calculated separately and added to the number found in the bound fraction. For example, in the representative immunized mouse unbound cell fraction, the total number of epitope specific CD four positive T cells was 8.97 times 10 to the third.
Thus, by adding the numbers in the bound and unbound fractions, there were 2.43 times 10 to the fourth total epitope specific CD four positive T cells in the whole immunized mouse sample. Indeed, if the epitope specific cell expansion is sufficiently robust, the enrichment process can be skipped. Following the enrichment procedure, self fixation and permeation steps can be performed to enable staining of intracellular proteins such as cytokines and transcription factors.
While this method was originally designed for studies of T cells from the spleen and lymph nodes, it can also be applied to other tissues like the thymus and intestine. The technique can also be used to study epitype specific T cells in human blood and tissue samples.
