Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Summary

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Abstract

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers¹. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody^2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope^4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.

The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this⁶, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues^3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

Protocol

1. Cell Isolation from Lymphoid Tissue

1. Add 1 ml of ice cold cEHAA (EHAA + 10% FBS, pen/strep, gentamycin, 2 mM L-glutamine, 55 mM 2-mercaptoethanol) or other equivalent T cell medium, to a 60 mm culture dish containing a small square of 100 μm nylon mesh and place on ice.
2. Euthanize mouse.
3. Remove the spleen and as many easily accessible lymph nodes as possible. These should include at least the inguinal, axillary, brachial, cervical, and mesenteric lymph nodes. Place them on top of the nylon mesh in the culture dish.
4. Using the flat top of a closed 1.5 ml microfuge tube, gently mash the lymphoid tissue over the nylon mesh to liberate lymphocytes. Add another 1 ml of cEHAA and pipet up and down to work cells into a suspension. Transfer the cells through another piece of nylon mesh placed over the top of a 15 ml polypropylene centrifuge tube. Rinse the dish and mesh with another 1 ml of ice cold cEHAA, pooling the volumes into the same tube. Repeat to achieve a final cell suspension volume of 4 ml.
5. Add cold sorter buffer (PBS + 2% FBS, 0.05% Azide) to a final volume of 15 ml and centrifuge the tube for 5 min at 300 x g, 4 °C.
6. Carefully aspirate the supernatant, making sure no droplets of liquid are left on the sides of the tube. Resuspend the cell pellet in Fc block (sorter buffer + 2.4G2 antibody) to a final volume equal to approximately twice that of the pellet itself. For example, the spleen and lymph nodes of a naive mouse usually produces a cell pellet of about 100 μl. In this case, add 100 μl of Fc block to bring the volume to 200 μl. If a large degree of cell clumping has occurred, carefully remove the cell clump at this point with a pipet tip.

2. Tetramer Staining

1. Add PE- or APC-labeled pMHC tetramer to a final concentration of 10 nM (or empirically optimized concentration).
2. Mix and incubate for 1 hr at room temperature (or empirically optimized time and temperature).
3. Add cold sorter buffer to a volume of 15 ml and centrifuge the tube for 5 min at 300 x g, 4 °C. Keep samples on ice or at 4 °C from now on.
4. Carefully aspirate the supernatant, making sure no droplets of liquid are left on the sides of the tube. Resuspend the cell pellet in sorter buffer to a final volume of 200 μl. For double tetramer enrichment, resuspend to a final volume of 150 μl.

3. Magnetic Enrichment

1. Add 50 μl of Miltenyi anti-PE or anti-APC microbeads. For double tetramer enrichment, add 50 μl of both.
2. Mix and incubate for 20 min at 4 °C.
3. Add cold sorter buffer to a volume of 15 ml and centrifuge the tube for 5 min at 300 x g, 4 °C.
4. While waiting, set up a Miltenyi LS magnetic column on a MidiMACS or QuadroMACS magnet. Position an open 15 ml polypropylene centrifuge tube on a rack directly underneath the column.
5. Add 3 ml of sorter buffer to the top of the column, allowing it to drain into the 15 ml tube.
6. Place a 100 μm nylon mesh square on top of the column.
7. When cells have finished spinning, carefully aspirate the supernatant and resuspend the pellet in 3 ml of sorter buffer.
8. Transfer the cell suspension through the nylon mesh onto the top of the column.
9. When the cell suspension has completely drained into the column, rinse the original tube with another 3 ml of sorter buffer and transfer the buffer through the nylon mesh into the column, rinsing the mesh in the process. Discard the nylon mesh.
10. When the buffer has completely drained into the column, add another 3 ml of sorter buffer to the column.
11. Repeat step 10 for a total of 3 x 3 ml washes.
12. Remove the column from the magnet and place over a new 15 ml polypropylene centrifuge tube.
13. Add 5 ml of sorter buffer to the column.
14. 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 the column into the tube.
15. Centrifuge the tubes containing the eluted bound fraction and the flow-through unbound fraction for 5 min at 300 x g, 4 °C.
16. Carefully aspirate the supernatant from the bound fraction, making sure no droplets of liquid are left on the sides of the tube. Resuspend the cell pellet in sorter buffer to a final volume of exactly 95 μl. Aspirate and resuspend the unbound fraction to a final volume of 2 ml.

4. Flow Cytometry

1. For each fraction, remove 5 μl and add to 200 μl of counting beads (adjusted to a concentration of 200,000/ml in sorter buffer) in a 5 ml FACS tube. Set aside at 4 °C for analysis later. To save time, beads can be pre-aliquoted to labeled tubes before the start of the experiment.
2. Prepare a master mix of antibodies to stain surface markers on the cells (Table 1). To save time, this can be done prior to the start of the experiment.
3. For the bound fraction, add a dose of antibody cocktail directly to the ~90 μl of cells in the tube. If analysis of the unbound fraction is desired, transfer 90 μl of cells to a 5 ml FACS tube and add a dose of antibody cocktail.
4. Set up a panel of single-color compensation controls for the flow cytometer. For each fluorochrome to be used, mix 50 μl of leftover unbound fraction cells in a 5 ml FACS tube with 1 μl of anti-CD4 antibody conjugated to the corresponding fluorochrome. Remember to set up an unstained control as well.
5. Vortex and incubate all samples for 30 min at 4 °C.
6. Add 5 ml of sorter buffer to each tube and centrifuge for 5 min at 300 x g, 4 °C.
7. For the bound fraction, carefully aspirate supernatant and resuspend the cells in 200 μl of sorter buffer. Transfer the cells into a 1.2 ml FACS microtube. Rinse the tube with another 200 μl of sorter buffer and pool into the same microtube. If cell clumps are apparent, pass the cells through a 50 μm filter.
8. For the unbound fraction and compensation controls, decant or aspirate the supernatant and resuspend in 2 ml sorter buffer.
9. Set up the flow cytometer using the single-color compensation controls. Select side scatter-width (SSC-W) as an additional parameter to be recorded.
10. Analyze the stained samples using a sequence of successive inclusion gates to identify CD4+ or CD8+ T cells as illustrated in Figures 1 and 2. For bound fractions, collect as many cells as possible up to a maximum of 2,000,000 total events. For unbound fractions, collect 1,000,000 total events. Keep the acquisition rate at or below 3,000 events per second.
11. Using the same machine settings, analyze the counting bead samples. Collect 10,000 total events.
12. Save all data as FCS files.

5. Data Analysis

1. Analyze FCS data files for the counting bead samples using FlowJo software. Plot forward scatter by FITC and set a gate aroung the counting beads (see Figures 1 and 2). Determine the total number of counting beads detected in each sample. Subtract from the total number of collected events to determine the number of cell events collected.
2. Calculate the total number of all cells in each sample using the equations outlined in Box 1.
3. Analyze FCS data files for the stained bound and unbound fraction samples. Set up a sequence of successive inclusion gates to identify lymphoid+, side-scatter-width^lo, dump-, CD3+, CD4+ or CD8+, tetramer+ T cells in each sample as illustrated in Figures 1 and 2.
4. Multiply the total number of cells in the sample by the frequencies of each of the inclusion gates used to define CD4+tetramer+ or CD8+tetramer+ cells to calculate the total number of epitope-specific T cells (Box 1).

Results

Figure 1 depicts representative flow cytometry plots of pMHCII tetramer enriched spleen and lymph node samples from naive mice, while Figure 2 depicts representative data for mice previously immunized with the relevant peptide+CFA. Serial gating removes autofluorescent and other unwanted events from the analysis of CD4+ T cell populations. The CD8+ T cell population serves as a useful internal negative control for pMHCII tetramer staining of CD4+ T cells. Note that bound fractions from t...

Discussion

The pMHC tetramer based cell enrichment method presented by this protocol is a powerful tool for studying epitope-specific T cells from endogenous T cell repertoires. The use of pMHC tetramers enables detection of epitope-specific T cells based directly on the ability of their TCRs to bind cognate pMHC ligands. The enrichment provides a level of sensitivity such that extremely rare populations of antigen-specific T cells can be detected from endogenous repertoires of T cells without any manipulation of their gene...

Materials

Name	Company	Catalog Number	Comments
PE or APC conjugated pMHC tetramer (or multimer)	Made by investigator, obtained from the NIH tetramer core, or purchased from commercial sources
Anti-PE conjugated magnetic microbeads	Miltenyi	130-048-801
Anti-APC conjugated magnetic microbeads	Miltenyi	130-090-855
LS magnetic columns	Miltenyi	130-042-401
MidiMACS or QuadroMACS magnet	Miltenyi	130-042-302 or 130-090-976
Cell counting beads	Life Technologies	PCB-100