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1Center for Immunology and Inflammatory Diseases, and Pulmonary and Critical Care Unit, Massachusetts General Hospital and Harvard Medical School
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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.
Legoux, F. P., Moon, J. J. Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells. J. Vis. Exp. (68), e4420, doi:10.3791/4420 (2012).
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 antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. 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 tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.
1. Cell Isolation from Lymphoid Tissue
2. Tetramer Staining
3. Magnetic Enrichment
4. Flow Cytometry
5. Data Analysis
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 the enrichment usually contain a significantly higher proportion of autofluorescent cells than the unbound fractions, making gating more challenging.
Absolute numbers of epitope-specific T cells in a sample are calculated by multiplying the total number of all 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+, as determined from the cell staining analysis (Box 1).
For the naive mouse in Figure 1, the concentration of all cells in the bound fraction of the sample is (4411/5589) (200,000) (0.200/0.005) = 6.31 x 106 /ml. The total number of all cells in the sample is (6.31 x 106 /ml) (0.095) = 6.00 x 105. Finally, the total number of epitope-specific CD4+ T cells is (6.00 x 105) (41.5%) (96.6%) (10.2%) (62.3%) (0.64%) = 98.
For the immunized mouse in Figure 2, the concentration of all cells in the bound fraction of the sample is (6410/3590) (200,000) (0.200/0.005) = 1.43 x 107 /ml. The total number of all cells in the sample is (1.43 x 107 /ml) (0.095) = 1.36 x 106. Finally, the total number of epitope-specific CD4+ T cells is (1.36 x 106) (40.9%) (93.9%) (9.54%) (72.0%) (42.7%) = 1.53 x 104.
The efficiency of enrichment declines as the number of epitope-specific T cells increases8, so tetramer+ 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. Therefore, in Figure 2, the concentration of all cells in the unbound fraction of the sample is (9031/969) (200,000) (0.200/0.005) = 7.46 x 107 /ml, the total number of all cells is (7.46 x 107 /ml) (2.0) = 1.49 x 108, and the total number of epitope-specific CD4+ T cells is (1.49 x 108) (62.7%) (96.4%) (44.5%) (54.7%) (0.0409%) = 8.97 x 103. Adding the numbers in the bound and unbound fractions, there are 1.53 x 104 + 8.97 x 103 = 2.43 x 104 total epitope-specific CD4+ T cells in the whole sample. Indeed, if epitope-specific cell expansion is sufficiently robust, the enrichment process can be skipped.
|Pacific Blue||dump (B220, CD11b, CD11c, F4/80)|
|PE||pMHC tetramer or phenotypic marker|
|APC||pMHC tetramer or phenotypic marker|
Table 1. Suggested antibody staining strategy
Figure 1. Flow cytometric analysis of epitope-specific CD4+ T cells in naive mice following pMHCII tetramer-based enrichment. Representative plots are shown for the bound (A) and unbound (B) fractions. A succession of gates are set to select lymphoid-scatter+, side-scatter-widthlo, dump-, CD3+ events. Of these, CD4+ or CD8+ events are gated for the analysis of epitope-specific T cells or background staining. Aliquots of unstained cells from the bound (C) and unbound (D) fraction were mixed with fluorescent counting beads and analyzed separately. Click here to view larger figure.
Figure 2. Flow cytometric analysis of epitope-specific CD4+ T cells in peptide-immunized mice following pMHCII tetramer-based enrichment. Representative plots are shown for the bound (A) and unbound (B) fractions. A succession of gates are set to select lymphoid-scatter+, side-scatter-widthlo, dump-, CD3+ events. Of these, CD4+ or CD8+ events are gated for the analysis of epitope-specific T cells or background staining. Aliquots of unstained cells from the bound (C) and unbound (D) fraction were mixed with fluorescent counting beads and analyzed separately. Click here to view larger figure.
Box 1. Calculation of epitope-specific T cell numbers
Absolute numbers of epitope-specific T cells are best calculated with the aid of fluorescent counting beads. An aliquot of unstained cells from each sample is mixed with a defined volume of counting beads set at a known concentration and then analyzed by flow cytometry. The concentration of cells in the sample can be inferred from a comparison of their frequency with the known concentration of fluorescent counting beads.
The total number of cells in the sample is then calculated by multiplying the cell concentration with the total sample volume.
The total number of epitope-specific T cells in the sample is simply the total number of all cells in the sample multiplied by the percentage of cells that are tetramer-positive.
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 genetic makeup or precursor frequency. As a result, this technique allows the investigator to directly track endogenous antigen-specific T cell populations from in vivo experimental systems from their naive levels through all stages of the immune response.
This protocol has been optimized for the use of pMHC class II (pMHCII) tetramers to enrich epitope-specific CD4+ T cells from the secondary lymphoid organs of mice. However, the technique is also applicable to pMHC class I (pMHCI) tetramers and CD8+ T cells9. Unlike CD4, the CD8 coreceptor plays a significant role in stabilizing TCR-MHC interactions, and this can have practical implications for pMHCI tetramer staining10. Most notably, the use of CD8 antibodies should be restricted to clones that do not impair CD8-tetramer binding, and they should be added to cells after tetramer staining. Indeed, some pMHCI tetramers have been engineered with mutated MHCI-CD8 binding sites to mitigate nonspecific binding to CD8+ T cells11,12.
Tetramer concentration, incubation time, and incubation temperature can greatly affect the efficiency of tetramer staining, and conditions should be optimized to achieve the best combination of high tetramer signal, low background signal, low tetramer internalization, and minimal changes to cell physiology. Ideally, these conditions should be empirically determined for each unique reagent. In our hands, however, a final concentration of 10 nM and an incubation of 1 hr at room temperature provides good generic conditions for most pMHCI or pMHCII tetramers. In general, pMHCI tetramers seem to stain more easily than pMHCII tetramers, and staining can often be performed at 4 °C for as little as 30 min.
The scale of this procedure is suited for the analysis of nearly all the secondary lymphoid organs of a mouse in a single sample. Therefore, each sample represents a fairly comprehensive analysis of the entire circulating peripheral T cell repertoire of a mouse. Epitope-specific T cells can also be enriched from other relevant tissues, including the thymus13,14. When thymii from 4-5 week old mice are analyzed, epitope-specific single positive thymocytes can be detected at numbers similar to those of peripheral naive T cells. Epitope-specific double-positive thymocytes, however, are very difficult to detect due to their low levels of TCR expression.
This protocol can also be adapted to detect epitope-specific human CD4+ or CD8+ T cells in blood or other tissues15-17. The frequency of epitope-specific T cells is roughly the same between mice and humans17, so the analysis of 50 - 100 ml of blood would yield comparable numbers of epitope-specific T cells as the pooled spleen and lymph nodes of a mouse18.
A major challenge in the flow cytometric analysis of cells following tetramer-based enrichment is distinguishing true cell events from background. This is largely due to the fact that many autofluorescent cells are also non-specifically enriched during the process. If not carefully gated out, these autofluorescent cells can appear as false tetramer-positive cells and throw off the accuracy of the analysis, particularly in the case of rare naive T cell populations. Our protocol employs a two-step gating strategy in which CD3+ events are first gated away from dump lineage+ events, and then CD4+ events are gated away from CD8+ events. In the process, autofluorescent cells, which tend to lie along the center diagonal of any FACS plot, are gated out of the analysis in two iterative steps. The effective removal of autofluorescent events comes at the cost of many fluorescent colors, so we highly recommend the use of flow cytometers capable of at least 6 fluorescent parameters.
Most pMHC tetramers are conjugated to PE and APC due to the brightness of these fluorochromes, and anti-PE and anti-APC magnetic microbeads are readily available to enable enrichment with them. However, other fluorochromes can also be used so long as corresponding magnetic microbeads are available. Indeed, multiple tetramers with different fluorochrome labels can be used together with the corresponding antibody-conjugated microbeads to simultaneously enrich multiple epitope-specific T cell populations from the same sample. We have outlined a very basic antibody-fluorochrome setup that is optimized for the use of PE- and APC-labeled tetramers (Table 1), but many other effective combinations are possible to increase flexibility in the study of phenotypic markers.
CD8+ T cell populations can be used as an internal negative control, since they should not bind to pMHCII ligands (and vice versa for epitope-specific CD8+ T cell enrichment). The frequency of tetramer+ CD8+ cells in a sample provides a good assessment of the level of background tetramer staining, although bona fide tetramer-positive CD8+ T cells with cross-restricted specificity to pMHCII epitopes may exist at very small frequencies19. Occasionally during very strong immune responses, these cells may exist at expanded frequencies. If desired, TCR transgenic T cells with or without relevant epitope specificity can be used as additional positive and negative controls. Note that for unknown reasons, some TCR transgenic cells and hybridomas do not stain well with their relevant tetramers.
Our protocol involves the use of fluorescent counting beads to assist in the calculation of cell numbers. While cell counts can also be achieved manually with a hemacytometer, we find that the use of counting beads results in much greater experimental precision, especially when multiple investigators are involved. Due to the small numbers and volumes of cells that are handled in this protocol, the minimization of experimental error should be a high priority.
Tetramer staining is compatible with cell fixation and permeabilization, and several studies have successfully analyzed intracellular cytokine and transcription factor expression in cells following tetramer-based cell enrichment20,21. However, the extra steps involved contribute to additional cell losses in the samples.
No conflicts of interest declared.
The authors would like to thank Andre Han and Lawrence Yen for technical assistance, and members of the Jenkins lab for help in the development of this protocol.
|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|
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