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 t....

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

List of materials used in this article

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