CD8 CTLs Can Be Tracked with MHC Tetramer Technology

MHC tetramers are laboratory-generated complexes of four MHC class I molecules bound to a specific peptide and linked to a fluorescent molecule. A given MHC-tetramer-peptide complex binds only CD8+ T cells that have TCRs specific for the particular peptide-MHC complex that makes up the tetramer. Thus, when a particular tetramer is added to a cell population containing T cells (spleen cells or lymph-node cells, for example), cells that bear TCRs specific for the tetramer become fluorescently labeled (Figure 14-3). Using flow cytometry, it is then possible to determine the proportion of cells in a population that have TCRs specific for a particular antigen by counting the number of fluores-cently labeled cells in a cell population. This very sensitive approach can detect antigen-specific T cells even when their frequency in the CD8+ population is as low as 0.1%. Furthermore, one can directly measure the increase in antigen-specific CD8+ T cells in response to exposure to pathogens such as viruses or cancer-associated antigens. In a related application, researchers infected mice with vesicular stomatitis virus (VSV) and systematically examined the distribution of CD8+ cells specific for a VSV-derived peptide-MHC complex throughout the entire body. This study demonstrated that during acute infection with VSV, the distribution of

Streptavidin

Streptavidin

Vsv Infection Mice
MHC tetramer
Cd8 Tetramer

Tetramer binds exclusively to TCR

complementary to selected peptide-MHC complex

Tetramer binds exclusively to TCR

complementary to selected peptide-MHC complex mouse strains carrying mutations that affect the ability of CTLs to induce death have led to the identification of the necessary molecules.

The primary events in CTL-mediated death are conjugate formation, membrane attack, CTL dissociation, and target-cell destruction (Figure 14-6). When antigen-specific CTLs are incubated with appropriate target cells, the two cell types interact and undergo conjugate formation. Formation of a CTL-target cell conjugate is followed within several minutes by a Ca2+-dependent, energy-requiring step in which the CTL programs the target cell for death. The CTL then dissociates from the target cell and goes on to bind to another target cell. Within a variable period of time (up to a few hours) after CTL dissociation, the target cell dies by apoptosis. Each of the steps in this process has been studied in detail with cloned CTLs.

The TCR-CD3 membrane complex on a CTL recognizes antigen in association with class I MHC molecules on a target cell. After this antigen-specific recognition, the integrin

Signal measured by flow cytometer

FIGURE 14-3

MHC tetramers. A homogeneous population of peptide-bound class I MHC molecules (HLA-A1 bound to an HIV-derived peptide, for example) is conjugated to biotin and mixed with fluorescently labeled Streptavidin. Four biotinylated MHC-peptide complexes bind to the high affinity binding sites of Streptavidin to form a tetramer. Addition of the tetramer to a population of T cells results in exclusive binding of the fluorescent tetramer to those CD8+ T cells with TCRs complementary to the peptide-MHC complexes of the tetramer. This results in the labeling of the subpopulation of T cells that are specific for the target antigen, making them readily detectable by flow cytometry. [Adapted in part from P. Klenerman, V. Cerundolo, and P. R. Dunbar, 2002, Nature Reviews/Immunology 2:264.]

VSV-specific CD8+ cells is far from uniform (Figure 14-4); large populations of antigen-specific cells are not limited to the lymphoid system, but can be found in the liver and kidney, too.

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