Ctl

Target cell

FIGURE 10-11 Steps in CTL-mediated lysis of target cells. A CTL recognizes the antigen-expressing target cell and is activated. Activation results in the release of granule contents from the CTL into the target cell through the area of contact (the immunologic synapse). Granule contents deliver a lethal hit to the target. The CTL may detach and kill other target cells. The formation of conjugates between a CTL and its target and activation of the CTL also require interactions between accessory molecules (LFA-1, CD8) on the CTL and their specific ligands on the target cell; these are not shown.

Antigen recognition and immune synapse formation

Granule exocytosis

Detachment of CTL

Target cell death

FIGURE 10-11 Steps in CTL-mediated lysis of target cells. A CTL recognizes the antigen-expressing target cell and is activated. Activation results in the release of granule contents from the CTL into the target cell through the area of contact (the immunologic synapse). Granule contents deliver a lethal hit to the target. The CTL may detach and kill other target cells. The formation of conjugates between a CTL and its target and activation of the CTL also require interactions between accessory molecules (LFA-1, CD8) on the CTL and their specific ligands on the target cell; these are not shown.

Recognition of Antigen and Activation of CTLs

The CTL binds and reacts to the target cell by using its antigen receptor, coreceptor (CD8), and adhesion molecules. To be efficiently recognized by CTLs, target cells must express class I MHC molecules complexed to a peptide (the complex serving as the ligand for the T cell receptor (TCR) and the CD8 coreceptor) and intercellular adhesion molecule 1 (ICAM-1, the principal ligand for the LFA-1 integrin). The CTLs and their target cells form tight conjugates (Fig. 10-12). This immunologic synapse (see Chapter 7) formed between the two cells is characterized by a ring of close apposition between the CTL and target cell membranes, mediated by LFA-1-ICAM-1 binding, and an enclosed gap or space inside the ring. Distinct regions of the CTL membrane can be observed by immunofluorescence microscopy within the ring, including a signaling patch, which includes the TCR, protein kinase C-0, and Lck, and a secretory domain, which appears as a gap to one side of the signaling patch. This interaction results in the initiation of biochemical signals that activate the CTL, which are essentially the same as the signals involved in the activation of helper T cells (see Chapter 7). Cytokines and costimulators provided by dendritic cells, which are required for the differentiation of naive CD8+ T cells into CTLs, are not necessary for triggering the effector function of CTLs (i.e., target cell killing). Therefore, once CD8+ T cells specific for an antigen have differentiated into fully functional CTLs, they can kill any nucleated cell that displays that antigen.

In addition to the T cell receptor, CD8+ CTLs express receptors that are also expressed by NK cells, which contribute to both regulation and activation of CTLs. Some of these receptors belong to the killer immunoglobulin receptor (KIR) family, discussed in Chapter 4, and recognize class I MHC molecules on target cells but are not specific for a particular peptide-MHC complex. These KIRs transduce inhibitory signals that may serve to prevent CTLs from killing normal cells. In addition, CTLs express the NKG2D receptor, described in Chapter 4, that recognizes class I MHC-like molecules MIC-A, MIC-B, and ULBP, expressed on infected or neoplastic cells. NKG2D may serve to deliver signals that act together with TCR recognition of antigen to enhance killing activity.

Killing of Target Cells by CTLs

Within a few minutes of a CTL's antigen receptor recognizing its antigen on a target cell, the target cell undergoes changes that induce it to die by apoptosis. Target cell death occurs during the next 2 to 6 hours and proceeds even if the CTL detaches. Thus, the CTL is said to deliver a lethal hit to the target cell. The principal mechanism of CTL-mediated target cell killing is the delivery of cyto-toxic proteins stored within cytoplasmic granules (also called secretory lysosomes) to the target cell, thereby triggering apoptosis of the target cell (Fig. 10-13). As discussed earlier, CTL recognition of the target leads to activation of the CTL, one consequence of which is cyto-skeleton reorganization such that the microtubule organizing center of the CTL moves to the area of the cytoplasm near the contact with the target cell. The cytoplasmic granules of the CTL are transported along micro-tubules and become concentrated in the region of the synapse, and the granule membrane fuses with the plasma membrane at the secretory domain. Membrane fusion results in exocytosis of the CTL's granule contents into the confined space within the synaptic ring, between the plasma membranes of the CTL and target cell.

The cytotoxic proteins in the granules of CTLs (and NK cells) include granzymes and perforin. Granzymes A, B, and C are serine proteases that cleave proteins after aspartate residues. Perforin is a membrane-perturbing molecule homologous to the C9 complement protein. The granules also contain and a sulfated proteoglycan, serglycin, which serves to assemble a complex containing granzymes and perforin. The main function of perfo-rin is to facilitate delivery of the granzymes into the cytosol of the target cell. How this is accomplished is still not well understood. Perforin may polymerize and form aqueous pores in the target cell membrane through which granzymes enter, but there is no proof that this is

CTL TC

FIGURE 10-12 Formation of conjugates between CTLs and a target cell. A, Electron micrograph of three CTLs from a cloned cell line specific for the human MHC molecule HLA-A2 binding to an HLA-A2-expressing target cell (TC) within 1 minute after the CTLs and targets are mixed. Note that in the CTL on the upper left, the granules have been redistributed toward the target cell. (Courtesy of Dr. P. Peters, Netherlands Cancer Institute, Amsterdam.) B, Electron micrograph of the point of membrane contact between a CTL (left) and target cell (right). Two CTL granules are near the synapse. Several mitochondria are also visible. (Reprinted from Stinchcombe JC, G Bossi, S Booth, and GM Griffiths. The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 8:751-761, 2001, © Cell Press, with permission from Elsevier). C, Confocal fluorescence micrograph of an immune synapse between a CTL (left) and target cell (right) stained with antibodies against cathepsins in a secretory granule (blue), LFA-1 (green), and the cytoskeletal protein talin (red). The image demonstrates the central location of the secretory granule and the peripheral location of the adhesion molecule LFA-1 and associated cytoskeletal protein talin. (Reprinted from Stinchcombe JC, and GM Griffiths. The role of the secretory immunological synapse in killing by CD8+ CTL. Seminars in Immunology 15:301-305, © 2003 Elsevier Science Ltd., with permission from Elsevier.)

critical for CTL-mediated cell killing. According to another current model, complexes of granzyme B, perforin, and serglycin are discharged from the CTL onto the target cell and are internalized into endosomes by receptor-mediated endocytosis. Perforin may act on the endosomal membrane to facilitate the release of the granzymes into the target cell cytoplasm. Once in the cytoplasm, the gran-zymes cleave various substrates, including caspases, and initiate apoptotic death of the cell. For example, granzyme B activates caspase-3 as well as the Bcl-2 family member Bid, which triggers the mitochondrial pathway of apoptosis (see Fig. 14-7, Chapter 14). Another protein found in human CTL (and NK cell) granules, called gra-nulysin, can alter the permeability of target cell and microbial membranes, but its importance in cell killing by CTLs is not established.

CTLs also use a granule-independent mechanism of killing that is mediated by interactions of membrane molecules on the CTLs and target cells. On activation, CTLs express a membrane protein, called Fas ligand (FasL), that binds to the death receptor Fas, which is expressed on many cell types. This interaction also results in activation of caspases and apoptosis of Fas-expressing targets (see Fig. 14-7, Chapter 14). Studies with knockout mice lacking perforin, granzyme B, or FasL indicate that granule proteins are the principal mediators of killing by CD8+ CTLs. Some CD4+ T cells are also capable of killing target cells (which, of course, must express class II MHC-associated peptides to be recognized by the CD4+ cells). CD4+ T cells are deficient in perforin and granzymes, and FasL may be more important for their killing activity.

After delivering the lethal hit, the CTL is released from its target cell, which usually occurs even before the target cell goes on to die. CTLs themselves are not injured during target cell killing, probably because the directed granule exocytosis process during CTL-mediated killing preferentially delivers granule contents into the target cell and away from the CTL. In addition, CTL granules

FIGURE 10-13 Mechanisms of CTL-mediated killing of target cells. CTLs kill target cells by two main mechanisms. A, Complexes of perforin and granzymes are released from the CTL by granule exocytosis and enter target cells. The granzymes are delivered into the cytoplasm of the target cells by a perforin-dependent mechanism, and they induce apoptosis. B, FasL is expressed on activated CTLs, engages Fas on the surface of target cells, and induces apoptosis.

Fas/Fas L-mediated cell killing

FasL on CTL interacts with Fas on target cell

Apoptosis of target cell

FIGURE 10-13 Mechanisms of CTL-mediated killing of target cells. CTLs kill target cells by two main mechanisms. A, Complexes of perforin and granzymes are released from the CTL by granule exocytosis and enter target cells. The granzymes are delivered into the cytoplasm of the target cells by a perforin-dependent mechanism, and they induce apoptosis. B, FasL is expressed on activated CTLs, engages Fas on the surface of target cells, and induces apoptosis.

contain a proteolytic enzyme called cathepsin B, which is delivered to the CTL surface on granule exocytosis, where it degrades errant perforin molecules that come into the vicinity of the CTL membrane.

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