CTL-mediated killing involves specific recognition of target cells and delivery of proteins into the target that induces cell death. CTLs kill targets that display the same peptide–MHC-I antigen that triggered the proliferation and differentiation of naive CD8+ T cells into functional CTLs. CTL-mediated killing is antigen-specific, and adjacent uninfected cells that do not present that peptide-MHC antigen are not harmed.
Killing specificity is achieved because a close region of contact between the CTL and the antigen-expressing target cell, called an immune synapse, is established, and the molecules that actually perform the killing are secreted into the synapse and do not diffuse to other nearby cells. The target cells are killed by apoptosis, which is a pathway of cell death that does not induce harmful inflammation, since the apoptotic cell is rapidly phagocytosed and removed before releasing its contents. Thus, there is often no collateral damage of adjacent normal tissues during CTL-mediated killing if the amount of killed cells is limited and does not overwhelm the capacity for cleanup by macrophages.
The process of CTL-mediated killing of targets consists of antigen recognition, activation of the CTLs, delivery of the lethal hit that kills the target cells, and release of the CTLs (Fig. 1). Each of these steps is controlled by specific molecular interactions.
Fig1. Steps in cytotoxic T lymphocyte (CTL)–mediated lysis of target cells. A CTL recognizes the antigen-expressing target cell and is activated. Activation results in the release (exocytosis) of granule con tents from the CTL into the target cell through the area of contact (the immune 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 (leukocyte function–associated antigen 1 [LFA-1], CD8) on the CTL and their specific ligands (inter cellular adhesion molecule 1 [ICAM-1] and MHC-I, respectively) on the target cell (not shown).
Recognition of Antigen and Activation of Cytotoxic T Lymphocytes
The CTL binds and reacts to the target cell by using its anti gen receptor, CD8 coreceptor, and adhesion molecules. To be efficiently recognized by CTLs, target cells must express MHC-I molecules displaying a peptide. The MHC molecule with a bound peptide serves as the ligand for the T-cell receptor (TCR) and recruits the CD8 coreceptor to initiate signaling. Signaling by the TCR promotes formation of the specialized immune synapse, with a central region containing TCRs and other signaling molecules and an outer ring of integrins, notably LFA-1 (leukocyte function–associated antigen 1) on the CTL binding to its ligand ICAM-1 (intercellular adhesion molecule 1) on the target cell (Fig. 2). An enclosed gap is present within the ring between the membranes of the two cells. Distinct regions of the CTL membrane can be observed by immunofluorescence microscopy within the ring, including a signaling patch, which includes the TCR, CD8, and signaling proteins (such as protein kinase C-θ and the tyrosine kinase LCK); and a secretory region, which appears as a gap on one side of the signaling patch. TCR engagement by antigen and CD8 engagement by the MHC molecule results in the initiation of biochemical signals that activate the CTL, leading to the process of cell killing, described in the following section. Cytokines and costimulators provided by DCs, as well as T-cell help, 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).
Fig2. Formation of conjugates between cytotoxic T lymphocytes (CTLs) and a target cell. (A) Electron micrograph of three CTLs from a cloned cell line specific for the human MHC molecule human leukocyte antigen-A2 (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 TC. (B) Electron micrograph of the point of membrane contact between a CTL (left) and the TC (right). Two CTL granules (secretory granules [SG]) are near the synapse. Several mitochondria are also visible. (C) Confocal fluorescence micrograph of an immune synapse between a CTL (left) and the TC (right) stained with antibodies against cathepsins in an SG (blue), leukocyte function–associated antigen 1 (LFA-1) (green), and the cytoskeletal protein talin (red). The image demonstrates the central location of the SG and the peripheral location of the adhesion molecule LFA-1 and associated cytoskeletal protein talin. A, Courtesy Dr. P. Peters, Netherlands Cancer Institute, Amsterdam; B, From Stinchcombe JC, Bossi G, Booth S, Griffiths GM. The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity. 2001;8:751–761; C, From Stinchcombe JC, Griffiths GM. The role of the secretory immunological synapse in killing by CD8+ CTL. Semin Immunol. 2003;15:301–305.
In addition to the TCR, 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 KIR (killer immunoglobulin receptor) family, discussed in Chapter 4, and recognize MHC-I 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, which recognizes the MHC-I-like molecules MIC-A, MIC-B, and ULBP, which are expressed on stressed (infected or transformed) cells. NKG2D may deliver signals that act together with TCR recognition of antigens to enhance killing activity.
Killing of Target Cells by Cytotoxic T Lymphocytes
The principal mechanism of CTL-mediated target cell killing is the delivery of cytotoxic proteins stored within cytoplasmic granules to the target cell, thereby triggering apoptosis of the target cell (Fig. 3). Within a few minutes after a CTL’s antigen receptor and coreceptor recognize a peptide-MHC complex on the target cell, the CTL granule proteins enter the target cell, and death occurs during the following 2 to 6 hours, even if the CTL detaches. Thus, the CTL is said to deliver a lethal hit to the target cell. When the CTL recognizes antigen, TCR signals lead to actin cytoskeleton reorganization. In this process, the micro tubule 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 microtubules and become concentrated in the region of the synapse, and the gran ule membrane fuses with the plasma membrane at the region of the synapse. 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.
Fig3. 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) FAS ligand (FAS-L) is expressed on activated CTLs, engages FAS on the surface of target cells, and induces apoptosis.
The major proteins in the granules of CTLs (and NK cells) that are required for cytotoxicity are granzymes and perforin. In human T cells, there are five different granzymes, named A, B, H, K, and M; of these, granzymes A, B, H, and K are expressed at high levels in CD8+ CTLs. All granzymes are serine proteases; granzyme B cleaves proteins after aspartate residues. It can cleave and thereby activate caspases, which induce apoptosis. The granules also contain a sulfated proteoglycan, serglycin, which holds granzymes and perforin in the granules in an inactive state. More recent studies have identified a subset of CD8+ T cells that is not cytotoxic but whose function may be to induce and amplify inflammation. These latter cells do not synthesize granzyme B, but upon reactivation they secrete gran zyme K. This protease can activate neighboring cells to release pro-inflammatory cytokines.
Perforin is a membrane-perturbing molecule that is homol ogous to the C9 complement protein. Its main function is to facilitate the delivery of granzymes into the cytosol of the target cell, by several possible mechanisms. Perforin can polymerize and form aqueous pores in the cholesterol-containing target cell plasma membrane; it is not clear if these pores are of sufficient size to allow granzymes to enter the target cell. Alternatively, complexes of granzyme B, perforin, and serglycin are discharged from the CTL onto the target cell, and perforin insertion into the target cell membrane elicits a membrane repair process that leads to internalization of both the perforin and granzymes into endosomes. Perforin may then act on the endosomal membrane to facilitate the release of the granzymes into the target cell cytosol. Once in the cytosol, the granzymes cleave various substrates, including caspases, and initiate apoptotic death of the cell. For example, granzyme B cleaves and activates caspase-3 as well as the BCL-2 family member BID, which triggers the mitochondrial pathway of apoptosis. Another protein found in human CTL (and NK cell) granules, called granulysin, can damage cholesterol-poor membranes, typical of bacteria but not mammalian cells. This leads to the delivery of granzymes into the microbes and induction of reactive oxygen species, resulting in killing intracellular microbes.
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 (FAS-L) 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. Studies with knockout mice lacking perforin, granzyme B, or FAS-L indicate that perforin and granzyme B are the principal mediators of killing by CD8+ CTLs.
After delivering the lethal hit, the CTL is released from its target cell, which usually occurs even before the target cell dies. CTLs themselves are not injured during target cell killing, and two mechanisms have been proposed for CTL protection. First, CTLs express a serine protease inhibitor in the cytosol called Spi6 that can antagonize granzymes, including granzyme B. Second, CTL granules contain a proteolytic enzyme called cathepsin B that is delivered to the CTL surface on granule exocytosis, where it degrades errant perforin molecules that come into the vicinity of the CTL membrane. How cathepsin B is presumably preferentially delivered to the surface of CTLs and not of target cells is not known.
