CTLs Kill Cells in Two Ways

The effector phase of a CTL-mediated response involves a carefully orchestrated sequence of events that begin with the embrace of the target cell by the attacking cell (Figure 14-5). Long-term cultures of CTL clones have been used to identify many of the membrane molecules and membrane events involved in this process. As described below, studies with

Blood and organs

Lung 30%

Peripheral blood 19%

Kidney 41%

Mesenteric lymph node 2.5%

Gut 28%

Bone marrow 7%

Mice Lymphoid Organs Scheme

Lymphoid tissues and organs

Peripheral lymph node 0.6%

Liver 30% Spleen 11%

Localizing antigen specific CD8+ T-cell populations in vivo. Mice were infected with vesicular stomatitis virus (VSV) and during the course of the acute stage of the infection, cell populations were isolated from the tissues indicated in the figure and incubated with tetramers containing VSV-peptide/MHC complexes. Flow cytometric analysis allowed determination of the percentages of CD8+ T cells that were VSV-specific in each of the populations examined. [Adapted from P. Klenerman, V. Cerundolo, and P. R. Dunbar, 2002, Nature Reviews/ Immunology 2:269.]

Blood and organs

Lung 30%

Peripheral blood 19%

Kidney 41%

Mesenteric lymph node 2.5%

Gut 28%

Bone marrow 7%

Lymphoid tissues and organs

Peripheral lymph node 0.6%

Liver 30% Spleen 11%

FIGURE 14-4

Localizing antigen specific CD8+ T-cell populations in vivo. Mice were infected with vesicular stomatitis virus (VSV) and during the course of the acute stage of the infection, cell populations were isolated from the tissues indicated in the figure and incubated with tetramers containing VSV-peptide/MHC complexes. Flow cytometric analysis allowed determination of the percentages of CD8+ T cells that were VSV-specific in each of the populations examined. [Adapted from P. Klenerman, V. Cerundolo, and P. R. Dunbar, 2002, Nature Reviews/ Immunology 2:269.]

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Cell Death

Animation

Scanning electron micrograph of tumor-cell attack by a CTL. The CTL makes contact with a smaller tumor cell. [From J. D. E. Young and Z. A. Cohn, 1988, Sci. Am. 258(1):38.]

FIGURE 14-5

Scanning electron micrograph of tumor-cell attack by a CTL. The CTL makes contact with a smaller tumor cell. [From J. D. E. Young and Z. A. Cohn, 1988, Sci. Am. 258(1):38.]

receptor LFA-1 on the CTL membrane binds to ICAMs on the target-cell membrane, resulting in the formation of a conjugate. Antigen-mediated CTL activation converts LFA-1 from a low-avidity state to a high-avidity state (Figure 14-7). Because of this phenomenon, CTLs adhere to and form conjugates only with appropriate target cells that display antigenic peptides associated with class I MHC molecules. LFA-1 persists in the high-avidity state for only 5-10 min after antigen-mediated activation, and then it returns to the low-avidity state. This downshift in LFA-1 avidity may facilitate dissociation of the CTL from the target cell.

Electron microscopy of cultured CTL clones reveals the presence of intracellular electron-dense storage granules. These granules have been isolated by fractionation and shown to mediate target-cell damage by themselves. Analysis of their contents revealed 65-kDa monomers of a pore-forming protein called perforin and several serine proteases called gran-zymes (or fragmentins). CTL-Ps lack cytoplasmic granules and perforin; upon activation, cytoplasmic granules appear, bearing newly expressed perforin monomers.

Immediately after formation of a CTL-target cell conjugate, the Golgi stacks and storage granules reorient within the cytoplasm of the CTL to concentrate near the junction with the target cell (Figure 14-8). Evidence suggests that perforin monomers and the granzyme proteases are then released from the granules by exocytosis into the junctional space between the two cells. As the perforin monomers contact the target-cell membrane, they undergo a conformational change, exposing an amphipathic domain that inserts into the target-cell membrane; the monomers then polymerize (in the presence of

Lymphocytes Kill The Target Cell

FIGURE 14-6

Stages in CTL-mediated killing of target cells. T-cell receptors on a CTL interact with processed antigen-class I MHC complexes on an appropriate target cell, leading to formation of a CTL/target-cell conjugate. The Golgi stacks and granules in the CTL

reorient towards the point of contact with the target cell, and the granule's contents are released by exocytosis. After dissociation of the conjugate, the CTL is recycled and the target cell dies by apopto-sis. [Adapted from P. A. Henkart, 1985, Annu. Rev. Immunol. 3:31]

FIGURE 14-6

Stages in CTL-mediated killing of target cells. T-cell receptors on a CTL interact with processed antigen-class I MHC complexes on an appropriate target cell, leading to formation of a CTL/target-cell conjugate. The Golgi stacks and granules in the CTL

reorient towards the point of contact with the target cell, and the granule's contents are released by exocytosis. After dissociation of the conjugate, the CTL is recycled and the target cell dies by apopto-sis. [Adapted from P. A. Henkart, 1985, Annu. Rev. Immunol. 3:31]

-

^^Antigen-activated CTLs

Resting

CTLs

+Ab to +Ab to

\

LFA-1 ICAMd

r

1—I—1 r~m

Effect of antigen activation on the ability of CTLs to bind to the intercellular cell-adhesion molecule ICAM-1. Resting mouse CTLs were first incubated with anti-CD3 antibodies. Crosslink-age of CD3 molecules on the CTL membrane by anti-CD3 has the same activating effect as interaction with antigen-class I MHC complexes on a target cell. Adhesion was assayed by binding radiolabeled CTLs to microwells coated with ICAM-1. Antigen activation increased CTL binding to ICAM-1 more than 10-fold. The presence of excess monoclonal antibody to LFA-1 or ICAM-1 in the microwell abolished binding, demonstrating that both molecules are necessary for adhesion. [Based on M. L. Dustin and T A. Springer, 1989, Nature 341:619.]

FIGURE 14-7

Effect of antigen activation on the ability of CTLs to bind to the intercellular cell-adhesion molecule ICAM-1. Resting mouse CTLs were first incubated with anti-CD3 antibodies. Crosslink-age of CD3 molecules on the CTL membrane by anti-CD3 has the same activating effect as interaction with antigen-class I MHC complexes on a target cell. Adhesion was assayed by binding radiolabeled CTLs to microwells coated with ICAM-1. Antigen activation increased CTL binding to ICAM-1 more than 10-fold. The presence of excess monoclonal antibody to LFA-1 or ICAM-1 in the microwell abolished binding, demonstrating that both molecules are necessary for adhesion. [Based on M. L. Dustin and T A. Springer, 1989, Nature 341:619.]

to form cylindrical pores with an internal diameter of

5-20 nm (Figure 14-9a). A large number of perforin pores are visible on the target-cell membrane in the region of conjugate formation (Figure 14-9b). Interestingly, perforin exhibits some sequence homology with the terminal C9 component of the complement system, and the membrane pores formed by perforin are similar to those observed in complement-mediated lysis. The importance of perforin to CTL-mediated killing is demonstrated by perforin-deficient knockout mice, which are unable to eliminate lymphocytic choriomeningitis virus (LCMV) even though they mount a significant CD8+ immune response to the virus.

Pore formation in the cell membrane of the target is one way that perforin mediates granzyme entry; another is the perforin-assisted pathway. Many target cells have a molecule known as the mannose 6-phosphate receptor on their surface that also binds to granzyme B. Granzyme B/mannose

6-phosphate receptor complexes are internalized and appear inside vesicles. In this case, perforin is necessary for releasing granzyme B from the vesicle into the cytoplasm of the target cell.

Once it enters the cytoplasm of the target cell, granzyme B initiates a cascade of reactions that result in the fragmenta tion of the target-cell DNA into oligomers of 200 bp; this type of DNA fragmentation is typical of apoptosis. Since granzymes are proteases, they cannot directly mediate DNA fragmentation. Rather, they activate an apoptotic pathway within the target cell. This apoptotic process does not require mRNA or protein synthesis in either the CTL or the target cell. Within 5 min of CTL contact, target cells begin to exhibit DNA fragmentation. Interestingly, viral DNA within infected target cells has also been shown to be fragmented during this process. This observation shows that CTL-mediated killing not only kills virus-infected cells but can also destroy the viral DNA in those cells. It has been suggested that the rapid onset of DNA fragmentation after CTL contact may prevent continued viral replication and assembly in the period before the target cell is destroyed.

Ctl Kill Assay

FIGURE 14-

Formation of a conjugate between a CTL and target cell and reorientation of CTL cytoplasmic granules as recorded by time-lapse cinematography. (a) A motile mouse CTL (thin arrow) approaches an appropriate target cell (TC). The thick arrow indicates direction of movement. (b) Initial contact of the CTL and target cell has occurred. (c) Within 2 min of initial contact, the membrane-contact region has broadened and the rearrangement of dark cytoplasmic granules within the CTL (thin arrow) is under way. (d) Further movement of dark granules toward the target cell is evident 10 min after initial contact. [From J. R. Yanelli et al., 1986, J. Immunol. 136:377].

FIGURE 14-

Formation of a conjugate between a CTL and target cell and reorientation of CTL cytoplasmic granules as recorded by time-lapse cinematography. (a) A motile mouse CTL (thin arrow) approaches an appropriate target cell (TC). The thick arrow indicates direction of movement. (b) Initial contact of the CTL and target cell has occurred. (c) Within 2 min of initial contact, the membrane-contact region has broadened and the rearrangement of dark cytoplasmic granules within the CTL (thin arrow) is under way. (d) Further movement of dark granules toward the target cell is evident 10 min after initial contact. [From J. R. Yanelli et al., 1986, J. Immunol. 136:377].

Perforin Deliver

Completed pore

Target cell

Polymerized perforin

FIGURE 14-9

Completed pore

Target cell

Polymerized perforin

FIGURE 14-9

CTL-mediated pore formation in target-cell membrane. (a) In this model, a rise in intracellular Ca2+ triggered by CTL-target cell interaction (1) induces exocytosis, in which the granules fuse with the CTL cell membrane (2) and release monomeric perforin into the small space between the two cells (3). The released perforin monomers undergo a Ca2+-induced conformational change that al lows them to insert into the target-cell membrane (4). In the presence of Ca2+, the monomers polymerize within the membrane (5), forming cylindrical pores (6). (b) Electron micrograph of perforin pores on the surface of a rabbit erythrocyte target cell. [Part (a) adapted from J. D. E. Young and Z. A. Cohn, 1988, Sci. Am. 258(1):38; part (b) from E. R. Podack and G. Dennert, 1983, Nature 301:442.]

Some potent CTL lines have been shown to lack perforin and granzymes. In these cases, cytotoxicity is mediated by Fas. As described in Chapter 10, this transmembrane protein, which is a member of the TNF-receptor family, can deliver a death signal when crosslinked by its natural ligand, a member of the tumor necrosis family called Fas ligand (see Figure 10-19). Fas ligand (FasL) is found on the membrane of CTLs, and the interaction of FasL with Fas on a target cell triggers apoptosis.

Key insight into the role of perforin and the Fas-FasL system in CTL-mediated cytolysis came from experiments with mutant mice. These experiments used two types of mutant mice, the perforin knockout mice mentioned above and a strain of mice known as lpr (Figure 14-10). Mice that are homozygous for the lpr mutation express little or no Fas and, consequently, cells from these mice cannot be killed by interaction with Fas ligand. If lymphocytes from normal H-2b mice are incubated with killed cells from H-2k mice, anti-H-2k CTLs are generated. These H-2b CTLs will kill target cells from normal H-2k mice or from H-2k animals that are homozygous for the lpr mutation. Incubation of H-2b cells of perforin knockout mice with killed cells from H-2k mice resulted in CTLs that killed wild-type target cells but failed to induce lysis in target cells from H-2k mice homozygous for the lpr mutation.

The results of these experiments taken together with other studies allowed the investigators to make the following inter pretation. CTLs raised from normal mice can kill target cells by a perforin-mediated mechanism, by a mechanism involving engagement of target-cell Fas with Fas ligand displayed on the CTL membrane, or, in some cases perhaps, by a combination of both mechanisms. Such CTLs can kill target cells that lack membrane Fas by using the perforin mechanism alone. On the other hand, CTLs from perforin-knockout mice can kill only by the Fas-FasL mechanism. Consequently, CTLs from perforin-knockout mice can kill Fas-bearing normal target cells but not lpr cells, which lack Fas. These workers also concluded that all of the CTL-mediated killing they observed could be traced to the action of perforin-dependent killing, Fas-mediated killing, or a combination of the two. No other mechanism was detected.

This experiment taken together with a number of other studies shows that two mechanisms are responsible for initiating all CTL-mediated apoptotic death of target cells:

■ Directional delivery of cytotoxic proteins (perforin and granzymes) that are released from CTLs and enter target cells

■ Interaction of the membrane-bound Fas ligand on CTLs with the Fas receptor on the surface of target cells

Either of these initiating events results in the activation of a signaling pathway that culminates in the death of the target cell

(a) Generation of CTLs

Normal H-2b

Normal H-2*

Normal H-2b

Lymphocytes

Normal H-2*

Lymphocytes

Mitomycin C

Killed lymphocytes

Culture Normal H-2b anti-H-2* CTLs

Perforin knockout H-2b Normal H-2^

Lymphocytes

Lymphocytes

Mitomycin C Killed lymphocytes

Culture

Perforin knockout H-2b anti-H-2* CTLs

(b) Interaction of CTLs with Fas+ and Fas targets

Target cells

Normal

lpr mutant H-2^

CTLs

H-2*

(no Fas)

Normal H-2b anti-H-2*

Killed

Killed

Perforin knockout H-2b anti-H-2^

Killed

Survive

Experimental demonstration that CTLs use Fas and perforin pathways. (a) Generation of CTLs. Lymphocytes were harvested from mice of H-2b and H-2k MHC haplotypes. H-2k haplo-type cells were killed by treatment with mitomycin C and co-cultured with H-2b haplotype cells to stimulate the generation of H-2k CTLs. If the H-2b lymphocytes were derived from normal mice, they gave rise to CTLs that had both perforin and Fas ligand. If the CTLs were raised

FIGURE 14-10

Experimental demonstration that CTLs use Fas and perforin pathways. (a) Generation of CTLs. Lymphocytes were harvested from mice of H-2b and H-2k MHC haplotypes. H-2k haplo-type cells were killed by treatment with mitomycin C and co-cultured with H-2b haplotype cells to stimulate the generation of H-2k CTLs. If the H-2b lymphocytes were derived from normal mice, they gave rise to CTLs that had both perforin and Fas ligand. If the CTLs were raised by stimulation of lymphocytes from perforin knockout (KO) mice, they expressed Fas ligand but not perforin. (b) Interaction of CTLs with Fas+ and Fas-targets. Normal H-2b anti-H-2^ CTLs that express both Fas ligand and perforin kill normal H-2^ target cells and H-2^ lpr mutant cells, which do not express Fas. In contrast, H-2b anti-H-2^ CTLs from perforin KO mice kill Fas+ normal cells by engagement of Fas with Fas ligand but are unable to kill the lpr cells, which lack Fas.

by apoptosis (Figure 14-11). A feature of cell death by apopto-sis is the involvement of the caspase family of cysteine proteases, which cleave after an aspartic acid residue. The name caspase incorporates all of these elements (cysteine, aspartate protease). Normally, caspases are present in the cell as inactive proenzymes—procaspases—that require proteolytic cleavage for conversion to the active forms. More than a dozen different caspases have been found, each with its own specificity. Cleavage of a procaspase produces an active initiator caspase, which cleaves other procaspases, thereby activating their proteolytic activity. The end result is the systematic and orderly disassembly of the cell that is the hallmark of apoptosis.

CTLs use granzymes and Fas ligand to initiate caspase cascades in their targets. The granzymes introduced into the target cell from the CTL mediate proteolytic events that activate an initiator caspase. Similarly, the engagement of Fas on a tar get cell by Fas ligand on the CTL causes the activation of an initiator caspase in the target cell. Fas is associated with a protein known as FADD (ias-associated protein with death domain), which in turn associates with a procaspase form of caspase 8. Upon Fas crosslinking, procaspase 8 is converted to caspase 8 and initiates an apoptotic caspase cascade. The end result of both the perforin/granzyme and Fas-mediated pathways is the activation of dormant death pathways that are present in the target cell. As one immunologist has so aptly put it, CTLs don't so much kill target cells as persuade them to commit suicide.

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