T-cell Mediated Cytotoxicity

BIO/MI 494G Fall 2008 T-cell Mediated Cytotoxicity I.

Introduction 1. Intracellular pathogens, e.g. viruses, certain bacteria 2. CD8 cytotoxic T-lymphocytes (CTL) 3. Activation of naïve CD8 T-cell

II.

Mechanism of activation of naïve CD8 T-cell 1. Three means of activation

III.

Steps in CTL mediated lysis of target cells 1. Initial binding 2. Conjugated formation 3. Delivery of lethal hit 4. Detachment of CTL 5. Target cell death

IV.

Cellular mechanism of killing 1. Apoptosis

V.

Polarization of CTL towards target cell 1. Formation of supramolecular activation complex (immunological synapse) VI.

Biochemical mechanism of CTL induced target cell apoptosis 1. Lytic granules a. Perforin b. Granzymes c. Granulysin 2. Mechanism of action of lytic granules 3. Fas/Fas ligand induced apoptosis

Antigen recognition

Lymphocyte activation

Differentiation Activation of macrophages, B cells, other cells

Killing of infected "target cells"; macrophage activation

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Memory CD8+ Teell Lymphoid organs

Peripheral tissues

FIGURE 9-2 Phases of T cell responses. Antigen recognition by T cells induces cytokine (e.g., IL-2) secretion, clonal expansion as a result of IL-2-induced autocrine cell proliferation, and differentiation of the T cells into effector cells or memory cells. In the effector phase of the response, the effector C04+ T cells respond to antigen by producing cytokines that have several actions, such as the activation of macrophages and B lymphocytes, and C08+ CTLs respond by killing other cells. APC, antigen-presenting cell.

Fig. 1.27 Mechanism of host defense against intracellular infection by viruses. Cells infected by viruses are recognized by specialized T cells called cytotoxic T cells, which kill the infected cells directly. The killing mechanism involves the activation of enzymes known as caspases, which contain cysteine in their active site and cleave after aspartic acid. These in turn activate a cytosolic nuclease in the infected cell, which cleaves host and viral DNA. Panel a is a transmission electron micrograph showing the plasma membrane of a cultured CHO cell (the Chinese hamster ovary cell line) infected with influenza virus. Many virus particles can be seen budding from the cell surface. Some of these have been labeled with a monoclonal antibody that is specific for a viral protein and is coupled to gold particles, which appear as the solid black dots in the micrograph. Panel b is a transmission electron micrograph of a virus-infected cell M surrounded by cytotoxic T lymphocytes. Note the close apposition of the membranes of the virus­ infected cell and the T cell (T) in the upper left corner of the micrograph, and the clustering of the cytoplasmic organelles in the T cell between its nucleus and the point of contact with the infected cell. Panel a courtesy of M. Sui and A. Helenius; panel b courtesy of N. Rooney.

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infected cell

Fig. 1.32 Cytotoxic C08 T cells recognize antigen presented by MHC class I molecules and kill the cell. The peptide:MHC class I complex on virus­ infected 6ells is detected by antigen­ specific cytotoxic T cells. Cytotoxic T cells are preprogrammed to kill the cells they recognize.

killed infected cell

CD4 T cells: peptide + MHC class II

CDS T cells: peptide + MHC ctass I .

Cytotoxic (killer) T cells

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virus·infected cell

macrophage

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apoptotic cell B lymphoblast

Fig. 8.27 There are three classes of effector T cell, specialized to deal with three classes of pathogen. COB cytotoxic cells (left panels) kill target cells that display peptide fragments of cytosolic pathogens, most notably viruses, bound to MHC class I molecules at the cell sur1ace. TH1 cells (middle panels) and TH2 cells (right panels) both express the CD4 co­ receptor and recognize fragments of antigens degraded within intracellular vesicles. displayed at the cell sur1ace by MHC class II molecules. TH1 cells . activate macrophages, enabling them to destroy intracellular microorganisms more efficiently. They can also activate B cells to produce strongly opsonizing antibodies belonging to certain IgG subclasses (lgG1 and IgG3 in humans, and their homologs IgG2a and IgG2b in the mouse). TH2 cells, in contrast, drive B cells to differentiate and produce immunoglobulins of ~II. other types, and are responsiblefor initiating B-cell responses by activating naive B cells to proliferate and secrete IgM. The various types of immunoglobulin together make up the effector molecules of the humoral immune response.

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Fig. 8,22 Armed effector T cells can respond 10 their target cells without co-stimulation. A naive T cell that recognizes antigen on the surface of an antigen-presenting cell and receives the required two signals (arrows 1 and 2, left panel) becomes activated, and both secretes and responds to IL-2. IL-2·drlven clonal expansion (center panel) is followed by the

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differentiation of the T cells to anned effector cell stalus. Once the cells have differentiated into effector T cells, any encounter with specific antigen triggers their effector actions without the need (or co-stimulation. Thus, as illustrated here, a cytotoxic T cell can kill targets that express only the pepMe:MHC ligand and not co-stimulatory signals (right panel).

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,Apc slimulates effcc1oi'CD4 T eel~ Whi,h In,fum_elivates ttle Apc , '

APC'aclivates CD4 T cell 10 make IL-2 and naive CDS T cellle'express It~2 receptors '

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Figure 6,22 Three ways of activating a naive COB T cell. The left panels show how a naive COB T cell can be activated directly by a virus-infected dendritic cell. The center and right panels show two ways in which antigen-presenting cells (APC) that offer suboptimal co-stimulation can interact with a CD4 T cell to stimulate a naive COB T cell. One way is for cytokines

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secreted by the CD4 T cell to improve the co-stimulation of the antigen-presenting cell, for example by the induction of B7 expression (center panels). A second way is for cytokines secreted by the CD4 T cell, for example It-2, to act directly on a neighboring COB T cell (right panels).

Fig. 8.28 Interactions ofT cells with their targets Initially Involve nonspecific adhesion molecules. The major initial interaction is between LFA-1 on the T cell, illustrated here as a cytotoxic CD8 T cell, and ICAM-1 or ICAM-2 on the target cell (top panel). This binding allows the T cell to remain in contact with the target cell and to scan its surface for the presence of specific peptide:MHC complexes. If the target cell

does not carry the specific antigen, the T cell disengages (second panel) and can scan other potential targets until it finds the specific antigen (third panel). Signaling through the T-cell receptor increases the strength of the adhesive interactions. prolonging the contact between the two cells and stimulating the T cel' to deliver its effector molecules. The T cell then disengages (bonom panel).

Cross­ presentation Dendritic cell

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Infected cells and viral antigens picked up by host APCs

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Virus,

infected cell

Figure 5-7 Cross-pres~ntationof antigens to CDS· T cells. . . Cells infected with intracellular microbes, such as viruses, are captured by profeSSional antigen· presenting cells (APCs), particularly dendritic cells, and the antigens o( the infectious microbes are broken down and presented in association with the MHC molecules of the APCs. T cells recognIZe the microbial antigens and costimulalors expressed on the APCs,. and th~ T cells are activated. This example shows COB· T cells recognizing class I MHC.... ssoclated antlgem; the same c~oss­ presenting APC may display cia" II MHC .... ssociated anligens (rom the microbe (or recogntliOn by C04' helper T cells,

CTL recognizes and binds vlrus-Infecled cell

ClL programs target for death, Inducing D.NA fragmentation

.Release of lytic granules at site of cell "'lnla.c;t.

CTL migrates to new target

Target cell dies by apoptosis

Fig. 8.34 Cytotoxic CD8 T cells can induce apoptosis in target cells_ Specific recognition of peptide:MHC complexes on a target cell (top panels) by a cytotoxic COB T cell (CTL) leads to the death of the target cell by apoptosis. Cytotoxic T cells can recycle to kill multiple targets. Each killing requires the same series of steps, including receptor binding and directed release of cytotoxic proteins stored in lytic granules. The process of apoptosis is shown in the micrographs (bollom panels), where panel a shows a healthy cell with a normal nucleus. Early in apoptosis (panel b) the chroma~n becomes condensed (red) and, although the cell sheds membrane vesicles, the integrity of the cell membrane is retained, in contrast to the necrotic cell in the upper part of the same field. In late stages of apoptosis (panel c), the cell nucleus (middle cell) is very condensed, no mitochondria are Visible, and the cell has lost much of its cytoplasm and membrane through the shedding of vesicles. Photographs (x 3500) courtesy of R. Windsor and E. Hirst.

Figure 6.28 When cytotoxic T cells recognize specific antigen the delivery of cytotoxins is aimed directly at the target cell. As shown in the panels on the left, initial adhesion to a target cell has no effect on the location of the lytic granules (LG) (top panel). Engagement of the T-cell receptor causes the T cell to become polarized: the cortical actin cytoskeleton at the site of contact reorganizes, enabling the microtubule-organizing center (MTOe). the Golgi apparatus (GA), and the lytic granules to align towards the target cell (center panel). Proteins stored in lytic granules are then directed onto the target cell (bottom panel). The photomicrograph in panel a shows an unbound. isolated cytotoxic T eel/. The microtubules are stained green and the lytic granules red. Note how the lytic granules are dispersed throughout the T cell. Panel b depicts a cytotoxic T cell bound to a (larger) target cell. The lytic granules are now clustered at the site of cell-eell contact in the bound T cell. The electron micrograph in panel c shows the release of granules from a cytotoxic T cell. Panels a and b courtesy of G. Griffiths. Panel c courtesy of E.R. Podack.

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Periorin

Aids in delivering contents of granules into the cytoplasm of target cell

Granzymes

Serine proteases, which activate apoplosis once in the cytoplasm of the target cell

Granulysin

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and can Induce apoplosis

Fig. 8.37 Cytotoxic effector proteins released by cytotoxic T cells.

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caspase-3

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Fig. 8.38 Perforin, granzymes and serglycin are released from cytotoxic granules and deliver granzymes into the cytosol of target cells to induce apoptosis. Recognition of its antigen on a virus-infected cell by a cytotoxic CD8 T cell induces the release of the contents of its cytotoxic granules in a directed fashion. Perforin and granzymes, complexed with the proteoglycan serglycin, are delivered as a complex to the membrane of the target cell (top panel). By an unknown mechanism, perforin directs the entry of the granule contents into the cytosol of the target cell

without apparent pore formation, and the introduced granzymes then act on specific intracellular targets such as the proteins BID and pro-caspase-3. Either directly or indirectly, the granzymes cause the cleavage of BID into truncated BID (tBID) and the cleavage of pro-caspas e­3 to an active caspase (second panel). tBID acts on mitochondria to release cytochrome c into the cytosol, and activated caspase-3 targets ICAD to release caspase-activated DNase (CAD) (third panel). Cytochrome c in the cytosOl promotes apoptosis, and CAD fragments the DNA (bottom panel).

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Ag. 6.24 Binding of Fas ligand 10 Fas initiates the process of apoplosls. When Fas ligand (FasL) blnds Fas this produces a signal which results In adaptor proteins binding to the clustered death domains the Fas trimer. One of these Is the protein FADD, which in tUm Interacts through a second death domain with the protease caspase a. Clustered caspase a can transaetivate, cleaving caspase 8 itself 10 release an active

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Activated caspase 3 cleaves !-CAD, lhe inhibitor 01 CAD, ..nlc!lls released 10 enter the nucleus .nd cleave OIIA

caspase domain that in tum can activate other caspases. The ensuing caspase cascade culminates In the activation of the caspase-activatable DNase (CAD), which Is present in all cells In an' 'Inactive cytoplasmic form bound to an inhibitory protein called I-CAD. Wheo I-CAD Is broken down by caspases, CAD can enter the nudeus. where h cleaves DNA into the 200-base­ pair fragments that are characteristic of apoptosis,