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Cell-mediated EffectorResponses

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The cell-mediated immunity (CMI)The antibody-mediated immunity (HumoralImmnity)Not completely independent evidenced by (1) Ab-dependent cytotoxicity (ADCC)

use Abs as receptors to recognize and target cells forkilling

(2) Chemotactic peptides generated by theactivation of C’in response to Ag-Abcomplexes can contribute to assembling thecell types required for a cell-mediated response.

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• Effector Responses• General Properties of Effector T cells• Cytotoxic T Cells• Natural Killer Cells• Antibody-Dependent Cell-Mediated Cytotoxicity• Experimental Assessment of Cell-Mediated Cytotoxicity

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Direct cytotoxic responseCTL-mediated cytotoxicity

Phase I: activation and differentiation of CTL-PPhase II: CTL-mediated destruction of target cells (perforin apoptosis)

NK-cell-mediated cytotoxicityNK-cell lineageMechanism of NK-cell killingRegulation of NK-cell cytotoxicity

Ab-dependent cell-mediated cytotoxicity(ADCC)

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Cell-mediated immunity Transfer of immune cells

macrophages, neutrophils, eosinophils,natural killer cells

Nonspecific cells

CTL (TC)TH / TDTHAg-specific cellsCD8+CD4+

Cytokines required

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Functionrecognizing altered self-cellseliminating them from the body

Clearance of intracellular pathogensVirus-infected cellsTumor cellsForeign grafts (allogeneic cells)Chemically conjugated cells

DiGeorge syndromewithout thymus, No CMIeven attenuated vaccines, life-threatening infection, repeated infections

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CD4+ TH1CD4+ TH2CD8+ (CTL)

CD45 has phosphastase activity, is involved in signal transduction.

Two isoforms: CD45RA and CD45 ROCD45RO better associates with CD4/CD8 molecule, so

more sensitive to signal 1, less stringent requirementfor signal 2.

↑2~4 x fold

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Functionmediate target cell destructionincrease Mφ activationB-cell activation

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Generation of CTLs from CTL-Ps appears to require at least three sequential signals.

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Three Sequential Signals

Contact signal: CD8, TCR (on TC) & epitope, MHC I (on target)

for activationCytokine signal: IL2 (IL4, IL6, γIFN)or co-stimulatory signal: CD28-B7

for proliferation and differentiation

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Generation of effector CTLs. Upon interaction with Ag-MHC I complexes on appropriate target cells, CTL-Ps begin to express IL-2R and lesser amounts of IL-2. Proliferation and differentiation of Ag-activated CTL-Ps generally require additional IL-2 secreted by TH1 cells resulting from antigen activation and proliferation of CD4+ T cells.

Contact signal

Cytokine signal

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All nucleated cells in the body express MHCI∴ CTLs can recognize and eliminate any

altered body cellsvirus-infected cellstumor cellsgraft-rejection reaction

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Q: Is requirement of CD4+ TH cells in CTL- Pproliferation and differentiation absolute?

Depletion of CD4+ TH with Mab anti-CD4+ →CTL response (+) in miceInteraction of accessory membrane molecules → cytokine signal

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Demonstration of alternative co-stimulatory signal in generation of CTL activity. (a) Incubation of CTL-Ps with melanoma cells in vitro generates an activating signal (1) that induces transcription of the gene encoding the IL-2 receptor (IL-2R). In the absence of IL-2, however, no co-stimulatory signal is produced and the CTL-Ps fail to proliferate and differentiate.

Activation signal (1) by contact

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When melanoma cells transfected with the gene encoding B7, a cell-surface ligand, are incubated with CTL-Ps, CTL activity is generated. Interaction of CD28 and B7 is thought to produce a co-stimulatory signal (2) that induces transcription of the IL-2 gene. Autostimulationby IL-2 then leads to the proliferation and differentiation of the CTL-Ps into CTLs.

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Memory CTL-Ps have lower activation requiremens than naive cells do.

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Proliferation of memory CTL-Ps may not require help from TH cells. (a) Antigen-activated memory CTL-Ps appear to secrete sufficient IL-2 to stimulate their own proliferation and differentiation into effector CTLs. They also may not require the CD28-B7 co-stimulatory signalfor activation.

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(b) A TH cell may provide the IL-2 necessary for proliferation of an antigen-activated naive CTL-P when it binds to the same APC as the CTL-P. Also, THcells may alter the behavior of APCs in a number of ways, such as increasing the display of co-stimulatory molecules by the APC.

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B7+- transfected tumor cells might be used to induce a CTL response in vivo

Linsley et al. (治療)

B7﹢ melanoma cells mice with melanoma 40% mice, melanomas regressed

Townsend & Allison (預防) B7+ melanoma cells healthy mice

protective

immunized

Challenge with malignant melanoma cells (unaltered)

inject

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Protective effect of vaccination with B7+ melanoma cells. Normal mice were treated with melanoma cells that had been transfected with the B7 gene. Subsequently, treated mice and untreated controls were challenged with unaltered malignant melanoma cells. Only about 11% of the treated mice developed tumors, whereas 80% of the controls did.

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MHC tetramers. A homogeneous population of peptide-bound class I MHC molecules 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 TCRscomplementary 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.

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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 percentage of CD8+ T cells that were VSV-specific in each of the populations examined.

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Scanning electron micrographs of tumor-cell destruction by a CTL.

(a)A CTL makes contact with a smaller tumor cell.

(b)Membrane damage to the tumor cell results in a visible cavity and allows an influx of water, resulting in cell swelling.

(c)Lysis of the tumor cell has occurred leaving only cell debris and the nucleus.

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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 apoptosis.

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Cytotoxic T-cell clones (CTL clones)1. long-term culture2. Identical receptor specificity

available to identify membrane molecules & events involved in the destructive processi.e. sensitized TC + target cells + IL2 high conc.

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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. Crosslinkage 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 CTLsto 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.

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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 approaches an appropriate target cell. 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 is under way. (d) Further movement of dark granules toward the target cell is evident 10 min after initial contact.

Target cell

CTL

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CTL-mediated pore formation in target-cell membrane. 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 perforininto the small space between the two cells (3). The released perforin monomers undergo a Ca+-induced conformational change that allows them to insert into the target-cell membrane (4). In the presence of Ca+, the monomers polymerize wthinthe membrane (5), forming cylindrical pores (6).

The pores facilitate entry of lytic substances released from the granules

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Electron micrograph of perforin pores on the surface of a rabbit erythrocyte target cell.

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Granules contains

Perforins (65kDa), some sequence homology with C`9Granzyme (or fragmentins): serine proteases DNA fragmentation of target cells viral infected cells & virusapoptosis within 5 minutes

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Q: Why CTL itself is not killed by itsown secreted perforin molecules?

Q: Why accidental killing of inappropriate target cells does not happen?

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CTL-mediated killing in which perforin is released in small membrane-bounded vesicles. The membrane molecules on the outer surface of these vesicles direct them toward the target cell where the TCR-CD3 complex and CD8 interact with antigen-class I MHC on the target-cell membrane.

Evidenced by EM. Ab conjugated with colloidal gold to express the TCR, CD3, CD8.

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The perforin-containing vesicles thought to be derived by endocytosis results in TCR/CD3/CD8 facing inward, but subsequent membrane invagination produces smaller vesicles with TCR/CD3/CD8 facing outward. [Peters et al., 1990, Immuno. Today 11:28.]

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Target-cell destruction by apoptosisExplained observations (negative evidences)

Some CTL lines have been isolated that are potent target-cell killers but lack detectable perforin and granzymesTarget-cell killing is accomplished by some CTL lines in the complete absence of Ca2+

High level of IL-2: CTL → NK-like cellsProbably, other cytolytic mechanisms, in additions to perforin-mediated lysis, are employed by the CTL to assure rapid and complete destruction of the target cell

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(positive evidence)Interaction of CTLs and target cells resulting in a killing process → target cells show all morphologic characteristics of apoptosisDNA fragmentation (oth target cells & virus)2 factors involved:TNF on the CTL membrane, TNFR on the target cell membraneCTL and target cell contact induces a transmembrane signal

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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

haplotype cells were killed by treatment with mitomycin C and co-cultured with H-2b

haplotype cells to stimulate the generation of H-2b CTLs against k haplotype of MHCI. 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.

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(b) Interaction of CTLs with Fas+ and Fas- targets. Normal H-2b anti-H-2k CTLs that express both Fas ligand and perforin kill normal H-2k target cells and H-2k lpr mutant cells, which do not express Fas. In contrast, H-2b anti-H-2k 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.