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by: Retta Mayert


Retta Mayert
GPA 3.54

Thomas Roszman

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Thomas Roszman
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This 20 page Class Notes was uploaded by Retta Mayert on Friday October 23, 2015. The Class Notes belongs to BIO 494G at University of Kentucky taught by Thomas Roszman in Fall. Since its upload, it has received 25 views. For similar materials see /class/228198/bio-494g-university-of-kentucky in Biology at University of Kentucky.




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Date Created: 10/23/15
BIOMl 494G Fall 2008 Tcell Mediated Cytotoxicity Introduction 1 Intracellular pathogens eg viruses certain bacteria 2 CD8 cytotoxic TIymphocytes CTL 3 Activation of naive CD8 Tcell Mechanism of activation of naive CD8 Tcell 1 Three means of activation Steps in CTL mediated Iysis oftarget 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 Mechanism of action of Iytic granules FasFas ligand induced apoptosis 9N Antigen Lymphocyte Clonal Effector recognition activation expansion D39 erem39atlon functions Activation of macrophages B cells other cells 0 DquotCytokines eg lL2 Killing of infected quottarget cellsquot Cytokines macrophage egy lL39Z CD8T cell activation CTL Memory CD8 T cell Lymphoid organs Peripheral tissues FIGURE 9 2 Phases ofT cell responses Antigen recognition by T cells induces cytokine eg lL 2 secretion clonal expansion as a result of lL 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 CD4 T cells respond to antigen by producing cytokines that have several actions such as the activation of macrophages and B lymphocytes and CD8 CTLs respond by killing other cells APC antigen presenting cell Fig 127 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 infected cell killed inlected cell 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 V 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 Bui and A Helenius panel b courtesy of N Rooney cytotoxic T cell MHC class I Fig 132 Cytotoxic CDB T cells recognize antigen presented by MHC class I molecules and kill the cell The peptideMHC class I complex on virus infected cells is detected by antigen specific cytotoxic T cells Cytotoxic T cells are preprogrammed to kill the cells they recognize CD8 T cells peptide MHC class l cm T oelis peptide MHC class it J Wtotoxlc killer T cells virusinfected cell apoptotic cell THt cells THZ cells Fig 827 There are three classes of effectorT cell specialized to deal with three classes of pathogen CD8 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 surface THt cells middle panels and TH2 cells right panels both express the CD4 co receptor and recognize tragments of antigens degraded within intracellular vesicles displayed at the cell surface by MHC class it molecules THt 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 lgG subclasses lth and tgea in humans and their homologs lgG2a and lgG2b in the mouse TH2 cells in contrast drive B cells to differentiate and produce immunoglobulins of all other types and are responsible for initiating B cell responses by activating naive B cells to proliferate and secrete lgM The various types of immunoglobulin together make bacterial toxin 390 antigenspeci c B cell antitoxin an Dsg 1205 5m dead lraoellular bade a B lymphoblast pp the effector molecules of the humoral Immune response quot5tlmulall9i other Tb39 tl 3 Promer39aun r cell 39 quot 39Active iaffector39 q39gltstm v 39 39 39 I I gt vim cte39di arget39rceuvs 39 MHC class I Tcell receptor RECOGNlTlON gt PROLlFERATlON Fig 822 Armed effectorT cells can respond to their target cells without co stimulation A naive T cell that recognizes antigen on the surface of an antigenpresenting cell and receives the required two signals arrows 1 and 2 left panel becomes activated and both secretes and responds to lL2 lL2driven clonal expansion center panel is followed by the EFFECTOR FUNCTION DIFFERENTIATK I differentiation of the T cells to armed effector cell status Once the cells have differentiated into effector T cells any encounter with speci c antigen triggers their effector actions without the need for co stimulation Thus as illustrated here a cytotoxic T cell can kill targets that express only the peptideMHC ligand and not co stimulatory signals right panel 39Dendritic cells39 iprje39ssquothlgh39levelsAoi B7 and 39can39 activate halve IDS T cells virusinlecled dandritic cell o W o W a o Atgtc stiniul t reflectorCD4 T cell which In AFC APc acti vate 39CD8 T Vi K 39sCDzl T cell to make llj2 and naive 39 39 tors cell to express lt 2 recap m APC an 45 Activated CD8 cell makes lL2 driving39its own proliferation and difletentlatiqn39 39 Activated AFC espresses B7 which 139 pig stimulates nai DST cell ll2 secretedby activated cog cell 39 39 is bound b yCD Tcell39 4 Figure 622 Three ways of activating a naive CD8 T cell The left panels show how a naive CD8 T cell can be activated directly by a virusinfected dendritic cell The center and right panels show two ways in which antigenpresenting cells APC that offer suboptimal costimulation can interact with a CD4 T Cell to stimulate a naive C08 T cell One way is for cytokines leCules Fig 828 Interactions ofT cells with their targets Initially Involve quot39 adhesion molecules The 6L 39bgnm stable pairing Ahuge39napeem39iec on andlocusw release deflector molecules major initial interaction is between LFA 1 on the T cell illustrated here as a cytotoxic 008 T cell and ICAM1 or lCAM 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 speci c peptidezMHC complexes If the target cell Antigen capture Infected cells and viral antigens picked secreted by the CD4 T cell to improve the costimulation of the antigenpresenting cell for example by the induction of 8 expression center panels A second way is for cytokines secreted by the CD4 T cell for example lL2 to act directly on a neighboring CD8 T cell right panels does not carry the specific antigen the T cell disengages second panel and can scan other potential targets until it nds the speci c 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 cell to deliver its effector molecules The T cell then disengages bottom panel Cross presentation Dendritic cell Phagocylosed Virus infected cell specific Costimulator Vira an gen Figure 5 7 Crosspresentation of antigens to CD8 T cells Cells inlected with intracellular microbes such as viruses are captured by prolesSIonal antigen presenting cells APCs particularly dendritic cells and the antigens ol the infectious microbes are broken down and presented in association with the MHC molecules of the APCs T cells recognize the microbial antigens and costimulators expressed on the APCs and the T cells are activated This example shows CD8 T cells recognizing class I MHC associated antigens the same cross presenting APC may display class II MHC associated antigens from the microbe tor recognition by CD4 helperT cells CDBt T cell T cell response vlruslnlected cell CTL recognizes and binds CTL programs target for death Inducing DNA fragmentation VCTL migrates to new target Target cell dies by apoptosls Collision and nonspecific adhesion cytotoxic T cell target cell 1 moo LG I a Specific recognition redistributes cytoskeleto and cytoplasmic components of T cel Fig 834 Cytotoxic CD8 T cells can induce apoptosis in target cells Specific recognition of peptideMHC complexes on a target cell top panels by a cytotoxic CD8 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 39 binding and directed release of cytotoxic proteins stored in lytic granules The process of apoptosis is shown in the micrographs bottom panels where panel a shows a healthy cell with a normal nucleus Early in apoptosis panel b the chromatin 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 oi the same eld 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 628 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 conical actin cytoskeleton at the site of contact reorganizes enabling the microtubuleorganizing center MTOC 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 cell The microtubdles are stained green and the lytic granules red Note how the Iytic granules are dispersed throughout the T cell Panel b depicts a cytotoxicT cell bound to a larger target cell The Iytic granules are now clustered at the site of cell cell 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 ER Podack Engagement of bygpeptideleHC complex causesgdireCtedr eleasfe otp rtorjn 39d 39 39 grammes indexed With 59ml l cytotoxic granule 5M TCR I serglycin granzyme MHC t y pertorin 7V virusinfected cell 39Gr39anzyme is delivered into the cytosot of infected cell and targets BID and procaspasee m procaspase 9 Truncated BID tBID disrdpts39 mitochondrial gout r39iriembrane and activated caspase3 39 cleaves lCAD releasing caSpaseactivated 39 r DNase tCADl e cytochrome c l b cw caspase3 IL Ftelease of cytochrome c into cytosoi activates39 apoptosis and CAD39 induces DNA fragmentation v A O cleaved lCAD quot 4 v 39 Actions 39o njtarget 7 Aids in delivering contents of granules into the cytoplasm of target cell Pertorin Serine proteases which activate apoplosis once in the cytoplasm of the target cell Has antimicrobial actions and can induce apoptosis Fig 837 Cytotoxic effector proteins released by cytotoxic T cells Granzymes Granulysin Fig 838 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 caspaseS Either directly or indirectly the granzymes cause the cleavage of BID into truncated BID tBlD and the cleavage of pro caspase 3 to an active caspase second panet YB acts on mitochondria to release cytochrome G into the cytosol and activated caspase 3 targets ICAD to release caspase activated DNase CAD third panel Cytochrome c in the cytOSO39 promotes apoptosis and CAD fragmems the DNA bottom panel IS Tzr ligandfasLthstoFas Mamtodst Fig 624 Binding of Fas ligand to Fas initiates the process of apoptosis When Fas ligand FasL binds Fas this produces a signal which results in adaptor proteins binding to the clustered death domains of the Fas lrimer One oi these is the protein FADD which in torn interacts through a second death domain with the protease caspase 8 Clustered caspase 8 can transactivate cleaving caspase 8 itself to release an active caspase domain that in turn can activate other caspases The ensuing caspase cascade culminates in the activation oi the caspaseactivatable DNase CAD which is present in all cells anquot hactlv cytoplasmic ionn bound to an inhibitory protein called lCAD When iCAD is broken down by caspases CAD can enter the nudeus where it cleaves DNA into the 200 base pair fragments that are dwaracteristic oi apoptosis T Cell Maturation I and II BIOMI 494G Oct 7 9 2009 Dr Joe McGillis MN 380 3236721 139 pmcgiOl ukyedu I II 111 Introduction 1 T cell subsets a yo T cells b OLB T cells i CD4 T cells ii CD8 T cells T cell receptors 1 Structure 2 CD3 complex 3 Accessory TCR and T cell proteins 4 Comparison to BCR T cell receptor genes 1 TCR gene rearrangement 2 TCR gene maps 3 Comparison to lg gene rearrangement Thymus Gland 1 Overview 2 Structure T cell development 1 Early T cell development 2 T cell selection a Positive selection b Negative selection Tcell contact g residue of peptide T cell receptor Polymorphi residue of MHC Antibody antigenFinding SI e antigenbinding site carbohydrate Tcell receptor antigenbinding site oz chain 6 chain variable region V constant region C transmembrane egion cytoplasmic tail Tcell Figure 31 The Immune System 2e Garland Science 2005 TCR s vs BCR s Characteristic BCR 39 TCR Gene Rearrangement Junctional Diversity N amp P nucleotides use RAG1 2 express TdT Have RSS s D regions H chain Bamp y chains Single Specificity Require Ag Presentation Secreted form Affinity maturation Isotype switch Plasma membrane 003 e lmrnunoreceptor tyrosinebase activation motif lTAM Disuliide bond 9 E sevler Abbas 91 av caluwar and Ma acular Immun lugv se 7 wwwmumuansuumm CD4 CD8 Fi uuuuuuuuuuuuuuu n s Human TCR Bchain locus 620 kb chromosome 7 n 67 L vm L Van D51 Ju1 CB1 D32 J52 W 1 arm Human TCR a 8 chain locus 1000 kb chromosome 14 n 54 L vuw L Van J n 61 cquot 5 7 u sil m enh 1quotquot 397 L V62 1 5 enh Human TCR 7 chain locus 200 kb chromosome 7 n 14 L v71 L vn Jyl 0Y1 Jyz 012 3 ysll yenh chromosome 14 achain locus LuVax7080 39Jax61 ca V5 D5X3 J5X3 C5 chromosome 7 ychain locus 17 V7x12 Figure 38 The Immune System 212 Garland Science 2005 I Cortex epithelium cortico Medulla iunction Hassall s corpuscle conical epithelial cell thymicorigin thymocyle bone marrow origin medullary epithelial cell lhymicorigin dendriliccell bone marrow origin macrophage bone marrow origin subcapsular re ion Immature CD3394 839 o ical doublenegative thymocytes epithelial cell mediates t ositive 00quot ex selection Immature CD3 4 8 It doublepositive thymocytes Dendrmc cortico cell mediates medullary ative junction selection medullary eplilihelial hy vi t 39 medulla 9 venule macrophage O igme 54 The immune System 212 m Galland Szienrg mus DN1 DN2 DN3 lt CD44Io CD25 cyt B CD44 CD25 CD44 CD2539 875 rearrangement DJB VDJB Cortex DN4 PreTCR pTociB 004mm CD25 CD3very Io DP DP SP C P C PreTCR TCRocB TCRocB pTa CD3Io CD3Io CD44 CD4 CD4 CD25 CD8 or CD3VI CD8 CD4 CD8 0t V 5 rearrangement Poste Selection Negative Selection F Medulla DN1 DN2 DN3 DP SP gt DN4 ltDCPgt


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