Pharm immuno7&8 cytokines r

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  • Fig 3-11
  • TAP1 & TAP2 are proteins encoded by MHC I gene TAP is necessary for the proper assembly of MHC I TAP: Transporter associated with antigen processing
  • Cofactor: Ubiquitin One of the most conserved proteins known Ubiquitin binding to proteins to be degraded Covalent Isopeptide bonds from the C-terminal glycine residue to  -amino groups of lysyl side chains Most abundant ubiquitin-protein conjugate Ubiquitin-Histone2A Not degraded Present primarily in actively transcribed chromosomal regions Ubiquitin: Homologous proteins BAG-1 : Binds to BCL-2; Inhibits apoptosis Ubiquitin cross-reactive protein (UCRP Overall process Multienzymatic Requires ATP at multiple steps Neutral or slightly basic pH Protein targeted for proteasome degradation: By binding to polyubiquitin chain Degradation usually by proteasome Covalently bound ubiquitin occasionally targets proteins for lysosomal degradation Some proteins may not require ubiquitination for proteasome degradation Short-lived enzyme ornithine decarboxylase Many denatured polypeptides (e.g. casein) Proteasome localization: Nucleus & Cytoplasm Proteins cleaved At selected amino acid sites: Arg, Phe, Tyr, Leu, Glu, Asp adjacent to leaving group By different β subunits with specific activities, acting together Chymotrypsin-like: After large hydrophobic residues; Rate-limiting site Trypsin-like: After basic residues Caspase-like (Peptidylglutamyl-peptide): After acidic residues; Aspartyl > Glutamate ?? After branched-chain or small neutral amino acids End products: Oligopeptides (4 to 24 residues) small enough to diffuse out of proteasome Substrates: Most short-lived regulatory proteins & misfolded proteins degraded by this system Inhibitors: Clastolactacystin β-lactone; Peptide-vinyl sulfones Role: Proteasome is primary site for degrading proteins (80% to 90%) in mammalian cells Ubiquitin binding process E1 : Activates carboxyl end of ubiquitin by conversion to thiol ester E2 : Carrier proteins (family) E3 (Ubiquitin-protein ligase) : Couples ubiquitin to protein substrate Ubiquitin binding site: Carboxyl group Protein substrate binding site:  -amino groups of lysines
  • Ubiquitin is a small (8.5kD) protein present in all eukaryotic cells. Its 76 amino acid sequence is so highly conserved that nearly identical versions exist in a variety of organisms; yeast and human ubiquitin, for example, differ at only 3 of the 76 residues. It is involved in multiple cellular functions: protein degradation, chromatin structure, and heat shock.
  • 1950- 1970: Antiviral interferons: IFN-  as T-cell-derived antiviral protein or activator of macrophage (Macrophage-activating factor) Pyrogens: IL-1 in association with bacterial infection Cytokines as regulators & effectors; need receptors; work at low concentrations
  • MCP-1: monocyte chemoattractant protein-1. MIP-1 α : Macrophage inflammatory protein RANTES A member of the interleukin-8 superfamily of cytokines. This cytokine is an 8-kD protein that is a selective chemoattractant for memory T lymphocytes and monocytes. Origin [ R egulated on a ctivation, n ormal T e xpressed and s ecreted]
  • interleukin-8 (IL-8) A cytokine (chemokine) derived from endothelial cells, fibroblasts, keratinocytes, macrophages, and monocytes which causes chemotaxis of neutrophils and T-cell lymphocytes. Syn: monocyte-derived neutrophil chemotactic factor, neutrophil-activating factor, neutrophil chemotactant factor, anionic neutrophil-activating peptide.
  • interferon (IFN) in-ter-fTrcon A class of small protein and glycoprotein cytokines (15–28 kD) produced by T cells, fibroblasts, and other cells in response to viral infection and other biological and synthetic stimuli. Interferons bind to specific receptors on cell membranes; their effects include inducing enzymes, suppressing cell proliferation, inhibiting viral proliferation, enhancing the phagocytic activity of macrophages, and augmenting the cytotoxic activity of T lymphocytes. Interferons are divided into five major classes (alpha, beta, gamma, tau, and omega) and several subclasses (indicated by Arabic numerals and letters) on the basis of physicochemical properties, cells of origin, mode of induction, and antibody reactions. The discovery in 1957 that viral infection of human cells induces the formation of natural antiviral agents raised the hope that these substances might have therapeutic potential. Early studies showed that, unlike antibodies, interferons are active against a broad range of viruses, but progress in applying this knowledge to human medicine was retarded by the difficulty of producing interferons in sufficient quantity. In the 1980s the development of recombinant DNA technology overcame this obstacle, and interferons now play an important role in the treatment not only of viral infections but also of certain malignancies. Commercially available interferons are produced by genetically altered colonies of Escherichia coli or Chinese hamster ovary cells, or are induced by controlled viral infection in pooled human leukocytes. Alpha interferons have found the widest application in medicine. (The spelling alpha is used with respect to naturally occurring interferons; in compliance with international conventions for generic drug names, the spelling alfa appears in names of pharmaceutical formulations.) Alpha interferons are used in the treatment of chronic hepatitis B and hepatitis C, hairy cell leukemia, chronic myelogenous leukemia, AIDS-related Kaposi sarcoma, malignant melanoma, condylomata acuminata and recurrent respiratory papillomatosis due to human papillomavirus, and infantile hemangiomatosis. About 50% of patients treated for chronic hepatitis B with interferon-alfa show disappearance of hepatitis Be antigen (HBeAg) and reversion of alanine aminotransferase to normal. The response rate in chronic hepatitis C is lower (15–25%), but better results are achieved by using more aggressive therapy (daily rather than thrice weekly administration) and continuing it longer (a minimum of 12 months). Modified formulations of interferon-alfa conjugated with polyethylene glycol (PEG), which have yielded promising results in hepatitis C with once-a-week dosing, are in phase III trials. Beta interferons reduce clinical recurrences and progression of myelin damage in multiple sclerosis. Gamma interferon is effective in retarding tissue changes in osteopetrosis and systemic scleroderma and in reducing the frequency and severity of infections in chronic granulomatous disease. Administration of interferons is parenteral (intravenous, intramuscular, subcutaneous, intranasal, intrathecal, or intralesional) and several weeks of treatment may be required before clinical response is noted. More than 50% of patients experience a flulike syndrome of fatigue, myalgia, and arthralgia. Gastrointestinal and CNS side effects are also common, and marrow suppression may occur with prolonged treatment. Origin [interfere + -on] antigen interferon fibroblast interferon immune interferon interferon alfa 2b interferon alpha interferon beta interferon beta 1b interferon gamma interferon-omega interferon-tau leukocyte interferon trophoblast interferon type I interferon type II interferon
  • Chemotactant = chemotractant = chemoattractant keratinocyte A cell of the living epidermis and certain oral epithelium that produces keratin in the process of differentiating into the dead and fully keratinized cells of the stratum corneum. CCL5 (earlier called RANTES ) is an 8kDa protein classified as a chemotactic cytokine or chemokine . CCL5 is chemotactic for T cells , eosinophils , and basophils , and plays an active role in recruiting leukocytes into inflammatory sites. With the help of particular cytokines (i.e., IL-2 and IFN-γ ) that are released by T cells , CCL5 also induces the proliferation and activation of certain natural-killer ( NK ) cells to form CHAK (CC-Chemokine-activated killer) cells. [1] It is also an HIV -suppressive factor released from CD8+ T cells . This chemokine has been localized to chromosome 17 in humans.
  • margination marcji-nQcsh\\n A phenomenon that occurs during the relatively early phases of inflammation; as a result of dilation of capillaries and slowing of the bloodstream, leukocytes tend to occupy the periphery of the cross-sectional lumen and adhere to the endothelial cells that line the vessels.
  • Lymphotoxin: A lymphokine from T lymphocytes that lyses or damages many cell types
  • TNF-alpha, IL-1 &LPS induce E-selectins and P-selectins Selectins interact with oligosaccharides on WBCs ICAM and Integrins are involved Cells migrate
  • function is achieved by interactions between ligands on the tumor cell and a variety of receptors on the NK cell, leading to the release of the NK cell’s cytotoxic granules . The various types of NK receptors, their ligands, and their contradictory effects on the cell’s ability to kill are the subject of this article. Figure 1. Crosslinking of the activation receptors leads to phosphorylation of the ITAM sequences on the associated DAP12 molecule by a src-family kinase. The phosphorylated ITAMs recruit and activate tyrosine kinases of the ZAP70/syk family. Crosslinking of the inhibitor receptor leads to ITIM phosphorylation and recruitment of the tyrosine phosphatase SHP-1, which then dephosphorylates the ZAP70/syk activation molecules. SRC v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) [ Homo sapiens ]
  • Natural Killer Cell Tolerance for Self Summary: Wayne Yokoyama studies natural killer (NK) cells, which are important components of the body's immune system, particularly in innate host defense against viruses and tumors. These cells possess potent effector functions, such as cellular killing and production of proinflammatory cytokines, that must be restrained to avoid inadvertent destruction of normal tissues; i.e., they must manifest tolerance for self. Recent studies indicate that this tolerance is achieved by a "licensing" process in which NK cell receptors recognize self–major histocompatibility complex class I molecules and acquire functional competence. A central theme in biology is the capacity to discriminate between self and nonself. From plants to vertebrates, a variety of distinct discrimination strategies have evolved for different biological processes. This discrimination is especially relevant for lymphocytes, vertebrate immune cells that express receptors capable of recognizing foreign invaders and also normal self-tissues. Natural killer (NK) cells comprise the third major lymphocyte population. As with other lymphocytes—B cells that produce antibodies, and T cells that are responsible for cell-mediated immunity—NK cells display potent effects, including the capacity to kill other cells and produce proinflammatory cytokines. For all lymphocytes, these effector functions must be regulated to prevent inadvertent destruction of normal tissues. This regulation— tolerance for self—is well understood for B and T cells, but much less is known about NK cell control. NK cells use unique mechanisms to control their reactivity against normal self-tissues. Each individual NK cell simultaneously expresses multiple germline-encoded receptors with differing ligand specificities. Some receptors recognize surface ligands on their targets and activate the effector functions. Stimulation through the activation receptors can be inhibited by other germline-encoded NK cell receptors with specificity for major histocompatibility complex class I (MHC-I) molecules expressed on target cells. The inhibitory receptors provide a molecular explanation for the "missing-self" hypothesis of Klas Kärre (Karolinska Institute), whereby NK cells are proposed to survey tissues for expression of MHC-I that prevents target killing. In the absence of MHC-I, due to a pathogenic event such as viral infection, the NK cell is then able to kill. Thus, the NK cell apparently discriminates between normal and abnormal cells by its inhibitory receptors for MHC-I molecules that are ubiquitously expressed on normal cells. Some predictions of the missing-self hypothesis, however, have not been experimentally observed. For example, the genetic absence of host MHC-I should lead to chronic activation of NK cells and tissue destruction due to absence of inhibition. Instead, NK cells from MHC-deficient hosts are defective in target killing. Another prediction is that all NK cells should express at least one receptor for self–MHC-I, but this appears not to be the case. Finally, MHC molecules are among the most polymorphic molecules known, with multiple genes in each individual and multiple alleles displaying differences between individuals. The inhibitory receptors are similarly highly polymorphic, with different MHC-I specificities. The dual polymorphisms of receptors and ligands raise the difficult question of how the right NK cell receptors can be appropriately paired with the relevant self-MHC molecules because the genes for the inhibitory receptors and their MHC ligands lie on different chromosomes and hence they are inherited independently. Therefore, how NK cell self-tolerance arises is poorly understood. Recently, our laboratory provided new insight into these dilemmas. Although the previously observed inability of NK cells from MHC-deficient hosts to kill targets could be due to many different reasons, further dissection with cellular targets was difficult because the universe of NK cell receptors involved in target recognition is still incompletely defined. We thus developed a target cell–free system for stimulation of resting NK cells through antibody cross-linking of the NK1.1 (Nkrp1c) molecule, an activation receptor expressed by all mature and developing CD3– NK cells in C57BL/6 (B6) mice. To assess the response of individual NK cells, we used flow cytometry and intracellular staining for interferon-γ production. This deceptively simple assay was very informative. When isolated from wild-type B6 mice, large numbers of NK cells were activated, whereas few NK cells were stimulated when derived from mice deficient in β2-microglobulin (β2m), the noncovalently associated light chain that is required for normal MHC-I expression. NK cells from mice lacking classical MHC-I heavy chains (Kb–/– Db–/–) were also unresponsive to NK1.1 cross-linking. Subsequent studies extended these results to other activation receptor pathways and indicated that the effects were intrinsic to the NK cell. Thus, NK cell functional maturation requires specific interaction with host classical MHC-I molecules; we have termed this process MHC-I–dependent "licensing" to distinguish it from MHC-I–dependent "education" that implies different events occurring during T cell development. In studies of MHC-congenic, -recombinant congenic, and -transgenic mice, we found that licensed NK cells are correlated with their expression of receptors with self–MHC-I specificity. Paradoxically, these receptors belong to the Ly49 family of NK cell receptors that were first characterized as inhibitory MHC-I–specific receptors in effector responses. Thus, our studies strongly suggested that the inhibitory receptors play a second role in NK cell response, i.e., the ability to license NK cell functions, ironically a positive effect. To show definitively that a Ly49 inhibitory receptor confers licensing, we took two complementary approaches. The first used a single-chain trimer (SCT) of the H2Kb MHC-I molecule that is a single, complete, appropriately folded MHC-I polypeptide developed by our collaborator, Ted Hansen (Washington University in St. Louis). SCT-Kb tetramers bound only one NK cell receptor on primary NK cells, the Ly49C inhibitory receptor. In a transgenic SCT-Kb mouse with concomitant deficiencies in H2Kb, HDb, and β2m, only one MHC-I molecule is expressed, SCT-Kb, and only Ly49C+ NK cells are licensed. In a second approach, we used retroviral transduction to express the Ly49A inhibitory receptor in bone marrow stem cells and reconstituted irradiated hosts. Transduced expression of Ly49A conferred enhanced functional capacity of NK cells but only in mice bearing the MHC-I ligand for Ly49A. Thus, the inhibitory MHC-I receptors confer the licensing effect on NK cells when their ligands are expressed as self–MHC-I molecules. To determine if the inhibitory receptors confer licensing directly or indirectly, we transduced Ly49A constructs with cytoplasmic mutations. Cytoplasmic domain–deficient Ly49A molecules did not confer licensing. The only known cytoplasmic signaling motif is the immunoreceptor tyrosine-based inhibitory motif (ITIM), responsible for inhibitory effects in effector responses. A point mutation in the Tyr residue of the ITIM abrogated the licensing effect. Thus, the receptor itself directly delivers the licensing effect to NK cells. (These studies were also supported by grants from the National Institutes of Health.) Licensing thus results in two types of self-tolerant NK cells, unlicensed NK cells and licensed NK cells with self–MHC-I inhibitory receptors. NK cells that do not express self–MHC-I–specific receptors do not become licensed and do not need to be inhibited by MHC-I because they are not functionally competent. MHC-I–dependent effector inhibition is therefore only relevant for licensed, competent NK cells; they are inhibited by the same self-MHC–specific receptors that confer licensing. Licensing thus pairs an inhibitory receptor with its cognate self–MHC-I ligand for functional development of NK cells. Our recent investigations and those of others suggest that human NK cells may undergo a similar process, resulting in the enhanced function of NK cells bearing a killer immunoglobulin-like receptor (KIR) specific for a given HLA (human MHC) class I molecule. These findings help explain clinical studies in which patient outcomes in several situations, such as resolution of chronic infections, are associated with certain pairs of KIR and HLA genotypes. Licensing considerations may also improve the outcome of bone marrow transplantations. Thus, NK cells may be harnessed for immune-based therapies by interventions that affect licensing. Our studies have other important implications. Other germline-encoded inhibitory receptors with ITIMs, related to the NK cell receptors, are broadly expressed on many leukocytes, suggesting that they may also be involved in self-tolerance mechanisms. Finally, we have described a unique biological strategy for self versus nonself discrimination. Last updated: December 14, 2006 HHMI INVESTIGATOR Wayne M. Yokoyama   AT HHMI Licensing to Kill (08.05.05) Learning How a Virus Evades the First Line of Immune Defense (06.24.04) How Natural Killer Cells Thwart Viral Infection (05.03.01) ON THE WEB The Yokoyama Lab (wustl.edu) Search PubMed Slide content These examples demonstrate how the expression of MHC class I protects against NK cell-mediated lysis, leading to Kärre’s “missing-self hypothesis” In this hypothesis, NK cells are constantly surveying tissues for normal expression of MHC class I. Because class I molecules are expressed on all tissues, NK cell cytotoxic activity is typically inhibited. However, when an NK cell finds a cell with down-regulated or mutated MHC class I, it is released from its inhibition and the target cell is lysed. This NK cell function is important because certain viruses are able to down-regulate MHC class I expression in the cell they’ve infected, protecting themselves from detection by cytotoxic T cells. Some tumors also have diminished MHC class I expression, and their recognition and lysis is the basis of “natural” killing.
  • The major surface molecules of CD4 + T cells involved in the activation of these cells (the receptor s), and the molecules on APCs (the ligand s) recognized by the receptors are shown CD8 + T cells use most of the same molecules, except that the TCR recognizes peptide-class I MHC complexes, and the coreceptor is CD8 , which recognizes class I Immunoreceptor tyrosine-based activation motifs ( IT AM s ) are the regions of signaling proteins that are phosphorylated on tyrosine residues and become docking sites for other signaling molecules CD3 is composed of three polypeptide chains
  • Fig 5-3B: Ligand-receptor pairs involved in T cell activation The important properties are summarized of the major " accessory" molecules of T cells, so called because they participate in responses to antigens but are not the receptors for antigen CTLA-4 (CD152) is a T cell receptor for B7 molecules that delivers inhibitory signals It shuts off T cell responses VLA molecules are integrins involved in leukocyte binding to endothelium LFA: Leukocyte function-associated AgMain Entry: LFA-1 Pronunciation: el-( )ef-( ) - w n Function: noun l ymphocyte f unction-associated a ntigen-1 : an integrin that is a heterodimeric protein on the surface of leukocytes and that functions in the adhesion of lymphocytes to other cells Learn more about "LFA-1" and related topics at Britannica.com Find Jobs in Your City Earn your Degree. Search top online and campus programs now. Sponsored Links Lfa 3 Get the Answers You're Looking For. Lfa 3 www.RightHealth.com /Chemotherapy Lexus Concept Vehicles Get an overview of the Lexus LF-A Concept Vehicle. www.Lexus.com Lva 11 Beautify Your Home on a Budget. Save on Lva 11! PriceGrabber.com/IndoorLiving Pronunciation Symbols
  • FasL = Fas ligand a molecule on the surface of cytotoxic T cells that binds to its receptor, Fas, on the surface of other cells, initiating apoptosis in the target cell. Fas A receptor present in cells that binds with Fas ligand to induce apoptosis
  • Granule-associated killing mechanisms Cytotoxic cell vesicles release Perforin Enzymes Leading to polymerization of perforins  Polyperforin channels in the membrane of the target cell Granules with enzymes (Granzymes) release enzymes that enter the target cell through polyperforin channels  death of target
  • FasL = Fas ligand a molecule on the surface of cytotoxic T cells that binds to its receptor, Fas, on the surface of other cells, initiating apoptosis in the target cell. Fas A receptor present in cells that binds with Fas ligand to induce apoptosis
  • Many functions start before the specific immunity starts
  • Pharm immuno7&8 cytokines r

    1. 1. Pharm-Immunology 7 & 8 7. Major Histocompatibility complex (MHC) 8. Cytokines &Cell Mediated Immunity Dr. Hussein
    2. 2. 7. MHC Objectives <ul><li>Extracellular antigens (bacterial infections) enter the MHC II pathway and presentation </li></ul><ul><li>Intracellular and cytoplasmic antigens such as virus, intracellular bacteria and tumor antigens enter the MHC I pathway and presentation </li></ul><ul><li>Role of class II invariant chain peptide (CLIP, Ii) in the development of MHC II </li></ul><ul><li>Role of the specialized transport molecule, transporter associated with antigen processing (TAP: TAP1 & TAP2), in MHC I development and presentation </li></ul>
    3. 3. The Role of MHC in Ag Presentation to T Cells <ul><li>Following Ag processing, the Ag is presented to lymphocytes in a form they can recognize </li></ul><ul><li>Ag presentation to CD4+ helper T cells is associated with MHC II </li></ul><ul><li>Ag presentation to CD8+ cytotoxic T cells is associated with MHC I. </li></ul><ul><li>APCs are usually macrophages, but any nucleated cell may serve as an APC </li></ul><ul><li>Ag processing is the series of events that occur between exposure to an Ag and eventual immune response: Ab production or T-cell activity. It includes fragmentation of the protein Ag into small peptides in the macrophage and the presentation to T cells as above. </li></ul>
    4. 4. Fig. 3-6: Human HLA (MHC) Genes <ul><li>Schematic maps of the human MHC (HLA complex) </li></ul><ul><li>illustrating the major genes that code for molecules </li></ul><ul><li>involved in immune responses </li></ul><ul><li>Sizes of genes and distances between them </li></ul><ul><li>are not drawn to scale . </li></ul>
    5. 5. The structure of class I MHC molecule <ul><li>The schematic diagrams and models of the crystal structures of MHC I and MHC II (next slide) molecules illustrate the domains of the molecules and the fundamental similarities between them </li></ul><ul><li>MHC I molecule contains: </li></ul><ul><ul><li>Peptide-binding clefts </li></ul></ul><ul><ul><li>Invariant portions that bind: </li></ul></ul><ul><ul><ul><li>CD8 (  3 domain) or </li></ul></ul></ul><ul><ul><li>ß2m: ß2-microglobulin </li></ul></ul>
    6. 6. The structure of class II MHC molecules <ul><li>The schematic diagrams and models of the crystal structures of MHC II molecules illustrate the domains of the molecule and the fundamental similarities to MHC I </li></ul><ul><li>MHC II molecules contain: </li></ul><ul><ul><li>Peptide-binding cleft </li></ul></ul><ul><ul><li>Invariant portion that binds: </li></ul></ul><ul><ul><ul><li>CD4 ( ß2 domain) </li></ul></ul></ul>
    7. 7. Figure 3-8 Properties of MHC molecules and genes
    8. 8. Fig.3-8 Properties of MHC molecules and genes
    9. 9. Fig 3-10: Features of peptide binding to MHC molecules
    10. 10. Fig 3-10
    11. 11. Pathways of intracellular processing of protein Ags <ul><li>MHC II pathway </li></ul><ul><li>converts protein </li></ul><ul><li>Ags that are </li></ul><ul><li>endocytosed into </li></ul><ul><li>vesicles of APCs </li></ul><ul><li>into peptides that </li></ul><ul><li>bind to MHC II </li></ul><ul><li>molecules for </li></ul><ul><li>recognition by </li></ul><ul><li>CD4 + T cells </li></ul><ul><li>MHC I pathway converts proteins in the cytoplasm into peptides that bind to MHC I molecules for recognition by CD8 + T cells . </li></ul><ul><li>ER, endoplasmic reticulum </li></ul>Fig 3-11 MHC II pathway MHC I pathway
    12. 12. Fig 3-12 Features of the pathways of antigen processing
    13. 13. Fig 3-12 Features of the pathways of antigen processing
    14. 14. MHC I pathway of processing of cytosolic antigens <ul><li>Proteins enter the cytoplasm of cells either from: </li></ul><ul><ul><li>phagocytosed microbes or </li></ul></ul><ul><ul><li>from endogenous synthesis by microbes , such as viruses, that reside in the cytoplasm of infected cells </li></ul></ul><ul><li>Cytoplasmic proteins are un-folded, ubiquitinated, and degraded in proteasomes </li></ul><ul><li>The peptides that are produced are transported by the TAP transporter into the ER, where the peptides bind to newly synthesized MHC I </li></ul><ul><li>The peptide-MHC I complexes are transported to the cell surface and are recognized by CD8 + T cells </li></ul>Fig 3-14
    15. 15. Transporter associated with antigen processing (TAP) & class II invariant chain peptide (CLIP) <ul><li>TAP  MHC I </li></ul><ul><li>TAP1 & TAP2 are proteins encoded by genes in the MHC “II” locus </li></ul><ul><li>TAP is necessary for the proper assembly of MHC I </li></ul><ul><li>TAP transport peptides actively into the ER where MHC I is assembled </li></ul><ul><li>MHC I without TAP molecule cannot be loaded with the peptide to be displayed on cell surface </li></ul><ul><li>MHC I without peptide is instable & would be destroyed by proteases </li></ul><ul><li>CLIP/Ii  MHC II </li></ul><ul><li>Newly synthesized MHC II carries CLIP or Ii peptide (class II invariant chain peptide)in the ER </li></ul><ul><li>If MHC II is found without peptide it will be degraded </li></ul><ul><li>DM (HLA-DM) is a peptide exchange molecule looks like MHC II </li></ul><ul><li>DM in the endosome removes CLIP from the cleft of MHC II </li></ul><ul><li>DM is not polymorphic , MHC II is </li></ul><ul><li>Ii will be replaced by the presentable, processed peptide and becomes stable, otherwise it will be degraded </li></ul>
    16. 16. The role of MHC-associated Ag presentation in the recognition of microbes by CD4 + T cells <ul><li>Protein antigens of microbes that are endocytosed from the extracellular environment by macrophages and B lymphocytes enter the MHC II pathway of antigen processing. </li></ul><ul><li>As a result, these proteins are recognized by CD4 + helper T cells, whose functions are to activate macrophages to destroy phagocytosed </li></ul><ul><li>microbes and </li></ul><ul><li>activate B cells </li></ul><ul><li>to produce Abs </li></ul><ul><li>against </li></ul><ul><li>extracellular </li></ul><ul><li>microbes and </li></ul><ul><li>toxins </li></ul>Fig 3-15A
    17. 17. The role of MHC-associated antigen presentation in the recognition of microbes by CD8 + T cells <ul><li>Protein Ags of microbes that live in the cytoplasm of infected cells enter the MHC I pathway of Ags processing </li></ul><ul><li>As a result, these proteins are recognized by CD8 + CTLs , whose function is to kill infected cells </li></ul>Fig 3-15B
    18. 18. Ubiquitination of proteins FYI
    19. 19. Ubiquitin <ul><li>Ubiquitin is a small (8.5kD) protein present in all eukaryotic cells. </li></ul><ul><li>Its 76 amino acid sequence is so highly conserved that nearly identical versions exist in a variety of organisms </li></ul><ul><ul><li>yeast and human ubiquitin differ at only 3 of the 76 residues </li></ul></ul><ul><li>It is involved in multiple cellular functions: </li></ul><ul><ul><li>protein degradation </li></ul></ul><ul><ul><li>chromatin structure </li></ul></ul><ul><ul><li>heat shock </li></ul></ul>FYI
    20. 20. Pharm-Immuno 8 Cytokines &Cell Mediated Immunity Dr. Saber Hussein
    21. 21. Objectives <ul><li>1. Define: Cytokine, lymphokine, chemokine </li></ul><ul><li>2. Biological characterization and Sources of cytokines </li></ul><ul><li>3. Role of cytokines in lymphocytes activation, growth and differentiation </li></ul><ul><li>4. Role of cytokines in immune-mediated inflammation </li></ul><ul><li>5. T-cell independent defense mechanisms: </li></ul><ul><li>Phagocytosis & chemotaxis </li></ul><ul><li>6. Central role of T helper cells in T-cell-dependent cell-mediated immunity </li></ul>
    22. 22. Objectives <ul><li>7. Cytotoxic T cells function & relation to T h </li></ul><ul><li>8. Cell-mediated cytotoxicity: </li></ul><ul><li>a. Ab-independent </li></ul><ul><li>i. MHC-presentation dependent </li></ul><ul><li>ii. MHC unrestricted: NK, LAK (Lymphokine Activated Killer) </li></ul><ul><li>b. Ab-dependent cell-mediated cytotoxicity (ADCC) </li></ul><ul><li>9. Role of macrophages in immune response </li></ul>
    23. 23. Types of intracellular microbes combated by T cell-mediated immunity <ul><li>A . Microbes may be ingested by phagocytes and survive within vesicles (phagolysosomes) or escape into the cytoplasm where they are not susceptible to the microbicidal mechanisms of the phagocytes </li></ul><ul><li>B . Viruses may bind to receptors on many cell types, including nonphagocytic cells, and replicate in the cytoplasm of the infected cells. Some viruses establish latent infections , in which viral proteins are produced in infected cells </li></ul>
    24. 27. Definitions <ul><li>Cytokine : </li></ul><ul><ul><li>Small protein , secreted by cells to influence behavior of other cells. </li></ul></ul><ul><ul><li>The effect is receptor-mediated </li></ul></ul><ul><li>Lymphokine : </li></ul><ul><ul><li>Cytokine made by lymphocytes; interleukins </li></ul></ul><ul><li>Chemokines : </li></ul><ul><ul><li>Chemotactic cytokines; bind heparin; lymphocytes & phagocytes migration; inflammatory responses </li></ul></ul><ul><li>Monokine : </li></ul><ul><ul><li>Cytokine produced by monocytes </li></ul></ul>
    25. 28. Biology of cytokines <ul><li>Antiviral interferons: </li></ul><ul><ul><li>IFN-  as T-cell-derived antiviral protein or </li></ul></ul><ul><ul><li>activator of macrophage (Macrophage-activating factor) </li></ul></ul><ul><li>Pyrogens: </li></ul><ul><ul><li>IL-1 in association with bacterial infection </li></ul></ul><ul><li>Cytokines as: </li></ul><ul><ul><li>Regulators </li></ul></ul><ul><ul><li>Effectors </li></ul></ul><ul><ul><li>need receptors </li></ul></ul><ul><ul><li>work at low concentrations like hormones </li></ul></ul><ul><ul><ul><li>Exocrine </li></ul></ul></ul><ul><ul><ul><li>Paracrine </li></ul></ul></ul><ul><ul><ul><li>Autocrine </li></ul></ul></ul>
    26. 29. Some common cytokines R egulated on a ctivation, n ormal T e xpressed and s ecreted CXCL8 (IL-8, )
    27. 32. Actions of IL2
    28. 33. NK
    29. 34. Lymphocyte activation <ul><li>Regulators of lymphocytes: </li></ul><ul><ul><li>IL-2 </li></ul></ul><ul><ul><li>IL-4 </li></ul></ul><ul><ul><li>TGF-  </li></ul></ul><ul><li>T H produce cytokines involved in regulation of acquired, specific immune response </li></ul>
    30. 35. IL-2 & IL-4 receptors <ul><li>IL-2 receptor is high-affinity, composed of 3 polypeptides: </li></ul><ul><ul><li>α & β bind to IL-2 </li></ul></ul><ul><ul><li>γ is involved in signaling to the cell in both receptors. </li></ul></ul><ul><li>IL-4 has only α chain with a binding site. </li></ul>
    31. 36. Cytokine Action <ul><li>Cytokine binds to its Receptor  Ligand-induced aggregation  Activation of intracellular signaling pathways (kinase cascade)  activation of transcription factors  Into nucleus  Binding to promoter or enhancer  Gene transcription </li></ul>
    32. 37. Cytokines & CD4 + T H Differentiation <ul><li>IL-12, IFN  , TGF  favor: T H0  T H1 </li></ul><ul><li>IL4 favors: T H0  T H2 The cytokine pattern influences the effector functions that are activated </li></ul>Ab  B cell Tc activation
    33. 38. IL-8 ( CXCL8 , RANTES) <ul><li>A cytokine (chemokine) derived from: </li></ul><ul><ul><li>endothelial cells, </li></ul></ul><ul><ul><li>fibroblasts, </li></ul></ul><ul><ul><li>keratinocytes, </li></ul></ul><ul><ul><li>macrophages, and </li></ul></ul><ul><ul><li>monocytes </li></ul></ul><ul><li>IL-8 causes chemotaxis of </li></ul><ul><ul><li>neutrophils and </li></ul></ul><ul><ul><li>T-cell lymphocytes . </li></ul></ul><ul><li>It is also called </li></ul><ul><ul><li>monocyte-derived neutrophil chemotactic factor, </li></ul></ul><ul><ul><li>neutrophil-activating factor, </li></ul></ul><ul><ul><li>neutrophil chemotactant factor, </li></ul></ul><ul><ul><li>anionic neutrophil-activating peptide </li></ul></ul><ul><ul><li>R egulated on A ctivation, N ormal T E xpressed and S ecreted </li></ul></ul>CCL5 RANTES Chemokine (C-C motif) ligand 5
    34. 39. Immune-mediated inflammation <ul><li>1. Recruitment of inflammatory cells via cytokine network </li></ul><ul><li>2. Specific receptors on target cells </li></ul><ul><li>3. Ag-activated CD4 & CD8 lymphocytes are main producer of cytokines that regulate immune-mediated inflammation </li></ul><ul><li>4. CD4 & CD8 cytokines are regulators & effectors </li></ul>
    35. 40. These cytokines are involved in Immune-mediated Inflammation (LT, TNF- β )
    36. 42. Cytotoxic T cells kill infected cells <ul><li>Cytotoxic T cells kill infected cells , preventing these cells from producing more pathogen. </li></ul><ul><li>Receptors on the surface of cytotoxic T cells detect fragments of the virus on the surfaces of infected cells. </li></ul><ul><li>A successful immune response against a virus means that we will make large numbers of virus specific cytotoxic T cells. </li></ul><ul><li>In an EBV infection , cytotoxic T cells can make up the vast majority of our white blood cells. </li></ul>
    37. 43. <ul><li>T cells contain a T cell receptor that is like the antibody of B cells. - Each T cell has only one kind of receptor with a unique specificity . - Analogous to the genetic events of antibody production, T cells rearrange a set of genes coding for the T cell receptor . - Each T cell ends up with a unique receptor , but the population of T cells contains billions of different receptors </li></ul>Th activates Tc in a receptor specific manner Step 1 Step 2 Step 3
    38. 44. Fig 5-2:Steps in the activation of T lymphocytes <ul><li>Naive T cells recognize MHC-associated peptide antigens displayed on APCs and other signals </li></ul><ul><li>The T cells respond by: </li></ul><ul><ul><li>Producing cytokines , such as IL-2 , and </li></ul></ul><ul><ul><li>Expressing receptors for these cytokines , leading to an autocrine pathway of cell proliferation </li></ul></ul><ul><li>The result is clonal expansion of the T cells </li></ul><ul><li>Some of the progeny differentiate into: </li></ul><ul><ul><li>Effector cells , which serve various functions in cell-mediated immunity, and </li></ul></ul><ul><ul><li>Memory cells , which survive for long periods </li></ul></ul>
    39. 45. T cell activation Fig 5-3 T cell activation
    40. 47. T-cell independent defense mechanisms <ul><li>Phagocytosis </li></ul><ul><li>Chemotaxis </li></ul>
    41. 49. Functions of KIRs <ul><li>KIR receptors recognize MHC I on the target cell </li></ul><ul><li>They signal inhibition of cytotoxicity </li></ul><ul><li>Other NK receptors identify the target cell positively for killing </li></ul><ul><li>Antigens recognizable include: </li></ul><ul><ul><li>CD2 </li></ul></ul><ul><ul><li>CD69 </li></ul></ul><ul><ul><li>Antibody bound to the Fc receptor (CD16) </li></ul></ul>
    42. 50. NK receptors crosslinking <ul><li>Crosslinking of the activation receptors leads to </li></ul><ul><ul><li>phosphorylation of the ITAM sequences on the associated DAP12 molecule by a src- family kinase </li></ul></ul><ul><ul><li>The phosphorylated ITAMs recruit and activate tyrosine kinases of the ZAP70/syk family </li></ul></ul><ul><li>Crosslinking of the inhibitor receptor leads to </li></ul><ul><ul><li>ITIM phosphorylation and recruitment of the tyrosine phosphatase SHP-1, which then dephosphorylate s the ZAP70/syk activation molecules </li></ul></ul>Tyrosine phosphorylation Activation Inhibition Tyrosine dephosphorylation
    43. 51. NK & the missing-self hypothesis <ul><li>Kärre’s “ missing-self hypothesis ” </li></ul><ul><ul><li>The expression of MHC I protects against NK cell-mediated lysis </li></ul></ul><ul><ul><ul><li>NK cells are constantly surveying tissues for normal expression of MHC I . Because class I molecules are expressed on all tissues, NK cell cytotoxic activity is typically inhibited. </li></ul></ul></ul><ul><ul><ul><li>NK cell is released from its inhibition when it finds a cell with down-regulated or mutated MHC I </li></ul></ul></ul><ul><ul><ul><ul><li>the target cell is lysed </li></ul></ul></ul></ul><ul><ul><ul><li>This NK cell function is important because certain viruses are able to down-regulate MHC I expression in the cell they’ve infected, protecting themselves from detection by cytotoxic T cells. </li></ul></ul></ul><ul><ul><ul><li>Some tumors also have diminished MHC I expression, and their recognition and lysis is the basis of “natural” killing </li></ul></ul></ul>
    44. 53. Fig 5-3: Ligand-receptor pairs involved in T cell activation <ul><li>Major surface molecules of CD4 + T cells involved in their activation and the ligand s on APCs </li></ul><ul><li>CD8 + T cells use most of the same molecules, except that the </li></ul><ul><ul><li>TCR recognizes peptide-MHC I complexes, and </li></ul></ul><ul><ul><li>the coreceptor is CD8 , which recognizes class I </li></ul></ul><ul><li>Immunoreceptor tyrosine-based activation motifs ( IT AM s ) are the regions of signaling proteins that are phosphorylated on tyrosine residues and become docking sites for other signaling molecules </li></ul><ul><li>CD3 is composed of three polypeptide chains </li></ul>
    45. 54. Fig 5-3 Lymphocyte functional Ag Very late Ag Intercellular Adh.Mol Vascular Adh.Mol Zeta
    46. 58. ADCC: A ntibody- D ependent C ell-mediated C ytotoxicity NK Neutrophil Eosinophil M  Ig FcR
    47. 59. Granule-associated killing mechanisms <ul><li>Cytotoxic cell vesicles release </li></ul><ul><ul><li>Perforin </li></ul></ul><ul><ul><li>Enzymes </li></ul></ul><ul><li>Leading to </li></ul><ul><li>polymerization </li></ul><ul><li>of perforins  </li></ul><ul><li>Polyperforin </li></ul><ul><li>channels in the </li></ul><ul><li>in the </li></ul><ul><li>membrane </li></ul><ul><li>of the target </li></ul><ul><li>cell </li></ul><ul><li>Granules with enzymes (Granzymes) release enzymes that enter the target cell through polyperforin channels  death of target </li></ul>(Tc, NK) Granzymes
    48. 60. Granule-associated killing mechanisms (Tc, NK) Granzymes
    49. 61. Receptor-mediated killing mechanisms <ul><li>FasL (Fas ligand) </li></ul><ul><ul><li>a molecule on the surface of cytotoxic T cells that binds to its receptor, Fas , on the surface of other cells  initiating apoptosis in the target cell. </li></ul></ul><ul><li>TNF -mediated killing </li></ul><ul><ul><li>TNF released by Tc binds TNF-Receptor on the target cell surface  Cell death </li></ul></ul>

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