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Chapter 5 Immunology

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Chapter 5 Immunology

  1. 1. Chapter 5 Antigen Recognition by T Lymphocytes
  2. 2. Chapter 5 <ul><li>T-cell receptor diversity </li></ul><ul><ul><li>-Formation of receptors </li></ul></ul><ul><li>Antigen processing and presentation </li></ul><ul><ul><li>-Antigen processing for T cell recognition </li></ul></ul><ul><li>The major histocompatibility complex </li></ul><ul><ul><li>-Polymorphism and MHC diversity </li></ul></ul>
  3. 3. B lymphocytes versus T lymphocytes: Similarities <ul><li>Igs and TCR structure </li></ul><ul><ul><li>Result of gene rearrangement. </li></ul></ul><ul><li>Variable and diverse antigen specificity </li></ul><ul><li>Clones express single species of antigen receptor. </li></ul><ul><li>Clonal distribution of diversity in receptors produced by genetic mechanisms. </li></ul>
  4. 4. Fab Domain structure of TCR is similar to Ig
  5. 5. B lymphocytes versus T lymphocytes: Differences <ul><li>Igs-variable binding sites (wide range of Ags.). </li></ul><ul><li>“ Ultimate” sole function to produce secreted antibodies. </li></ul><ul><li>Bind epitopes on “intact molecules” </li></ul><ul><ul><li>Proteins, carbohydrates & lipids on surfaces of bacteria, viruses and parasites </li></ul></ul><ul><ul><li>Soluble toxins </li></ul></ul><ul><li>TCR-variable binding sites (one type of Ag). </li></ul><ul><li>More diverse roles with other cells. </li></ul><ul><li>Important differences in the “type of Ags” </li></ul><ul><ul><li>One type of antigen </li></ul></ul><ul><ul><li>Requires presentation by another human cell </li></ul></ul><ul><ul><li>Ag-presenting glycoproteins ( MHC molecules ) </li></ul></ul>
  6. 6. Key Difference between Ig and TCR antigen recognition <ul><li>T cells bind one type of antigen which must be presented to them on the surface of another human cell. </li></ul>
  7. 7. Major histocompatibility complex (MHC) <ul><li>Ag presenting molecules are glycoproteins. </li></ul><ul><li>Expressed on almost all cells. </li></ul><ul><li>Large number genetically determined variants in human population. </li></ul><ul><ul><li>i.e. differences between individuals in the MHC molecules. </li></ul></ul><ul><ul><ul><li>MHC determinants of tissue incompatibility before AP role was known. </li></ul></ul></ul>
  8. 8. T-cell receptor <ul><li>Membrane bound glycoprotein </li></ul><ul><li>Resembles a single antigen-binding arm of immunoglobulin. </li></ul><ul><li>Consists of two polypeptides the  and  chains ; one Ag-binding site . </li></ul><ul><ul><li>“ membrane bound ”; no secreted form </li></ul></ul><ul><ul><li>Each chain has variable region (binds Ag) and a constant region just like Igs . </li></ul></ul>
  9. 9. T-cell development <ul><li>T-cell development </li></ul><ul><ul><li>Gene rearrangement = sequence variability in V regions (similar to B-cell) </li></ul></ul><ul><ul><li>No further mutation in Ag-binding site after Ag stimulation. </li></ul></ul><ul><ul><li>No switching of constant-region isotype. </li></ul></ul><ul><li>TCR function - used only to recognize Ag. </li></ul><ul><li>Ig functions </li></ul><ul><ul><li>Recognition (Fab region) </li></ul></ul><ul><ul><li>Effector (Fc region) </li></ul></ul>
  10. 10. The T-cell receptor resembles a membrane associated Fab fragment of Ig <ul><li>2 different polypeptide chains = TCR  and TCR  </li></ul><ul><li>Genes encoding  and  chains </li></ul><ul><ul><li>consist of segments that must be rearranged to form a functional gene (like Ig). </li></ul></ul><ul><li>Rearrangements occur during T cell development ; mature T cell has one functional  , one functional  - chain together they define a unique TCR. </li></ul><ul><li>An individual’s population of T-cells = many millions of different TCR , each defines a clone of T-cells with a single Ag-binding specificity. </li></ul>
  11. 11. Organization of TCR  and  chains <ul><li>TCR  and  chains </li></ul><ul><ul><li>V regions and C regions. </li></ul></ul><ul><ul><li>Folded into discrete protein domains. </li></ul></ul><ul><ul><li>Each chain has an amino-terminal V domain, followed by C domain , and then a membrane-anchoring domain. </li></ul></ul>
  12. 12. Comparison of a.a. sequences of V domains from different clones of T-cells <ul><li>Sequence variation in the  and  chains clustered into regions of “ hypervariability”. </li></ul><ul><li>Correspond to loops of the polypeptide farthest from the T-cell membrane. </li></ul><ul><li>Loops = Ag-binding site ; complementarity-determining regions ( CDR1,CDR2,CDR 3) </li></ul>
  13. 13. T-cell receptor binding <ul><li>TCRs possess single binding site for antigen. </li></ul><ul><li>Used only as “ cell-surface” receptors for Ag. </li></ul><ul><ul><li>Never as soluble Ag-binding molecules </li></ul></ul><ul><li>Occurs in context of two opposing cell surfaces. </li></ul><ul><li>Multiple copies of TCR bind to multiple copies of Ag:MHC complex on the opposing cell. </li></ul><ul><li>Multipoint attachment </li></ul>
  14. 14. T-cell receptor diversity is generated by gene rearrangement <ul><li>B-cell mechanisms: </li></ul><ul><li>Prior to Ag encounter </li></ul><ul><ul><li>Gene rearrangement = V-region sequence </li></ul></ul><ul><li>Post Ag encounter </li></ul><ul><ul><li>mRNA splicing =secreted Ig </li></ul></ul><ul><ul><li>C-region DNA rearrangements = isotype switching </li></ul></ul><ul><ul><li>Somatic hypermutation = Ab of higher affinity </li></ul></ul><ul><li>T-cell mechanisms: </li></ul><ul><li>Prior to Ag stimulation </li></ul><ul><ul><li>Gene rearrangement = V-region </li></ul></ul><ul><li>Post Ag stimulation </li></ul><ul><ul><li>Genes encoding TCR remain unchanged </li></ul></ul>
  15. 15. T-cell receptor is used only for the recognition of Ag <ul><li>Effector functions handled by other T-cell molecules. </li></ul><ul><li>Effector functions of B-cells = solely dependent on secreted Abs. </li></ul><ul><ul><li>Ab’s different C-region isotypes trigger different effector functions. </li></ul></ul>
  16. 16. Human TCR  chain locus (Chr 14) Human TCR  -chain locus (Chr 7) <ul><li>Organization of TCR C-region simpler than Ig: </li></ul><ul><ul><li>only one C  gene and </li></ul></ul><ul><ul><li>two C  genes (no functional distinction known) </li></ul></ul><ul><li>The  chain contains V and J segments (like Ig L-chain) </li></ul><ul><li>The  chain contains V, J, and D segments (like Ig H-chain) </li></ul>
  17. 17. TCR rearrangement occurs during T cell development in the thymus <ul><li>The V domain of  is encoded by V and J. </li></ul><ul><li>The V domain of  is encoded by V, D, and J. </li></ul><ul><li>TCR gene segments flanked by recombination signal sequences (RSS). </li></ul><ul><li>RAG complex and other DNA-modifying enzymes involved in the recombination process. </li></ul><ul><li>In addition to V, D, J recombination junctional diversity is also attained by insertion of additional, non-templated P and N nucleotides. </li></ul>
  18. 18.
  19. 19. Rare genetic defect in a RAG gene = Severe combined immunodeficiency disease (SCID) <ul><li>One of the RAG genes does not work. </li></ul><ul><li>Combined = Functional B and T cells both absent. </li></ul><ul><li>SCID children die in infancy from common infections (unless they have bone marrow transplants ) </li></ul>Candida albicans infection
  20. 20. Missense mutations that produce RAG proteins with partial enzymatic activity = Omenn syndrome <ul><li>Rapidly fatal immunodeficiency </li></ul><ul><ul><li>Differs from SCID in symptoms </li></ul></ul>Red rash on face and shoulders
  21. 21. <ul><li>TCR =  :  heterodimer </li></ul><ul><ul><li>Alone can not leave ER </li></ul></ul><ul><ul><li> :  heterodimer stable association with 4 “invariant” membrane proteins </li></ul></ul><ul><ul><ul><li>CD3 complex (Chr 11, homologous CD3  , CD3  & CD3  ). </li></ul></ul></ul><ul><ul><ul><li> chain (Chr 1). </li></ul></ul></ul><ul><ul><ul><li>TCR has short cytoplasmic tails . </li></ul></ul></ul>Expression of the TCR on the cell surface requires association with additional proteins <ul><li> = gamma </li></ul><ul><li>= delta </li></ul><ul><li>= epsilon </li></ul><ul><li> = zeta </li></ul>
  22. 22. Expression of the TCR on the cell surface requires association with additional proteins (cont.) <ul><li>CD3 complex &  chain, longer sequence , transduce signals to cell’s interior after Ag recognition by TCR. </li></ul><ul><li>Lack of functional CD3  & CD3  = low TCR expression and impaired signal transduction = immunodeficiency. </li></ul>
  23. 23. TCR complex <ul><li>The TCR complex is composed of 8 polypeptides . </li></ul><ul><li> chains form core. </li></ul><ul><li>the  chains interact with intracellular signaling molecules </li></ul>
  24. 24.  And  chains form a second class of TCR expressed by a distinct population of T-cells =  :  TCR <ul><li> :  TCR structure similar to  :  TCR </li></ul><ul><ul><li> chain resembles  chain </li></ul></ul><ul><ul><li> chain resemble  chain </li></ul></ul><ul><li>A cell can express either  :  or  :  TCRs , never both. </li></ul><ul><li>Cells expressing  :  TCR are called  :  T cells and cells expressing  :  TCRs are called  :  T cells. </li></ul><ul><li>Cells expressing  :  receptors form a small subset of all T cells (only 1-5%). </li></ul><ul><li>Can be dominant T cell population in epithelial tissue. </li></ul><ul><li> :  T-cells immune functions and Ags less well defined. </li></ul><ul><li>Not restricted to recognition of Ag associated with MHC. </li></ul>
  25. 25. Ag presented by MHC No MHC
  26. 26. The germline organization of the  and  loci resembles that of the  and  loci <ul><li>The  gene segments are situated “within” the  -chain locus on chromosome 14. </li></ul><ul><ul><li>Between the V  and J  </li></ul></ul><ul><ul><li>Rearrangement of the  -gene = deletion and inactivation of the  -chain. </li></ul></ul><ul><li>The  chain is on chromosome 7. </li></ul><ul><li>The  and  chain loci have fewer V gene segments than the  - or  -chain loci. </li></ul><ul><ul><li>Might produce less diverse receptors. </li></ul></ul><ul><ul><li>The  chain compensates by having an increase in junctional diversity (pg 72 typo. book states “  chain compensates”) . </li></ul></ul>
  27. 27. <ul><li>Organization of the human T-cell receptor γ - and  -chain loci </li></ul><ul><li> gene segments are situated within the  -chain locus on chromosome 14 </li></ul>
  28. 28. Rearrangement at the  and  loci resembles that of the  and  loci <ul><li>Exception: during  -gene rearrangement two D segments can be incorporated into the final gene sequence. </li></ul><ul><ul><li>= increased variability of the  chain </li></ul></ul><ul><ul><ul><li>1. Increase in the potential numbers of recombinations. </li></ul></ul></ul><ul><ul><ul><li>2 . Extra N nucleotides can be added at the junction between the two D segments, as well as at the VD and DJ junctions. </li></ul></ul></ul>
  29. 29. Antigen processing and presentation
  30. 30. B cells can recognize a wide range of molecules in their native form.
  31. 31.
  32. 32. TCR recognize Ag as a peptide bound to MHC on human cell surface <ul><li>Pathogen-derived protein must first be broken down (Ag processing) and displayed on the surface of cells bound to MHC (Ag presentation) </li></ul>
  33. 33. Microorganisms that infect the human body can be broadly divided into two intracellular and extracellular <ul><li>Microorganisms that propagate within cells. </li></ul><ul><ul><li>Example - Viruses </li></ul></ul><ul><li>Microorganisms that live in the extracellular spaces. </li></ul><ul><ul><li>Most bacteria </li></ul></ul><ul><ul><li>Virus particles present in the EC fluid after release from infected cells. </li></ul></ul>
  34. 34. Two class of T cells are specialized to respond to intracellular and extracellular sources of infection <ul><li>Circulating  :  T-cells = 2 “mutually exclusive” classes </li></ul><ul><ul><li>Defined by CD4 glycoprotein expression </li></ul></ul><ul><ul><li>Defined by CD8 glycoprotein expression </li></ul></ul><ul><ul><ul><li>Different functions </li></ul></ul></ul><ul><ul><ul><li>Different types of target pathogens </li></ul></ul></ul><ul><li>CD8 T-cells are cytotoxic and kill cells that are infected with a virus or other intracellular pathogen. </li></ul><ul><ul><li>Prevents pathogen replication and further infection of healthy cells. </li></ul></ul><ul><li>CD4 T-cells - help other immune cells respond to extracellular sources of infection. </li></ul>
  35. 35. The structures of CD4 and CD8 glycoproteins <ul><li>CD4 and CD8 molecules are called T cell co-receptors . </li></ul>Ig-like domains
  36. 36. CD4 T-cells are helper T-cells <ul><li>Two subclasses: </li></ul><ul><li>T H 2 cells stimulate B cell (plasma cells) to make antibody. </li></ul><ul><li>T H 1 cells activate macrophages to phagocytose and kill extracellular pathogens secrete cytokines & other biologically active molecules to affect the course of the immune response. </li></ul>
  37. 37. T-cells function by making contact with other cells
  38. 38. Two classes of MHC molecule present antigen to CD8 and CD4 T-cells respectively <ul><li>MHC molecules function to ensure that the appropriate class of T cells is activated in response to a particular source of infection. </li></ul><ul><li>MHC Class I </li></ul><ul><ul><li>Presents intracellular Ags to CD8 T-cells (ex. Virus infected cell) . </li></ul></ul><ul><li>MHC Class II </li></ul><ul><ul><li>Presents extracellular Ags to CD4 T-cells (ex. phagocytosed or endocytosed antigens) . </li></ul></ul>
  39. 39. The two classes of MHC membrane glycoprotein molecules have similar 3-D structures <ul><li>MHC Class I = transmembrane heavy chain, or  chain noncovalently complexed to  -microglobulin . </li></ul><ul><li> -chain has 3 extracellular domains (  1,  2 and  3) encoded by a gene in the MHC loci. </li></ul><ul><li> -microglobulin is not coded by a gene in MHC loci. </li></ul><ul><li>Folding of  1 and  2 = peptide-binding site farthest from the cell membrane, supported by  3 and  -microglobulin. </li></ul>
  40. 40. <ul><li>MHC Class II consists of two transmembrane chains,  and  chain . </li></ul><ul><ul><li>Each contributes one domain to the peptide-binding site and one Ig-like supporting domain. </li></ul></ul><ul><ul><li>Both  and  chains are encoded by genes in the MHC. </li></ul></ul>The two classes of MHC membrane glycoprotein molecules have similar 3-D structures
  41. 41. The similar 3-D structures of MHC I and MHC II molecules consist of two pairs of extracellular domains <ul><li>The paired domains farthest form the membrane resemble each other and form the peptide-binding site. </li></ul><ul><li>The domains supporting the peptide-binding domains are Ig-like domains:  3 and  -microglobulin in MHC I and  2 and  2 chain in MHC II. </li></ul><ul><li>Ig-like domains of MHC class I and II are not just a support for the peptide-binding site. </li></ul><ul><li>They provide binding sites for the CD4 and CD8 co-receptors. </li></ul><ul><li>Allows the simultaneous engagement of both T-cell receptor and co-receptor by an MHC molecules . </li></ul>
  42. 42. MHC class I binds CD8 and MHC class II binds CD4
  43. 43. MHC bind a variety of peptides <ul><li>MHC molecules have degenerate binding-capable of binding peptides of many different amino acid sequences. </li></ul><ul><li>Peptide-binding site = deep groove on the surface of the MHC molecule, a single peptide is tightly noncovalently bound. </li></ul><ul><li>Length and amino acid sequence constraints. </li></ul>
  44. 44. MHC I molecule binding <ul><li>MHC class I </li></ul><ul><ul><li>Length limited because the two ends of the peptide are grasped by pockets situated at the ends of the peptide-binding groove. </li></ul></ul><ul><ul><ul><li>8, 9, 10 a.a. (slight kinking to accommodate) </li></ul></ul></ul><ul><ul><ul><li>Also may have a hydrophobic or basic residue at the carboxyl terminus complementary to pocket present in MHC I binding groove. </li></ul></ul></ul>
  45. 45. MHC II molecule binding <ul><ul><li>MHC class II </li></ul></ul><ul><ul><ul><li>Two ends of the peptide not pinned down into pockets at each end </li></ul></ul></ul><ul><ul><ul><li>Extend out at each end of the groove </li></ul></ul></ul><ul><ul><ul><li>Longer and more variable in length than peptides bound by MHC class I </li></ul></ul></ul><ul><ul><ul><li>13-25 a.a. in length or longer </li></ul></ul></ul>
  46. 46. There are 2 major compartments within cells, separated by membranes <ul><li>Peptide Ags are bound and presented by MHC. </li></ul><ul><li>Proteins derived from “intracellular” and “extracellular” antigens are: </li></ul><ul><ul><li>In 2 “ different” intracellular compartments. </li></ul></ul><ul><ul><li>Processed by 2 different intracellular pathways of degradation. </li></ul></ul><ul><ul><li>Bind to 2 different classes of MHC molecule. </li></ul></ul>
  47. 47. There are 2 major compartments within cells, separated by membranes <ul><li>Peptide Ags are bound and presented by MHC inside the cells. </li></ul><ul><li>Proteins derived from “intracellular” and “extracellular” antigens are: </li></ul><ul><ul><li>In 2 “ different” intracellular compartments. </li></ul></ul><ul><ul><li>Processed by 2 different intracellular pathways of degradation. </li></ul></ul><ul><ul><li>Bind to 2 different classes of MHC molecule. </li></ul></ul>(2) Vesicular system (ER, Golgi, vesicles) is contiguous with extracellular fluid (1) Cytosol is contiguous with the nucleus through nuclear pores. Extracellular fluid
  48. 48. Peptides Generated in Cytosol are Transported into the ER where they bind MHC class I molecules <ul><li>Peptides derived from degradation of intracellular proteins or pathogens are: </li></ul><ul><ul><li>Formed in the cytosol </li></ul></ul><ul><ul><li>Delivered to the ER </li></ul></ul><ul><ul><li>Bound by MHC class I </li></ul></ul>
  49. 49. Peptides presented by MHC class II molecules are generated in acidified intracellular vesicles <ul><li>Peptides derived from degradation of extracellular proteins or pathogens are: </li></ul><ul><ul><li>Taken up by cellular phagocytosis and endocytosis. </li></ul></ul><ul><ul><li>Degraded in the lysosomes and other vesicles of the endocytic pathways. </li></ul></ul><ul><ul><li>Bind to MHC class II molecules in these endocytic vesicles. </li></ul></ul>
  50. 50. The processing pathway determines which class of MHC molecule interacts with a peptide that originates from extracellular or intracellular pathogen. Intracellular pathogen Extracellular pathogen
  51. 51. The processing pathway determines which class of MHC molecule interacts with a peptide that originates from extracellular or intracellular pathogen. Intracellular pathogen Extracellular pathogen
  52. 52. Viral infection of human cells <ul><li>Viruses exploit the cell’s protein synthesis machinery to synthesize viral proteins. </li></ul><ul><ul><li>Viral proteins are found in the cytosol prior to being assembled into viral particles (peptides). </li></ul></ul><ul><li>In response, the cell uses its normal processes of breakdown and turnover of cellular proteins. </li></ul><ul><ul><li>To degrade some of the viral proteins into peptides. </li></ul></ul><ul><ul><li>Peptides are bound by MHC class I molecules. </li></ul></ul><ul><ul><li>Presented to cytotoxic CD8 cells. </li></ul></ul>
  53. 53. Formation and transport of peptides that bind to MHC class I molecules <ul><li>Proteins in the cytosol are degraded by the proteasome protein complex </li></ul><ul><ul><li>28 polypeptide subunits (20-30 kDa) </li></ul></ul>Tap-2 Tap-1
  54. 54. Formation and transport of peptides that bind to MHC class I molecules <ul><li>Ag peptides are then transported into the ER </li></ul><ul><ul><li>By TAP (the membrane embedded transporter associated with antigen processing) </li></ul></ul><ul><ul><li>TAP = heterodimer </li></ul></ul><ul><ul><ul><li>TAP-1 and TAP-2 </li></ul></ul></ul><ul><ul><ul><li>Transport dependent on the binding and hydrolysis of ATP </li></ul></ul></ul><ul><ul><ul><li>TAP transports peptides of eight or more amino acids having hydrophobic or basic residues at the carboxy terminus </li></ul></ul></ul>Tap-1 Tap-2
  55. 55. Newly synthesized MHC class I molecules <ul><li>Newly synthesized MHC class I H-chains and  -microglobulin translocate to the ER. </li></ul><ul><ul><li>Partially complete folding </li></ul></ul><ul><ul><li>Associate together </li></ul></ul><ul><ul><li>Bind peptide to complete folding </li></ul></ul><ul><ul><ul><li>Chaperones = proteins that assist in correct folding of proteins and assembly of other proteins, protection until they enter their respective cellular pathways and to carry out their intended functions </li></ul></ul></ul>
  56. 56. When MHC class I heavy chains enter the ER they bind a membrane protein-calnexin <ul><li>Calnexin retains the partly folded  -chain in the ER </li></ul><ul><ul><li>Calnexin is a Ca 2+ dependent lectin </li></ul></ul><ul><ul><ul><li>Lectins are carbohydrate-binding proteins that retains many multisubunit glycoproteins (TCRs and Igs) in the ER until they fold correctly. </li></ul></ul></ul><ul><ul><li>MHC class I  -chain binds  2-microglobulin and calnexin is released from the  :  -microglobulin heterodimer . </li></ul></ul>
  57. 57. MHC class I H-chain binds  2-microglobulin and calnexin is released from the  :  -microglobulin <ul><li>Calreticulin and tapasin bind the TAP-1 subunit of the peptide transporter to position the partly folded  :  -microglobulin heterodimer to wait for a suitable peptide from the cytosol. </li></ul><ul><li>A peptide delivered by TAP binds to class I heavy or  -chain , forming mature MHC class I molecule. </li></ul><ul><li>The class I molecule dissociates from calreticulin, tapasin, and TAP and is exported from the ER to cell surface. </li></ul>
  58. 58. Chaperone proteins aid the assembly and peptide loading of MHC class I molecules in the endoplasmic reticulum Golgi stacks  cell membrane
  59. 59. Bare lymphocyte syndrome <ul><li>TAP is non-functional??? </li></ul><ul><li>No peptides enter ER … MHC class I does not reach surface </li></ul><ul><li>Patients have less than 1% of normal MHC class I </li></ul><ul><li>Patients have poor CD8 T cell responses to viruses and suffer chronic respiratory infections </li></ul>
  60. 60. Peptides Presented by MHC Class II are Generated in Acidified Vesicles <ul><li>Recall: Extracellular bacteria , extracellular virus particles and soluble protein Ags are processed by a different intracellular pathway than intracellular bacteria (cytosolic proteins) and viral proteins. </li></ul>
  61. 61. Peptides Presented by MHC Class II are Generated in Acidified Vesicles (cont.) <ul><li>Vesicles travel inwards from the plasma membrane , their interiors become acidified by the action of proton pumps in the vesicle membrane. </li></ul><ul><li>Vesicle membranes fuse with other vesicles ( lysosomes ) to form phagolysosomes that contain proteases and hydrolases that are activated at low pH. </li></ul><ul><li>Enzymes degrade the vesicles contents to produce peptides from proteins and glycoproteins. </li></ul>
  62. 62. Internalization of extracellular Ags by receptor-mediated endocytosis <ul><li>B cells also bind specific Ags via surface Ig , internalize the Ags by receptor-mediated endocytosis. </li></ul><ul><li>These antigens are similarly degraded within the vesicular system as by endocytosis mechanism. </li></ul><ul><li>Peptides within endosomes bind to MHC class II complexes and are carried to the cell surface by outgoing vesicles. </li></ul><ul><li>Take home message: </li></ul><ul><ul><li>MHC class I pathway samples the intracellular environment </li></ul></ul><ul><ul><li>MHC class II pathway samples the extracellular environment </li></ul></ul>
  63. 63. The pathways of the MHC class I and II
  64. 64. MHC Class II Molecules are Prevented from Binding Peptides in the ER by the Invariant Chain <ul><li>Newly synthesized MHC Class II  and  chains translocated from ribosome  ER. </li></ul><ul><li> and  chains ( vary from person) associate in ER with an “invariant” chain (identical in all persons). </li></ul><ul><ul><li>Invariant chain blocks peptide binding site (formed by  and  chain) from binding peptides present in the ER. </li></ul></ul><ul><ul><li>Result is that all peptides in ER bind MHC class I molecules only. </li></ul></ul><ul><ul><li>Invariant chain also delivers class II molecules to endocytic vesicles (called MHC class II compartments or MIIC ). </li></ul></ul>
  65. 65. MHC class II compartments or MIIC (Endocytic vesicles ) <ul><li>MIIC contain proteases ( cathepsin S ) that selectively cleave invariant chain leaving a small fragment of the invariant chain to cover the MHC class II peptide-binding site </li></ul><ul><ul><li>Class II-associated invariant-chain peptide ( CLIP fragment) </li></ul></ul>
  66. 66. MHC class II compartments or MIIC (Endocytic vesicles ) <ul><li>Removal of CLIP and binding of peptide the MHC class II molecule is aided by interaction HLA-DM glycoprotein. </li></ul><ul><li>HLA-DM catalyzes the release of CLIP and allows MHC class II molecule to sample other peptides until it finds one that binds strongly. </li></ul><ul><li>MHC class II molecule binds appropriate peptide and is transported to cell surface by outward going vesicles . </li></ul>
  67. 67. The invariant chain prevents peptides from binding to a MHC class II molecule UNTIL…it reaches the site of extracellular protein breakdown
  68. 68. The TCR Specifically Recognizes Both Peptide and MHC Molecules <ul><li>TCR binds to a peptide - MHC complex . </li></ul><ul><ul><li>TCR contacts both peptide and MHC surface </li></ul></ul><ul><ul><li>Each peptide-MHC complex forms unique TCR ligand. </li></ul></ul>TCR MHC class I peptide
  69. 69. The TCR Specifically Recognizes Both Peptide and MHC Molecules <ul><li>Floor of the peptide-binding groove of both classes of MHC molecule is formed by eight strands of antiparallel  -sheet , with two antiparallel  -helices </li></ul><ul><li>Peptide lies between the  -helices and parallel to them. </li></ul>
  70. 70. The TCR Specifically Recognizes Both Peptide and MHC Molecules <ul><li>Peptide residues that bind to the MHC molecule are deep within the peptide-binding groove making them inaccessible to the TCR. </li></ul><ul><li>S ide chains of other peptide amino acids stick out of the binding site to bind the TCR. </li></ul>Interact with T cell Interact with MHC
  71. 71. TCR-ligand:MHC molecules visualized by X-ray crystallography <ul><li>Overall organization of the </li></ul><ul><li>TCR Ag-binding site </li></ul><ul><li>resembles that of an Ab </li></ul>TCR Ag-binding site of an Ab
  72. 72. Similar interactions for peptides bound to either MHC class I or class II <ul><li>TCR binds to MHC class I:peptide complex with long axis of its binding site oriented diagonally across the MHC class I molecule peptide-binding groove </li></ul><ul><li>TCR binds to an MHC class II:peptide complex in a similar orientation. </li></ul>
  73. 73. The CDR3 loops of the TCR  and  chains form the central part of the binding site <ul><li>CDR3 loops grasp the side chain of one of the amino acids in the middle of the peptide. </li></ul><ul><li>CDR1 and CDR2 loops form the periphery of the binding site and contact the  -helices of the MHC molecule. </li></ul><ul><li>CDR3 loops directly contacts peptide Ag and they are the most variable part of the T-cell receptor Ag-recognition site. </li></ul><ul><li>The  -chain CDR3 includes the joint between the V and J sequences. </li></ul><ul><li>The  -chain CDR3 includes the </li></ul><ul><ul><li>joints between V and D, </li></ul></ul><ul><ul><li>the whole of the D segment and </li></ul></ul><ul><ul><li>the joint between D and J </li></ul></ul>
  74. 74. TCR does not interact symmetrically with the peptide and the helices of the MHC molecule. <ul><li>CDR1 and CDR2 loops of the  -chain make stronger contact with the peptide:MHC complex that do the CDR1 and CDR2 loops of the  -chain TCR’s. </li></ul><ul><ul><li> -chain CDR1 and CDR2 are light and dark blue respectively. </li></ul></ul><ul><ul><li> -chain CDR1 and CDR2 are light and dark purple respectively. </li></ul></ul><ul><ul><li>The  -chain CDR3 is yellow and the  -chain CDR3 is dark yellow. </li></ul></ul><ul><ul><li>The 8 amino acid peptide is colored yellow with the first (P1) and the last (P8) amino acids indicated . </li></ul></ul>
  75. 75. The TCR Specifically Recognizes Both Peptide and MHC Molecules <ul><li>When the TCR binds to a peptide-MHC complex, it contacts both peptide and MHC surface. </li></ul><ul><li>Each peptide-MHC therefore forms unique TCR ligand. </li></ul>
  76. 76. Most cells express MHC class I; few express MHC class II <ul><li>MHC class I molecules are expressed on almost all nucleated cells. </li></ul><ul><li>MHC class I are most highly expressed in hematopoietic cells. </li></ul>
  77. 77. Most cells express MHC class I; few express MHC class II <ul><li>MHC class II molecular are normally expressed only by a subset of hematopoietic cells (antigen presenting cells) and by stromal cells in the thymus. </li></ul><ul><li>MHC class II molecules can be produced by other cell types on exposure to the cytokine interferon-  . </li></ul>
  78. 78. The major histocompatibility complex
  79. 79. Major histocompatibility complex <ul><li>MHC molecules and other proteins involved in Ag presentation are encoded by cluster of closely linked genes called the major histocompatibility complex . </li></ul><ul><li>Large number of genetic variants for some MHC class I and class II; evolved to permit MHC molecules to bind large variety of peptide sequences. </li></ul><ul><li>Differences in MHC molecules responsible for graft rejection in organ transplantation. </li></ul>
  80. 80. Diversity of MHC molecules in the human population is due to multigene families and genetic polymorphism <ul><li>Unlike Ig and TCR, MHC genes are stable and do not undergo rearrangement or somatic structural change. </li></ul><ul><li>Inherited diversity is achieved in 2 ways: </li></ul><ul><ul><li>Multiple similar gene families </li></ul></ul><ul><ul><ul><li>encoding MHC class I heavy or  chains and </li></ul></ul></ul><ul><ul><ul><li>encoding MHC class II  and  chains </li></ul></ul></ul><ul><ul><li>Polymorphism: existence within the population of a great many alternative forms of a MHC class I or class II gene. </li></ul></ul><ul><ul><li>Individuals are therefore heterozygous for MHC genes </li></ul></ul>
  81. 81. <ul><li>The genetic loci that makes up the MHC is highly polymorphic, that is many different forms of the gene or alleles exist at each locus in a population. Their encoded proteins are allotypes. </li></ul><ul><li>Genes for the MHC loci lie close together and individuals inherit the alleles encoded by these closely linked loci as two sets, one from each parent – called haplotype. </li></ul><ul><li>Alleles are co-dominantly expressed. </li></ul><ul><li>Heterozygous = individual inherits different forms of a gene from each parent. </li></ul><ul><li>Homozygous = an individual inherits the same form of a gene from both parents . </li></ul>
  82. 82. MHC class I and class II genes are linked in the MHC complex <ul><li>Genes that encode MHC class I heavy chain and MHC class II  and  chains are closely linked. </li></ul><ul><li> -microglobulin and invariant chain are not polymorphic. </li></ul><ul><ul><li>located on chromosomes 15 and 5 , respectively </li></ul></ul><ul><li>Region called the MHC complex because first identified as region containing polymorphisms underlying graft rejection. </li></ul>
  83. 83. In humans the MHC is called the HLA complex <ul><li>HLA = human leukocyte antigen complex </li></ul><ul><li>Antibodies used to identify human MHC molecules react with white cells (leukocytes) but not with red cells. </li></ul><ul><li>The isotypes differ in function and the extent of their polymorphism. </li></ul>6 MHC class I isotypes 5 MHC class II isotypes
  84. 84. Human MHC class I and II isotypes differ in function and the extent of their polymorphism <ul><li>Human MHC class I isotypes </li></ul><ul><ul><li>HLA-A, HLA-B and HLA-C present peptide Ags to CD8 T cells and form ligands for NK-cell receptors </li></ul></ul><ul><ul><li>HLA-E and HLA-G are oligomorphic and form ligands for NK-cell receptors </li></ul></ul><ul><ul><li>HLA-F is intracellular and of unknown function </li></ul></ul><ul><li>Human MHC class II isotypes </li></ul><ul><ul><li>HLA-DP, HLA-DQ and HLA-DR present peptide Ags to CD4 T cells. </li></ul></ul><ul><ul><li>HLA-DM and HLA-DO are intracellular and regulate peptide loading of HLA-DP, HLA-DQ and HLA-DR. </li></ul></ul>
  85. 85. The polymorphism of HLA class I and class II genes <ul><li>The number of known functional alleles in the human population for each HLA class I ( greater diversity ) and class II genes. </li></ul><ul><li>Class II, diversity contributed by  and  chains </li></ul><ul><li>Class I, H-chain = polymorphism </li></ul><ul><li>Notice no gene for  2 invariant light chain of HLA class I - (Chr 15) </li></ul>
  86. 86. For each HLA class II isotype, the genes encoding the  and  chains are called A and B, respectively <ul><li>Human MHCs differ in the number of DR genes. </li></ul><ul><li>The MHC on every human chromosome 6 carries one gene (DRA) for the HLA class II DR  chain but four different genes (DRB1,3,4 or 5) for the DR  chain. </li></ul><ul><li>In addition, some MHCs carry other DRB3, DRB4 or DRB5. </li></ul><ul><li>Any DR  chain can pair with the DR  chain to form a class II molecule. </li></ul>
  87. 87. <ul><li>The MHC class I and class II genes occupy different regions of the MHC. </li></ul><ul><li>The positions within the HLA complex of the HLA class I and II genes. </li></ul><ul><li>The class I genes are all contained in the class I region . </li></ul><ul><li>The class II genes are all contained in the class II region . </li></ul>The MHC class I and class II genes occupy different regions of the MHC on Chromosome 6
  88. 88. <ul><li>Class III region (central MHC) contains a variety of genes (not shown) involved in other immune functions. </li></ul><ul><li>HLA-DP, HLA-DQ and HLA-DR , the  and  chain genes, are close together and shown as a single yellow block . </li></ul><ul><li>HLA-DO (  and  genes, DOA and DOB) are separated by the HLA-DM (  and  genes together) genes. </li></ul>The MHC class I and class II genes occupy different regions of the MHC on Chromosome 6
  89. 89. Other proteins involved in antigen processing an presentation are encoded in the MHC class II region <ul><li>HLA complex contains more than 200 genes of which the HLA class I and II genes constitute a minority. </li></ul><ul><li>Other genes embrace a variety of functions (several important to the immune system). </li></ul><ul><li>Class II region of the MHC is almost entirely dedicated to genes involved in processing and presenting Ag to T cells. </li></ul><ul><ul><li>Genes encoding the  and  chains of the 5 HLA class II isotypes. </li></ul></ul><ul><ul><li>Two polypeptides of the TAP peptide transporter. </li></ul></ul><ul><ul><li>Gene for tapasin. </li></ul></ul><ul><ul><li>Genes encoding proteasome subunits ( LMP2 and LMP7). </li></ul></ul>
  90. 90. Almost all of the genes in the HLA class II region are involved in Ag processing & presentation to T cells <ul><li>Genes shown in dark gray are pseudogenes that are related to functional genes but are not expressed and unnamed genes in light gray are not involved in immune system function. </li></ul><ul><li>The class II region includes genes for the peptide transporter (TAP), proteasome components (LMP) and tapasin. </li></ul><ul><li>Notice no gene for the invariant chain gene (chromosome 5). </li></ul>
  91. 91. Variations between MHC allotypes is concentrated in the sites that bind peptide and T-cell Receptor <ul><li>HLA class I molecule allotype variability is clustered in specific sites within the  1 and  2 domains. </li></ul><ul><li>These sites line the peptide-binding groove, lying either in the floor of the groove where they influence peptide binding or in the  helices that form the walls, which are also involved in binding the TCR. </li></ul><ul><li>In HLA class II which is a DR molecule, variability is found only in the  1 domain because the  chain is monomorphic. </li></ul>
  92. 92. Peptide-binding motifs and the sequence of peptides bound for some MHC isoforms <ul><li>HLA-A and HLA-B isoforms </li></ul><ul><ul><li>Isoform peptide-binding motif with complete aa sequence of 1 peptide presented by the isoform </li></ul></ul><ul><ul><li>Blank boxes in the peptide-binding motifs are position at which the identity of the aa can vary </li></ul></ul>
  93. 93. Peptide-binding motifs and the sequence of peptides bound for some MHC isoforms <ul><li>HLA-DR and HLA-DQ isoforms </li></ul><ul><ul><li>only the sequence of a self-peptid e that is bound by the isoform is shown </li></ul></ul><ul><ul><li>Anchor residues are in green circles </li></ul></ul><ul><ul><li>MHC class II peptide-binding motifs are not readily defined </li></ul></ul>
  94. 94. Polymorphism of MHC Classes I and II affects antigen recognition by T cells <ul><li>Variation between MHC allotypes is concentrated in the sites that bind peptide and TCR. </li></ul><ul><li>T cells that responds to peptide presented by one MHC allotype will not respond to that peptide bound to another MHC allotype – </li></ul><ul><li>MHC restriction </li></ul>
  95. 95. T-cell recognition of Ags is MHC restricted <ul><li>TCR of the CD8 T cell is specific for the complex of the peptide X with the class I molecule HLA-A*0201. </li></ul><ul><li>Because of this co-recognition, which is called MHC restriction , the TCR does not recognize the same peptide when it is bound to a different class I molecule, HLA-B*5201. </li></ul><ul><li>TCR does not recognize the complex of HLA-A*0201 with a different peptide. </li></ul>
  96. 96. Haplotype <ul><li>The term was first used in connection with the genes of the MHC </li></ul><ul><li>With respect of a linked cluster of polymorphic genes, the “set of alleles” carried on a single chromosome </li></ul><ul><li>Every person inherits 2 haplotypes, one from each parent </li></ul>
  97. 97. MHC polymorphism is the primary cause of alloreactions that reject transplants <ul><li>Rejection of transplant caused by immune response in which B cells and T cells of the recipient respond to differences in structure between host and recipient MHC molecules. </li></ul><ul><li>Differences are allogeneic: the immune response they provoke is an alloreaction. </li></ul><ul><li>The different MHC molecules are alloantigens and the antibodies they provoke are alloantibodies. </li></ul>
  98. 98. Random combination of maternal and paternal haplotypes produces millions of combinations <ul><li>Immense diversity of HLA type means clinical transplants performed across range of HLA matches and mismatches </li></ul><ul><li>Solid organs (heart/kidney) HLA mismatch overcome by immunosuppressive drugs </li></ul><ul><li>Bone marrow transplant more sensitive to HLA mismatch; have an alloreaction of the transplanted lymphocytes against recipient’s tissue </li></ul><ul><li>This graft vs. host disease can be fatal </li></ul><ul><li>Look for match among siblings </li></ul>
  99. 99. MHC heterozygosity delays the progression to AIDS in people infected with HIV-1 <ul><li>Seroconversion = HIV-1 infected person begins to make detectable antibodies to the virus </li></ul><ul><ul><li>The onset of overt symptoms of AIDs occurs years after seroconversion </li></ul></ul><ul><ul><li>The rate of progress to AIDs decreases with the extent of HLA heterozygosity as compared for individuals who are polymorphic for all the HLA class I and II loci to homozygous for 1,2 or 3 </li></ul></ul>Heterozygous for all HLA class I and II loci homozygous for 1 locus homozygous for 2 or 3 loci

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