micr 304 immunology & serology
DESCRIPTION
MICR 304 Immunology & Serology. Lecture 8A Antibodies Part II Chapter 4.2- 4.5, 4.12 – 4.20, 9.14 – 9.20. Overview of Today’s Lecture. Generation of antibodies Secondary antibody modifications Effector functions of antibodies. Key Players in Immunology. - PowerPoint PPT PresentationTRANSCRIPT
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MICR 304 Immunology &
Serology
MICR 304 Immunology &
Serology
Lecture 8A Antibodies Part II
Chapter 4.2- 4.5, 4.12 – 4.20, 9.14 – 9.20
Lecture 8A Antibodies Part II
Chapter 4.2- 4.5, 4.12 – 4.20, 9.14 – 9.20
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Overview of Today’s Lecture
• Generation of antibodies• Secondary antibody modifications• Effector functions of antibodies
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Key Players in Immunology
Innate Adaptive
Cells PhagocytesEpithelial Cells
NK Cells
Lymphocytes(B-Ly, T-Ly)
Effector molecules
ComplementAntimicrobial (Poly)PeptidesAntimicrobial
lipids?
Antibodies
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Variable Regions Are Constructed From Gene Segments
• Variable regions of light chain– V (variable) gene segments– J (joining) gene segments
• Variable regions of heavy chain– V (variable) gene segments– D (diversity) gene segments– J (joining) gene segments
• From multiple genes, randomly one each of V and J gene segments for the light chain and one each of V, D and J gene segments for the heavy chain are recombined.
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Construction of the Antibody V Regions
L: Leader sequence; directs protein to cell’s secretory pathway: Hinge region
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Recombination of a Limited Number of Gene Segments Results in Millions of
Variations~
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Germline Organization of the Light and Heavy Chain
Includes a significant number of non-functional segments (pseudogenes)
on chromosome 22
on chromosome 2
on chromosome 14
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Gene Rearrangement and Recombination in Variable Regions
• Somatic gene recombination
• Lead to unique antibodies• Conserved regions flank
gene segments (blue, orange)– Recombination signal
sequences (RSS)– Aid in rearrangement
• Performed by lymphocyte specific recombinases and ubiquitous DNA modifying enzymes– RAG1 and RAG2
• Rearrangement followed by looping out and joining by recombination
• Irreversible changes!
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Recombination Signal Sequences
• Consist of– Conserved heptamer– Spacer (12 OR 23 base pairs)– Conserved nonamer
• 12/23 rule
In heavy chain, V cannot be directly joined with J.
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Antibodies Can be Membrane Bound Or
Secreted• 2 exons located at the end of constant
regions from heavy chain– Code for hydrophobic amino acid stretch for
membrane spanning domain– Code for more for hydrophilic amino acids
for secretion
• Both are initially transcribed• Either membrane coding or the
secretion coding sequences are removed by splicing prior to translation
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Transmembrane and Secreted Forms of Antibodies
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Secondary Modifications of Antibodies
• In activated B cells (after first antigen contact)
• Driven by antigen• Initiated by activation-induced
cytidine deaminase (AID)• Somatic hypermutation• Gene conversion• Class switching
Variable region
Constant region
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Diversification of the Antibody Repertoire by Three Major
Processes
Involves heavy chain only
Involves variable region on heavy and light chain
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Somatic Hypermutation
• In mice and humans in germinal centers
• Requires also signals from activated T cells
• Random point mutations in V regions
• Alter affinity of the antibody for its antigen– Reduced antigen binding leads to
negative selection and cell death– Improved antigen binding leads to
positive selection, proliferation and finally plasma cell development
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Successful Somatic Hypermutations Occur
Predominantly in CDR Regions
Mutations in the framework tend to disrupt the antibody structure and are selected against.
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Gene Conversion
• Modification of re-arranged variable region
• Introduction of sequences derived from V gene segement pseudogenes
• Creates additional antibody specificities
• Occurs in some species – Birds, rabbits, cows, pigs, sheep, horses– Little or no germ line diversity
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Immunoglobulin Class Switch
• Same as isotype switch• Initial heavy chain is replaced by heavy
chain regions of another isotype–
• Aided by switch regions• Induced by cytokines (T cell derived) or
mitogenic signals from pathogen• Modification of antibody effector function• Does not change antigen specificity• B cell can undergo multiple rounds of
switching
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Germline Organization of Constant Region of the Heavy
Chain
• Naïve but mature B cells co-express IgM and IgD• IgM is always secreted first• IgM indicates acute infection/antigen challenge
Pseudogene
Carbohydrate group
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IgM and IgA Can Form Multimers
IgM Pentamer (5mer)Excellent agglutination~960 kDa
Monomers are cross-linked by disulfide bridges that connect each other and J-chain
IgA Dimer (2mer)Epithelial transcytosis~320 kDa
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Cytokine-Induced Switching of Isotypes
• IL4 induces IgE• IL5 augments IgA• IFN induces IgG• TNF induces IgG and IgA
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Distribution of Antibodies in the Body
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Basic Functions and Distribution of Antibodies
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Neutralization (IgG and IgA)
Toxin
Virus
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Antibodies Can Block Bacterial Adherence to Host
Cells
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OpsonizationAntibody only
In conjunction with complement
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Complement Activation
• Antigen:Antibody complexes
• Antibody conformation change
• Activate classical pathway of complement
• Binding of C1q to the Fc piece
• Pentameric IgM is more efficient
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Complement Receptors are Important in the Removal of Antigen:Antibody
Complexes
• Immune complexes activate C1q• Deposition of C3b• Binding to CR1 on erythrocytes via
bound C3b• Transport to liver and spleen• Removal from circulation by
phagocytes
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Anti-Parasitic Function of IgE
• Helminths are too bnig to be phagocytosed
• IgE binds to helminths• Eosinophils have Fc
receptors• After crosslinking
degranulation– Granules contain anti-
helminthic proteins
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IgG Mediated Sensitization for Killing by NK Cells
Antibody Dependent Cell Mediated Cytotoxicity
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Today’s Take Home Message• The variable regions of an antibody molecule are
coded by V and J gene segments in the light chain and by V, J, and D gene segments in the heavy chain.
• There are a limited number of V, J, and D gene segments.
• Gene rearrangement, looping out and gene recombination lead to the antibody diversity.
• Isotype switch is greatly influenced cytokines and dictates the functions of antibodies
• The major functions of antibodies include: neutralization, opsonization, complement activation, NK cell activation.