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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.

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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
Overview of Today’s Lecture
  • Generation of antibodies
  • Secondary antibody modifications
  • Effector functions of antibodies
variable regions are constructed from gene segments
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.
construction of the antibody v regions
Construction of the Antibody V Regions

L: Leader sequence; directs protein to cell’s secretory pathway

: Hinge region

germline organization of the light and heavy chain
Germline Organization of the Light and Heavy Chain

on chromosome 22

on chromosome 2

on chromosome 14

Includes a significant number of non-functional segments (pseudogenes)

gene rearrangement and recombination in variable regions
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 specificrecombinases and ubiquitous DNA modifying enzymes
    • RAG1 and RAG2
  • Rearrangement followed by looping out and joining by recombination
  • Irreversible changes!
recombination signal sequences
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.

antibodies can be membrane bound or secreted
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
secondary modifications of antibodies
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

diversification of the antibody repertoire by three major processes
Diversification of the Antibody Repertoire by Three Major Processes

Involves heavy chain only

Involves variable region on heavy and light chain

somatic hypermutation
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
successful somatic hypermutations occur predominantly in cdr regions
Successful Somatic Hypermutations Occur Predominantly in CDR Regions

Mutations in the framework tend to disrupt the antibody structure and are selected against.

gene conversion
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
immunoglobulin class switch
Immunoglobulin Class Switch
  • Same as isotype switch
  • Initial m heavy chain is replaced by heavy chain regions of another isotype
    • d, g, a, e
  • 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
germline organization of constant region of the heavy chain
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

Carbohydrate group

Pseudogene

igm and iga can form multimers
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

cytokine induced switching of isotypes
IL4 induces IgE

IL5 augments IgA

IFNg induces IgG

TNFb induces IgG and IgA

Cytokine-Induced Switching of Isotypes
opsonization
Opsonization

Antibody only

In conjunction with complement

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

Complement Activation
complement receptors are important in the removal of antigen antibody complexes
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
anti parasitic function of ige
Anti-Parasitic Function of IgE
  • Helminths are too bnig to be phagocytosed
  • IgE binds to helminths
  • Eosinophils have Fce receptors
  • After crosslinking degranulation
    • Granules contain anti-helminthic proteins
igg mediated sensitization for killing by nk cells
IgG Mediated Sensitization for Killing by NK Cells

Antibody Dependent Cell Mediated Cytotoxicity

today s take home message
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.