MICR 304 Immunology & Serology
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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

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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


Overview of today s lecture

Overview of Today’s Lecture

  • Generation of antibodies

  • Secondary antibody modifications

  • Effector functions of antibodies


Key players in immunology

Key Players in Immunology


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


Recombination of a limited number of gene segments results in millions of variations

Recombination of a Limited Number of Gene Segments Results in Millions of Variations

~


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


Transmembrane and secreted forms of antibodies

Transmembrane and Secreted Forms of Antibodies


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


Distribution of antibodies in the body

Distribution of Antibodies in the Body


Basic functions and distribution of antibodies

Basic Functions and Distribution of Antibodies


Neutralization igg and iga

Neutralization (IgG and IgA)

Toxin

Virus


Antibodies can block bacterial adherence to host cells

Antibodies Can Block Bacterial Adherence to Host Cells


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.


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