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

Overview of Today’s Lecture

• Generation of antibodies• Secondary antibody modifications• Effector functions of antibodies

Key Players in Immunology

Innate Adaptive

Cells PhagocytesEpithelial Cells

NK Cells

Lymphocytes(B-Ly, T-Ly)

Effector molecules

ComplementAntimicrobial (Poly)PeptidesAntimicrobial

lipids?

Antibodies

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

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~

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

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!

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• 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

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

Involves heavy chain only

Involves variable region on heavy and light chain

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

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

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

• 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

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

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• IFN induces IgG• TNF induces IgG and IgA

Distribution of Antibodies in the Body

Basic Functions and Distribution of Antibodies

Neutralization (IgG and IgA)

Toxin

Virus

Antibodies Can Block Bacterial Adherence to Host

Cells

OpsonizationAntibody 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 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

• Helminths are too bnig to be phagocytosed

• IgE binds to helminths• Eosinophils have Fc

receptors• After crosslinking

degranulation– Granules contain anti-

helminthic proteins

IgG Mediated Sensitization for Killing by NK Cells

Antibody Dependent Cell Mediated Cytotoxicity

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.