Lecture 27 - Immunity II

Question 1

What are antibodies?

Answer 1

Antibodies (aka immunoglobulins or Ig) are essential for adult survival and makeup around 20% of the protein in the blood plasma

Question 2

What is the basic structure of antibodies?

Answer 2

2 antigen binding sites and consists of 2 light and 2 heavy chains

Question 3

What are the different classes of antibodies?

Answer 3

5 classes - IgG, IgM, IgA, IgD and IgE where each has its OWN HEAVY CHAIN.

Also sub-classes (e.g. IgG1, IgG2, IgG3, IgG4) - each have different hinge and tail structures giving unique characteristics.

Question 4

What occurs when an antigen binds to the antigen receptor of a B-cell?

Answer 4

Proliferation and differentiation - antibodies are secreted from plasma cells

Question 5

What are features of IgM?

Answer 5

• IgMs are the first class of antibody that a developing B cell makes and major class secreted during the into blood on first exposure to an antigen.
• IgM is a pentamer composed of five 4-chain units, giving it a total of 10 antigen-binding sites.
• When antigens with multiple antigenic determinants bind to IgM, it alters the structures of the pentamer, allowing it to activate the complement system. This system is a molecular process that is activated by the constant domain that does the killing of the cells.

Question 6

What are features of IgGs?

Answer 6

• the most abundant antibody found in the body
• made of 2 copies of 2 proteins (4 in total), linked by covalent di-sulphide bonds: 2x Heavy chains (around 440aa) and 2x Light chains (around 220aa)
• each chain consists of variable and constant domains

Question 7

What does the constant domains interact with?

Answer 7

Other parts of the immune system - e.g. innate immune & complement systems)

Question 8

What do variable domains make up?

Answer 8

the antigen binding sites

Question 9

What are both light & heavy chains made up of?

Answer 9

made up of repeating 110aa domains as immunoglobulin domains each of which contains an internal di-sulphide bond. Likely to be the result of gene duplication during evolution

Question 10

Describe the size of the variable and constant regions of heavy and light chains.

Answer 10

Variable region - same size for light and heavy & chains

Constant region larger in heavy chain than light

Question 11

What does the antigen binding region consist of?

Answer 11

2 variable domains made up of a single modified Ig domain and containing 3 hyper variable regions.

Question 12

What is the Ig domains that make up the constant part of the heavy chain encoded by?

Answer 12

A single exon

Question 13

How many antibody molecules can a naive, unchallenged human immune system generate?

Answer 13

1 x 10^12 (a primary antibody repertoire)

Question 14

What can a mature immune system do?

Answer 14

Can make antibodies able to bind to essentially any antigen essentially infinite flexibility - this is able to occur through generation of antibody diversity

Question 15

How many genes does the entire genome encode?

Answer 15

25,000

Question 16

How does generation of antibody diversity occur?

Answer 16

• The Ig domains that make up the constant part of the heavy chain are each encoded by a single exon.
• the DNA encoding the variable domain is a bit more complicated.
• in the germ line a k-light chain gene variable domain contains 40x V domains, 5x J domains and a single C domain
• and a heavy chain gene variable domain contains 40x V domains, 25x D domains, 6x J domains and 5x C domains

Question 17

How are the domains recombined in developing B-cells?

Answer 17

In developing B-cells these are recombined in a process known as VJ or V(D)J recombination

Question 18

What is the process of VJ-recombination (light chain)?

Answer 18

• developing B cells join together separate gene segments in DNA in order to create the genes that encode the primary repertoire of low-affinity antibodies
• during the development of a B cell, a coding sequence joining a V to a J segment is assembled by removing the intervening genomic DNA (e.g. joining V3 to J3)
• transcription starts immediately upstream of the fused V segment, which lies immediately upstream of a J region. Extra downstream J segments are transcribed but edited out of the mRNA transcript

Question 19

What is the process of V(D)J recombination (heavy chain)?

Answer 19

• The process of V(D)J recombination joins separate antibody gene segments together to form a functional VL- of VH- region coding sequence
• DNA splicing is driven by the V(D)J recombinase enzyme (encoded by RAG1 & RAG2 genes)
• RAG1 or RAG2 mutations have a severe combined immunodeficiency phenotype (SCIP). Such mice are often used in xenograft experiments
• during joining of gene segments, a variable number of nucleotides are often lost or inserted from the ends of the recombination gene segments. This is called JUNCTIONAL DIVERSIFICATION
• in many cases this will shift the reading frame to produce a nonfunctional gene. These developing B cells never make a functional antibody molecule and die in the bone marrow.
• Developing B&T-cells are diploid (one maternal & one paternal copy), but choose just one allele to recombine - this is known as allelic exclusion.

ALL OF THIS JUST GENERATES THE PRIMARY ANTIBODY REPERTOIRE.

Question 20

How does the generation of mature antibodies?

Answer 20

• a naive, unchallenged human immune system contains around 1 x 10^12 different antibody molecules (the primary antibody repertoire
• a mature immune system can make antibodies able to bind to essentially antigen (essentially infinite flexibility)

Question 21

Where do you get 1 x 10^12 antibodies from?

Answer 21

Antigen-driven somatic hypermutation

Question 22

What occurs to the affinity of the antibodies after initial immunization?

Answer 22

Over time after initial immunization, there is a progressive increase in affinity of the antibodies towards antigens - this is known as affinity maturation.

Question 23

What is the process of somatic hypermutation?

Answer 23

Accumulation of point mutations in both heavy and light chain V-region coding sequences. This happens AFTER VJ/V(D)J recombination has assembled the gene segments to generate the primary repertoire.

Go from low affinity to high affinity over time. Happens via feedback loop - cells that produce best antibodies proliferate the most.

Question 24

What is somatic hypermutation?

Answer 24

• developing B-cells present their antibodies on their surface and binding of antigens stimulate their proliferation.
• most somatic mutations will either have no effect or will the antibody worse. This will stop antigen binding. Remove stimulus.
• cells containing mutations that increase affinity of the antibody to the antigen will increase the stimulus. These clones will survive and proliferate (especially as antigen levels get very low)
• in vivo evolution that selects cells with beneficial mutations

Question 25

Does somatic hypermutations occur in all cells?

Answer 25

No, somatic hypermutation does not occur in all B cells. It specifically occurs in germinal center B cells during an immune response.

Question 26

Describe the generation of mature antibodies

Answer 26

• affinity maturation occurs via the accumulation of point mutations in both heavy and light chain variable region (V-region) coding sequencing.
• after B cells have been stimulated by antigen and helper T cells in a peripheral lymphoid organ, some of the activated B cells proliferate rapidly in the lymphoid follicles and form structures called GERMINAL CENTRES.
• B cells mutate at the rate of about 1 mutation per variable region coding sequence per cell generation.
• Approximately 1 million times faster than ‘background’ mutation rate
• this is termed SOMATIC HYPERMUTATION
• mutations driven by ACTIVATED-INDUCED DEAMINASE (AID) expressed in germinal centres (GCs).

Question 27

How does BCL-6 inhibits the p53 pathway to prevent apoptosis in germinal center B cells?

Answer 27

• normally cells experiencing double stranded breaks (e.g. during V(D)J recombination) and high levels of DNA damage (e.g. somatic hyper mutation) will go through apoptosis via the p53 pathway that act as a “watch keeper” to kill cells with potentially oncogenic mutations.
• BCL-6 is a transcriptional repressor expressed in germinal centres
• BCL-6 binds to sites in the p53 promoter switching off expression

Leaves germinal centres without ‘watch keeper’

This process is critical in allowing B cells to undergo processes like somatic hypermutation and affinity maturation without being prematurely eliminated due to DNA damage.

Question 28

What is a summary for B-cells & antibodies?

Answer 28

• VJ and V(D)J recombination shuffles the light & heavy chain genes in the region encoding the variable domains in the genome, and cuts out intervening pieces to join together a random selection of exons that together (1v + 1D + 1J + 1C) make up 1 variable domain - approximately 110 amino acids.
• nucleotide lost/gain in recombining gene segments creates junctional diversification.
• initial ‘primary repertoire’ library of 1x10^12 B-cells
• affinity maturation via ‘in vivo evolution’ based on binding of antigens
• this selects amongst variations induced by somatic hypermutation

Question 29

What is the process of how B cells generate a diverse and effective antibody repertoire during an immune response.

Answer 29

• Combinatorial joining of gene segments –>
• Junctional diversification during gene segment joining –>
• Combinatorial joined of L & H chains –>
• Somatic hypermutation + class-switch recombination

Question 30

What is class-switch recombination?

Answer 30

class-switch recombination changes the antibody’s constant region to alter its class (e.g., IgM to IgG) without affecting antigen specificity.