Humoral Immunity: Generation of Antibody Diversity

Question 1

what is an antibody?

Answer 1

a Y shaped molecule expressed by immune cells to target pathogens, eg. bacteria

work by preventing bacteria from entering the cells by binding to their docking site, or by neutralising them and their toxins

Question 2

Antibody (Immunoglobulin) structure?

Answer 2

made up of 2 heavy chains and 2 light chains

heavy chain is made up 4 domains, 1 variable domain and 3 constant regions

heavy chain (μ,δ,γ,α or ε) has a hinge region between (CH1 and CH2) - stretch of polypeptides that makes the antibody flex so to expose the active site so immune cells can bind

light chain (κ or λ) has 1 variable region and 1 constant region

constant regions are the same for each antibody of the same class. Eg. all gammas will have the gamma constant region.

The variable region binds to specific epitopes of specific pathogens (via CDR’s)
The constant region has effector functions (activating complement, binding phagocytes)

Question 3

antibodies have 2 forms - what are they?

Answer 3

first form is anchored to the B cell receptor membrane.

the final form is the secreted form, used to fight off pathogens
- secreted in a monomeric form, and it’s capable of combining and forming multimeric forms like IgM after its been secreted

Question 4

Life cycle of B cells

Answer 4

split into 2 stages: antigen independent (bone marrow) and antigen dependent (blood and lymph nodes)

Stem cell differentiates into a pro-B cell, pro B cell undergoes 1st round of recombination called VDJ recombination which codes in the heavy chain variable region - the cell becomes a pre B cell once it makes valid heavy chain. Variable region will be co expressed with the mui constant region.

Junctional flexibility - P and N nucleotide addition

Undergoes v-J recombination to code in the light chain variable and constant region. It is now an immature B cell, and it will continue to mature until it expresses both IgM and IgD through differential mRNA splicing

Now a mature recirculating B cell, which will be activated when the body encounters a pathogen.

B cells then migrate into the germinal centre and undergo special forcal selection - affinity maturation and class switching.

B cell differentiates into plasma cells (secrete antibodies) or memory B cells.

Question 5

Somatic recombination vs Differential splicing

Answer 5

Somatic recombination (changes at DNA level):
V(D)J recombination
Tdt nucleotide addition
Somatic hypermutation
Class switching

Differential splicing (changes at mRNA level):
IgM and IgD
Membrane bound and secreted Ig

Question 6

what are CDR’s

Answer 6

Complementarity
Determining
Regions

3 finger-like protrusions in the variable region, interact with the antigen

Question 7

how many B cells does the body make?

Answer 7

1,000,000,000 resting B cells, each contains unique ‘random’ BCR

Question 8

when is a B cell classified as a mature/naïve B cell?

Answer 8

once it can express IgM and IgD ie. able to alternatively splice mRNA

Question 9

Three genetic loci encoding Ig - what are they?

Answer 9

Two for light chain: kappa (κ) and lambda (λ) locus

One for heavy chain

Located on different chromosomes

Question 10

How many antibody genes are inherited?

Answer 10

NONE! No complete genes are inherited, only gene segments

Arranging these gene segments in different combinations generate many Ig sequences

Question 11

what does VDJ stand for?

Answer 11

Variable Diversity Joining

J or D/J codes for CDR3 – most variable region of Ab

Question 12

VJ recombination of kappa light chain genes (Chromosome 2)

slide 20

Answer 12

40 Variable (V) segments

5 Joining (J) segments

Constant region (C) segment

Question 13

VDJ recombination of gamma heavy chain genes (Chromosome 14)

slide 22

Answer 13

51 Variable (V) segments, 27 Diversity (D) segments, 6 Joining (J) segments, Constant region (C) segments

cmui and cdelta are added, and then alternative splicing cuts one out - B cell can make 2 different classes of BCR via alternative splicing

Recombination signal sequences (RSS) – conserved sequences upstream or downstream of gene segments

One-turn/two-turn rule (12/23 rule)
Recombination only occurs between a segment with a 12bp spacer and a 23bp spacer

Question 14

Generation of antibody diversity

Answer 14

Multiple germline V, D and J gene segments
Combination V-J and V-D-J joining
Junctional flexibility
P-nucleotide addition
N-nucleotide addition
Combinatorial association of heavy and light chains
Somatic hypermutation during affinity maturation

Question 15

what happens in hairpin opening and joining

Answer 15

hairpin opening, end-processing, joining

Artermis randomly generates the knick between 2 A’s which is now the site of hairpin cleavage.

Exonucleases and TdT will interact with free ends and add base pairs (N nucleotides added)

At the end, you have V and J (yellow and green), but also P and N nucleotides in between in pink. The addition of the nucleotides causes a frame shift and increases diversity as you have new amino acids being produced.

Question 16

Junctional flexibility

Answer 16

Precise mechanisms unknown, occurs during flexibility during V(D)J recombination, P and N nucleotide additions

Involves Exonuclease - removes mismatched nucleotides

Good: Antibody diversity

Bad: Non-productive rearrangements (incorrect reading frame) – wasteful process

Question 17

how nay copies of Ig gene do we have?

Answer 17

2 copies, one from mother and one from father

Allelic exclusion - one is silenced, only one heavy chain allele and one light chain allele is expressed