W8L9 - The Antibody Paradox

Question 1

Functions of Antibodies

Answer 1

On surface of B cells they act as an antigen receptor
- stimulate B cell to make antibodies to antigen
In circulation antibodies bind antigen:
- agglutination of antigen by crosslinking
- neutralisation by blocking (virus, toxin) attachment to receptor
- opsonisation where phagocytes with Fc receptors engulf the antigen
- activate complement

Question 2

Structure of Antibodies

Answer 2

Antibodies are Y shaped molecules
Fc portion is where phagocytic cells bind
Further near the hinge region is where complement binds
Ends of Y made of both heavy and light chains are the two “antigen binding sites”.
Constant (C) and Variable (V) regions are coded by different genes
V region different for all antibodies and bind to different antigens

Question 3

Variable Region of Antibodies

Answer 3

Contain hypervariable regions where the amino acid sequence is very different between different antibodies
- called complementary determining regions (CDR)
- allow for binding specifically to antigen
Constant region does not have these

Question 4

The Antibody Paradox

Answer 4

The immune system repertoire needs a massive number of specificities and if one antibody is coded for by one gene, how are so many antibodies made when the whole human genome contains only about 30,000 genes?
- not enough genes in human genome to make all antibodies needed
The repertoire must be universal
- the immune system is set up to potentially recognise anything
Antibody diversity cannot be generated by germ line cells and must be generated by somatic cells
- new antibody specificities are created during life

Question 5

Somatic Cell Recombination

Answer 5

Genetic information for antibodies is transmitted in segments
- not a single gene for a protein
- multiple gene segments
- different segments combine giving different antibodies
- the segments make a whole complete gene
Only antibodies and T-cell receptor genes do this

Question 6

Strange Genetics

Answer 6

Variable and constant regions of immunoglobulin heavy and light chains
- encoded by different regions of DNA in germ-line cells
These join together to give a single gene in B cells
- make whole heavy or light chain (complete functional antibody)

Question 7

Somatic Cell Recombination - How do V and C regions Join Together to form Complete Functional Antibody

Answer 7

V and C genes different from germ cells (present in separate DNA fragments)
Combination to form zygote
Cross over to form new V & C combination
New chain
Combination not used is excluded in a process called allelic exclusion

Question 8

Antibody Gene Segments for Heavy and Light Chains

Answer 8

For light chains:
- variable (approx 30)
- joining (4-5)
- constant (2: kappa or lambda - with some variation)
For heavy chains
- variable (approx 40)
- diversity (25)
- joining (6)
- constant (5: ɣ, μ, ε, α, δ - with some variation)

Question 9

Recombination of Segments

Answer 9

Each of the segments for heavy and light chains can be recombined in different combinations
Recombination can join any V with any J (light chain)
- generating approx 300 combinations
Recombination of V, D and J (heavy chain)
- generating approx 6,000 combinations

Question 10

Unwanted and Wanted Genes

Answer 10

Unwanted genes need to be removed
Wanted genes need to be combined
Done by:
1. Recombination signal sequences (RSS)
- at ends of segments
- allows for cutting and re-attachment
2. Recombination activating gene (RAG) enzyme
- cuts at RSS
- loops out section
- re-combination of genes
- two RAG-1 and RAG-2
Other enzymes as well e.g. V(D)J Recombinase

Question 11

P-Region and N-Region Nucleotide Additions

Answer 11

Somatic recombination is not the only mechanism that greats antibody diversity
P-region nucleotide insertions
- palindromic (P) sequences found at V region junctions, generated by hairpin loops
N-region nucleotide additions
- up to 15 nucleotides can be inserted into the junctions of gene segments by the terminal deoxynucleotidyl transferase (TdT) enzyme

Question 12

Somatic Hypermutation

Answer 12

Occurs within the germinal centres of the lymph node
Not in germ cells (not inherited)
Generates B cells with higher affinity Ig receptors
- most occur in CDR1 & 2 of variable region
Occurs after antigen stimulation
- induces point mutations
Antibodies become very specific for antigen

Question 13

Receptor Editing

Answer 13

Occurs in bone marrow
When an antibody directed against a self antigen is not deleted
Modifies the sequence of light chain V and J genes
- components of the antigen receptor
- light chain goes around again for rearrangement
Different specificity - no longer recognise self antigens

Question 14

B Cell Development - Heavy Chain Genes

Answer 14

Stem cell - germline
Early pro-B cell - D-J rearranging
Late pro-B cell - V-DJ rearranging
Large pre-B cell to mature B cell - VDJ rearranged

Question 15

B Cell Development - Light Chain Genes

Answer 15

From stem cell to large pre-B cell - germline
Small pre-B cell - V-J rearranging
Immature B cell and mature B cell - VJ rearranged

Question 16

B Cell Development - Surface Ig

Answer 16

From stem cell to late pro-B cell - absent
Large pre-B cell - u chain transiently at surface as part of pre-B cell receptor (mainly intracellular)
Small pre-B cell - intracellular u chain
Immature B cell - IgM expressed on cell surface
Mature B cell - IgM and IgD made from alternatively spliced H-chain transcripts

Question 17

B Cell Development - Additional Information

Answer 17

First heavy chain rearranges then light chain
If B cell fails to rearrange H or L chains it dies
Mature B cells leaves bone marrow and travels to spleen and lymph nodes

Question 18

Clonal Deletion and Clonal Selection

Answer 18

Clonal deletion = in bone marrow, B cells that have surface Ig that react to self antigens are removed
Clonal selection = in the periphery, B cells that react with foreign antigen get activated and proliferate