51. Humoral Immunity, Generation of Antibody Diversity Flashcards
What does an antibody consist of?
- 2 heavy, 2 light chains (heavy: g, a, m; light: kappa, lambda)
- Variable region binds antigen, CH1 support
- Constant region- effector functions (activating complement, binding phagocytes)
- Disulphide bonds stabilize
- Hinge region
What are the different forms of an antibody?
1) Membrane bound
2) Secreted form
•Secreted as a monomer and then it combines to form multimeric forms
Life cycle of B cells
- First in the BM – antigen independent stage
- Blood and lymph node - antigen dependent stage
Stem cell-> Pro-B cell -> Pre-B cell (valid and functional heavy chain) -> immature B cell -> mature recirculating B cell (IgM and IgD expression) -> in blood and in spleen lymph node
GC (germinal center) B cells then differentiate into Memory cells (in the blood) or plasma cells (in the lymph node).
What is somatic recombination and differential splicing?
SOMATIC RECOMBINATION ~ DNA level changes
- 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
What are the 3 genetic loci encoding Ig?
- Two for light chain: kappa (κ) and lambda (λ) locus
- One for heavy chain
- Located on different chromosomes
How many antibody genes are inherited?
- NONE! No complete genes are inherited, only gene segments
* Arranging these gene segments in different combinations generate many Ig sequences
Describe V(D)J recombination
LIGHT CHAIN
•v = variable region
•J = gene segments (light chain)?
•C = constant segments
HEAVY CHAIN •v = variable region •D = diversity segment •J = gene segments (light chain)? •Cμ = constant segment
•These segments are rearranged at DNA level. This will produce different regions
•J or D/J codes for CDR3 ~
Most variable region of Ab
•C regions encodes for constant region
Expand on VJ recombination of kappa light chain genes (Chromosome 2)
- 40 Variable (V) segments
- 5 Joining (J) segments
- Constant region (C) segment
- V23/J4 are selected at random
- This will be rearranged at a DNA level
- This will then be transcribed at RNA transcript. – the region between the leader and the variable region, and the region between the J bit and the C bit will be spliced out and removed ~ forms a mature mRNA
- Once the sequence has been corrected folded and gone to where it needs to be, the leader segment will be cleaved off
- Whatever is left will be the light chain of the B-cell R or the secreted antibody
Expand on VDJ recombination of gamma heavy chain genes (Chromosome 14)
- 51 Variable (V) segments
- 27 Diversity (D) segments
- 6 Joining (J) segments
- Constant region (C) segments
- D7/J3 are selected at random and then the V20 is selected at random to join the D7/J3
- The Cμ and Cδ will be transcribed with the L-VDJ
- With alternative splicing, you can get rid of the Cμ or Cδ
- You splice out the L region after the chain has reached where it needs to be.
- μ heavy chain = VDJ Cμ
- δ heavy chain = VDJ Cδ
Generation of antibody diversity
- 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
V(D)J recombination mechanisms
- Recombination signal sequences (RSS) – conserved sequences upstream or downstream of gene segments
- ‘Turns’ consisting heptamer and nonamer with a 12 or 23 bp spacer
What is the one-turn/two-turn rule (12/23 rule)?
Recombination only occurs between a segment with a 12bp spacer and a 23bp spacer
Mechanisms for recombintion
- RAG1 and RAG2 have to clamp onto the RSS at both ends - This forms a major hairpin
- Then it will create nicks in DNA - Then the DNA will form a minor hair pin
- There are 2 types of hairpins – between the upper and lower strands, and the major one is the whole structure folded on itself
- Then there are enzymes involved that allow the coding joint (V17-J3) and for there to also be a circular signal joint
Mechanism of hairpin opening and joining
1) Hairpin opening ~ Artemis comes and opens the hairpin
2) End-processing ~ The exonucleases and the TdT will mess around with the free ends – e.g. adding of deleting bps
3) Joining by enzymes. There are P and N (heavy chain) nucleotides added
What is junctional flexibility?
- Precise mechanisms unknown
- Involves Exonuclease - removes mismatched nucleotides
- Coding joints are impercise
- Signal joints are always precise
- Junctional flexibility during V(D)J recombination, P and N nucleotide additions
+ Antibody diversity
- Non-productive rearrangements (incorrect reading frame) – wasteful process
