Lecture 2 Flashcards
Specific/adaptive immunity:
- is induced by exposure to a particular infection
- shows a high degree of specificity
- exhibits “memory”
Antibodies
Number of different possible antibodies in humans:
>1011
< 30,000 (3 x104) genes in human genome
(approx. 170 human H/L chain genes)
How does the number of antibosies work?
How can there be 3 x106 more (1011/3 x104) possible BCR/antibodies than there are genes present in the human genome?
A range of mechanisms allows the generation of an enormous antibody repertoire from relatively few (~200) genes
(and similar (but not identical) mechanisms exist for generation of equally large repertoire of different TCRs)
- very fine tuned
Multiple genes encode a single polypeptide chain:
• V and C regions of antibody and TCR polypeptide chains are encoded by separate gene segments that rearrange during lymphocyte differentiation
• H chain and TCR:
V region encoded by three gene segments:
V, D and J (V is the biggest)
• L chain and TCR :
V region encoded by two gene segments: V and J
What are the V & C regions of antibody & TCR polypeptide chains are encoded by?
Separate gene segments that rearrange during lymphocyte differentiation
Domains are encoded by very small mini genes
There are bits in-between V & C
What are the bits in-between V & C?
D = diversity
J = joining
What is the V region of H chains & TCR-beta encoded by?
V
D
J
What is the V region of L chains & TCR-alpha encoded by?
V
J
What do V, D & J encode?
V region of H chains & TCR-beta
What do V & J encode?
V region of L chains & TCR-alpha
Rearrangement of Ig genes in B cells
Genes rearrange during B cell development to form a functional gene
The genes in B cells are closer than in every other type of cell in the body
DNA is different in B cells than every other cell
Non-homologous end joining (NHEJ) recombination
B cells break their DNA in a way no other cells do (apart from T cells) & move genes so they are closer
How do B cells produce a functional immunoglobulin?
Involves recombination to bring gene segments together
Light chain of an antibody
After DNA breaks, a single V & a single J gene segment are joined together to encode the V region of the light chain
The 2 exons come together
DNA is broken randomly to bring 2 of each together randomly
Leads to different BCRs in every cell
B cell has to decide to break its DNA at one locus – either lambda or kappa
What happens once V & J are next to each other for the light chain?
It gets transcribed into RNA like a normal gene making the light chain – can then fold up to form a unique antigen binding site
Heavy chain of an antibody
Similar mechanism but extra sequence section with the D region
2 breaks needed
A single V, D & J gene fragment are joined together to encode the V region of the heavy chain
Order of breakage in the heavy chain
Each B cell first breaks between D & J to bring them together
Then is sequentially broken again & the DJ joins the V
Lots of combinations – every V can go with every D & J
Hierarchy of rearrangements in light and heavy chains
- greater variability in H chain as V,D and J
- first H chain genes (D-J then V-D)
- then light chain genes: κ first (V-J)
- if κ rearrangement unsuccessful then λ genes rearrange
What forms the binding site?
Variable regions of H & L chain
What chain has greater variability?
Greater variability in H chain as V, D & J
What loci are the H, kappa & lambda chains encoded?
H chain – chromosome 14
Kappa chain – chromosome 2
Lambda chain – chromosome 22
Recombination signal sequences (RSS):
- DNA rearrangement is guided by special sequences flanking the V, D and J regions = recombination signal sequences
- involves a complex of enzymes = V(D)J recombinase
- (recombination activating gene)
- RAG-1 and RAG-2 genes encode lymphoid-specific components of the recombinase
- mutations in RAG genes result in immunodeficiency
Which gene segments encode the variable region of the Ig heavy chain?
V, D, J regions
Which gene segments encode the variable region of the light chain of an antibody?
V region and a J region
How are the heavy (H) chain of an antibody and the beta (b) chain of the TCR similar?
Both have a V region, D region and a J region
When does rearrangements of Ig genes occur?
Genes rearrange during B cell development to form a functional gene
What are the 2 types of light chain found in antibodies?
Kappa and lambda
Allelic exclusion
In a single B cell only one allele of H chain expressed, similarly for L chain
light chain isotype exclusion
a single B cell expresses either k or l ,never both
These mechanisms ensure that an individual B cell produces only one specificity of antibody
Mechanisms for generation of antibody diversity:
1) multiple germline genes
2) combinatorial diversity
3) junctional diversity
4) combinations of heavy and light chains
5) somatic hypermutation
multiple germline genes
Multiple VH, Vk and Vl
also, multiple D and J (no D for k and l)
combinatorial diversity
Different V, D and J segments recombine to produce different sequences
e.g. 40V x 25D x 6J would give 6,000 combinations
(note, due to D segments, H chains potentially more diverse than L chains)
junctional diversity
Includes: imprecise joining (small differences in position of V-D and D-J join)
N regions (random addition of nucleotides at junctions of V-D and D-J by terminal transferase)
combinations of heavy and light chains
e.g. 106 H and 104 L would give 1010 possible antibodies
(Further) mechanisms for generation of antibody diversity
somatic hypermutation
mutation frequency in antibody VH genes orders of magnitude higher than normal spontaneous mutation rate
occurs in germinal centres as B cells recognize Ag and proliferate/become activated
Somatic hypermutation:
- Involves the enzyme, activation-induced deaminase (AID)
- AID acts on DNA to de-aminate cytosine to uracil
- uracil is then recognized by error-prone DNA repair pathways leading to mutations
Membrane (BCR) vs. secreted antibody:
- following Ag recognition as each B cell differentiates, it will start to secrete its unique BCR as an antibody
- the secreted form made by each B cell has an alternative constant region that lacks a transmembrane region. As the original re-arranged VDJ regions are not altered, the secreted antibody has the same antigen specificity as the membrane BCR.
- the membrane and secreted forms are produced by alternative RNA processing
Antibody classes:
IgM - Heavy chain μ IgD - Heavy chain δ IgG - Heavy chain γ IgA - Heavy chain α IgE - Heavy chain ε
Heavy chain constant region genes and class-switching:
- constant region of each heavy chain is encoded by a different C region gene segment (i.e. Cμ, Cδ, Cγ, Cε, Cα)
- four g chain genes correspond to the four IgG subclasses: two α chain genes
- As the Cμ is physically the closest to the V, D and J genes at the heavy chain locus this is why IgM is first class/isotype BCR/antibody expressed by each developing B cell
Summary
- the BCR/antibody polypeptide in each B cell is encoded by multiple genes segments
- these rearrange during B cell development (randomly so each B cell will assembly a different BCR)
- the random gene segments used will determined antibody specificity
- the joining process introduces further variation
After B cells have assembled a random BCR
After leaving bone marrow, V region genes may undergo somatic hypermutation in specialized areas of secondary lymph organs following the binding of Ag. and B cell activation
this leads to further BCR specificities being generated
activated B cells start to secrete their BCR (antibody)
class-switch recombination may then occur resulting from the presence of cytokines
