Lecture 11: Generation of diversity Flashcards
What is the estimated number of antibody molecules that an individual is born with?
10^11
Antibody repertoire
More base pairs than in entire genome, how is this so? - lecture explains
What is clonal selection theory?
Each and every b lymphocyte produced from bone marrow has only one antibody specificity
What does the clonal selection theory say about antigen generation?
- antibody repertoire is generated randomly before birth with no antigen knowledge.
- specifications are selected by antigenic stimulation
- activated clones expand and produce antibody producing plasma cells or memory cells
What else may the clonal selection theory apply to?
T cells with at TcR.
Describe clonal selection theory
Naive B cells
- clonal expansion
- affinity maturation
- plasma cell (AB) or memory cell
In broad terms describe B cell origins
Immunoglobulin gene locus
Arise in bone marrow and mature in the spleen
Describe in broad terms T cell development
T cell receptor gene locus
Arise in bone marrow and mature in the thymus
What are the seven key elements in adaptive immunity and memory?
(In B cell development)
- Repertoire Production
- Antigen stimulation
- Clonal expansion
- Somatic hyper mutation
- Affinity maturation
- Class switching and antibody production
- Memory
Describe what is meant be repertoire production in B cell development?
Many B cells with randomly arranged Ig gene locus are produced in the bone marrow.
This is random (stochastic)
IgM is the default B cell surface antigen receptor
Describe what is meant by antigen stimulation in B cell development;
Small number of B cells in lymphoid tissue encounter antigen.
Naive B cells have weak affinity toward antigen, respond and proliferate (antigen presentation is essential for this step)
Each has a unique antigen specificity
Describe what is meant by clonal expansion in B cell development;
Proliferating B cells expand rapidly and form a germinal centre in a follicle.
Antigen drives this activation, expansion and later maturation
Describe what is meant by somatic hyper mutation in B cell development;
During B cell development, errors occur called somatic hypermaturation, some of these will improve affinity toward and antigen.
Describe what is meant by B cell maturation in B cell development;
Higher affinity B cell clones expand at a greater rate because they require less antigenic stimulation. Thus overtaking their slower growing predecessors
I.e morehigher affinity B cells are produced
Describe what is meant by class switching and antibody production in B cell development
Highest affinity clones undergo a change which induces class switching from default IgM to IgG.
Majority because plasma cells producing massive amounts of IgG
A small number of IgM become memory cells. Essential for adaptive immunity
What happens to the Ig gene loci during B cell development?
Undergoes somatic recombination ( same as TcR)
Describe the Ig gene loci
It is segmented into germline V, D and J exons
What happens to the germ line segments (V,D,J) during B cell development?
Germ line segments in the heavy and light chain loci rearrange to produce an entirely new gene.
This is completely random.
Recombination events are not inherited.
What cells does recombination occur in!
Only t and B cells because of their recombinant enzymes (RAG1 & RAG2)
What is recombination controlled by?
The order and process of rearrangement is strictly controlled by two important rules
Describe the hypervariable regions and the germ line structures;
Ig and TcR have three hypervariable regions;
CDR/HV1 = v segment CDR/HV2 = v segment CDR/HV3 = VDJ segment ( junction diversity)
What are five mechanisms that generate diversity?
- Random H-L pairing
- Segmentation of H and L gene locus
- Somatic recombination
- N region diversity
- Somatic hyper mutation
What does random HL pairing cause?
Random H L chain pairing provides a degree of diversity (of antibodies)
How does H and L loci segmentation create AB diversity?
Gene duplication of individual V,d,j,c segments creates a large repertoire of different segments.
How does somatic recombination contribute to antigen diversity?
Somatic recombination of VJDC segments to create new coding genes.
D joins V
V joints D
J joints C
Describe what n region diversity is and how it contributes to antigen diversity
N region diversity results from the imprecise joint between V,D,J germ line gene segments when they combine.
Thus allowing greater diversity as the possible alterations are limitless
What creates the junction all diversity of the n region?
- d segment can be read in all three reading frames and sometimes in reverse.
- imprecise joining leaves p nucleotides that would otherwise be removed
- n nucleotides are added or removed to the ends of D or V segments (TgT)
Describe how somatic hypermutation contributes to antibody diversity
Continued mutations at hot spots within CD-R regions of Ig gene during clonal expansion.
Key for affinity maturation
What gene does somatic hypermaturation not occur in?
TcR genes
Describe the temporal process of Ig heavy chain rearrangement (look at book page 88)
In the genome, somatic recombination occurs twice. Firstly it reorganises genome so that d and j segments join. Then this occurs again that d and v join.
VDJ (now one segment, no other random DNA between them) joins the c region (constant) via mRNA splicing not recombination.
C(m) region is typical at this stage. During affinity maturation isotope switching can occur and c regions can be switched.
Describe the temporal process of Ig light chain rearrangement
No D segments!
Somatic rearrangement v and j.
mRNA splicing joins VJ and C(kappa) (
What is essential for recombination?
RAG 1 & 2. These are recombination genes and are tightly regulated to ensure that one cell produces one antibody
Describe the RAG origin.
RAg 1 & 2 originally transposing inter grated with PRR gene,
Transposable elements genes relocated to different part of genome but left recognition sequence behind, (Rs) could operate in trans
Trans recombination events - gene duplication and segmentation
What recognises the RS sequence left behind?
RAG 1 and 2
What is the key to recombination events?
The preserved signal sequence at the flank
3’ end of all v segments
5’ and 3’ of d segments
5’ end of j segments
With 12 or 23 base pair intervening sequence (2 or 1 helix turns)
What is recombination regulated by?
12/23 base pair joining rule.
I.e 12/12 and 23/23 are not allowed.
Considering the 12/23 rule, what happens in segment binding?
- D regions can be joined in both orientations (by j) as 12bp either end, j is 23
- H chains only allow v-d or d-j. But no v-j as j and v both 23 bp for RS
- L chains allow v j binding as no d segment. J(l) unlike j(h) has 12 bo at 5 end instead
What are the three joining mechanisms of recombination?
- Loop out deletion(I,e v-3’ joins 5’-D and intervening DNA is excised)
- Inversion. V and j brought into parallel and same result, but intervening DNA ends up 5’ end of bind, not excised
- Sister chromatid exchange of loci
What Ig class has four sub classes?
IgG
Describe how the initial splicing of mRNA creates and IgM molecule
VDJ is spliced (joined) to c(m) region segment. (Default) though IgD can occur too
How does c class switching occur?
Following antigenic stimulation signals are received by the B cell which initiates c region class switching.
Exact mechanism of how VDJ is spliced or moved to C(g) is unknown but CD40 is required…
What is allelic exclusion?
It’s believed that one allele is methylated preventing its rearrangement.
This occurs if Ig (heavy) and TcR beta. (Not IgL or TcR a)
Thus preventing two IgG molecules.
How does affinity maturation occur?
Results from somatic hyper mutation of h and l chains post B cell selection.
Mutations in variable regions
As antigen depletes in germinal centre, higher affinity B cells are selected (boosting)
A very high affinity antibody is obtained eventually and this is the memory cell.
