3 Antibody Diversity I Flashcards
where do B cells come from in adults and foetus’?
adult mammals - directly from the bone marrow
foetal liver at 8-9 weeks gestation
what are primary lymphoid organs?
bone marrow and thymus
Bone marrow - Pluripotent stem cell that develops to a T or B cell. The pluripotent stem cells then enter the thymus where they are educated not to react to self antigens.
The B cells come out of the bone marrow and enter the secondary lymphoid organs such as blood, spleen, lymph or lymph nodes.
examples of secondary lymphoid organs
Blood
Spleen
Lymph
Lymph nodes
Have circulating B and T lymphocytes
B cell development in the bone marrow
- Progenitor cells line the bone endosteum.
- They travel through the bone marrow and go through stromal reticular cells (mixed phenotype - fibroblast; endothelial; myofibroblasts). These cells provide all the necessary signals for the B cell to survive and differentiate, predominantly IL-7
- As they pass through the reticular cells they develop and mature
- The ones that don’t make it get taken by macrophages and discarded
- Each progenitor can produce up to 64 progeny
- Progeny migrate to the centre of spongy bone and then adventitial reticular cells - these aid the cells to enter circulation via venous sinusoid
- The cells that have a productively rearranged receptor on their surface are taken up by the adventitial reticular cells to later enter circulation
B cell selection
~75% of B cells don’t make it out of the bone - have an unproductive rearrangement of their receptor so undergo apoptosis - phagocytosed by macrophages
Those B cells that survive have rearranged their Ig gene successfully
Autoreactive B cells are deleted by –ve selection
Mechanisms of diversity
Somatic recombination: this allows the joining of one segment of the gene to another.
Somatic mutation: this allows for ‘sloppy’ joining of those segments.
Together these generate the heavy and light chains of Ig.
The pairing of unique heavy and light chains adds another level of diversity
Order of diversity generation
First Heavy Chain rearrangement
If successful get κ light chain
If no κ, then λ chain rearranges
Heavy chain V, D & J
ch 14
-components of the Ig diversity region
V region - 80 gene segments, approx 50 are functional
D region - 23 DH segments
J region - 6 segments
Recombination of any one V with any one D and any one J forms the functional heavy chain VDJ region
Each V region codes for a signal peptide which directs the polypeptide to the RER, Golgi and then out of the cell.
Heavy chain rearrangement
- First get a joining of the JH to the DH
- DH are highly variable, both in number of codons and sequence
- 3 possible reading frames
- Can be used singly or in combination!
- The VDJ- region constitutes the highly diverse 3rd hypervariable region (CDR3) of the Ig molecule
If this is a productive rearrangement then
the DHJH signals the rearrangement to a VH gene segment.
This rearrangement forms a contiguous sequence encoding the VHDHJH protein sequence.
VDJ recombines to Cμ if naïve B-cell or one of the other 8 C regions after antigen experience
Light chain rearrangement
-Human κ light chain locus is on Chromosome 2
-31-35 functional Vκ gene segments, a promoter sequence 5’ to each one: no germline transcription
-No Dκ segments
-5 Jκ segments
-1 Cκ region
Additional diversity generated by ‘sloppy’ joining!
- Primary ‘nuclear’ RNA contains additional J regions which are spliced out to form the mRNA
- If a non-productive VJ rearrangement is created then the other κ allele is used.
- If this is also non-productive then the cell moves to the λ chain
λ chain
λ locus on Chromosome 22
Has 29-33 functional V λ gene segments
Has 7-11 J λ segments each linked to a C λ region (the number of J λ C λ sequences is dependent on the haplotype).
Extra diversity is generated by imprecise joining.
After V λ and J λ have combined there is an intron (non-coding sequence) between VJ and C λ. This region is cleaved out from the primary RNA transcript.
How does recombination happen?
Essential part of B cell and T cell receptor generation
Utilises highly specific signal sequences
Enzymes involved include RAG 1 and 2 complex (Recombination-Activating Genes) - cleave the DNA at specific signal sequences
N-region junctional diversity by TdT (Terminal-deoxynucleotidyl Transferase) - adds the extra nucleotides for further diversity
Recombination Signal Sequences (RSS)
Flanking all V, D and J segments
CACAGTG heptamer is downstream (3’) of VH, VL and DH (or analogue)
Followed by a spacer of 12 or 23 non-conserved bases
Then a ACAAAAACC nonamer (or analogue)
Upstream (5’) of JH, DH and JL is a corresponding nonamer, spacer (12 or 23) and heptamer sequence
The 12 and 23 spacers correspond to one or two turns of the double helix.
Only 12 will combine with 23, not with another 12 (12/23 rule)
Mechanism of recombination (Lymphoid specific)
Firstly the portions of the gene are made available to the recombination machinery. Two selected coding segments and their RSSs are brought together. Chromosomal loop.
Double stranded breaks are generated at the RSS-sequence junctions by RAG1/RAG2 complex (VDJ recombinase). This creates a hairpin end on the coding region.
Opening of the hairpins (Artemis) and addition or removal of bases (TdT) to add extra diversity.
The coding ends are then rejoined by a number of factors (inc DNA ligase.
Diversity summary
Combinational diversity
V, (D) and J regions can combine randomly creating many antigen specificities.
The random pairing of VH and VL chains
Not all pairings will form functional receptors
Junctional diversity
Addition and removal of bases at the V,D,J junctions
Largest contribution to diversity
