Week 6 Lecture 8 - Genetics of B and T Cell Receptors Flashcards
Antibody Structure
The hypervariable loops from each domain create a single hypervariable site at the tip of each arm of the molecule:
- antigen-binding site
The Light chain V region has three hypervariable (HV) regions flanked by four framework regions
Hypervariable regions are in the three loops at the tip of the antibody molecule
Framework regions correspond to the beta-strands and the remaining loops
The hypervariable regions are called:
- Complementarity-Determining Regions (CDRs)
- three CDRs from the light chains: CDR1, CDR2 and CDR3
The Heavy chain also has three hypervariable regions: HV1, HV2 and HV3, flanked by four flanking regions
Antibody Repertoire
Virtually any substance can be a target of an antibody response → huge numbers of different antibodies each with different antibody specificities available to an individual
This is called Antibody repertoire
Antibody repertoire in humans is >10^11 , the number of antibody specificities at any one time is limited by:
1. The total number of B cells, and
2. The individual’s previous encounters with antigen
How are new Specificities Created During Life
- DNA sequence encoding each V region is generated by the rearrangement of a small group of inherited gene segments (somatic recombination)
- Diversity is enhanced by somatic hypermutation in mature activated B cells
Organisation of Ig Genes
Light Chain (λ): ~ 30 V gene segments & 4 pairs of functional J gene segments & C genes
Light Chain (κ): ~ 40 V gene segments & a cluster of 5 J gene segments, but one C gene
Heavy Chain: ~ 40 V gene segments & cluster of ~ 25 D segments & 6 J segments & a cluster of C genes
Light Chain Gene Segments - Overview
L chain V region is encoded by two separate gene segments:
- variable segments, V
- small joining segments, J
In the germline DNA one V and one J are joined to form a complete light chain V-region exon
The V gene segment is preceded by an exon encoding a leader peptide (L), to direct the peptide to a secretion pathway and is then cleaved
Light chain C region is encoded by a separate exon and is joined to the V-region exon by splicing the mRNA to remove the introns
κ Light Chain Rearrangement & Synthesis
An enzyme complex: V(D)J recombinase mediates joining - it recognises recombination signal sequences (RSS) located at the ends of the V and J gene segments
Recognition of these conserved RSS motifs ensures the gene segments join in the correct order
The products of the two recombination-activating genes: RAG-1 and RAG-2 are critical in initiating recombination
Both the RAG-1 and RAG-2 proteins are involved in cleaving DNA at the junction of the RSS and the gene coding sequences
λ Light Chain Rearrangement & Synthesis
The λ light chain also undergoes rearrangement of DNA in much the same way as the κ light chain:
- Vλ gene segment encoding for the N-terminal gene segment joins a Jλ gene segment, but……
- as each Jλ gene is associated with a different Cλ gene a λ-chain will have one of four possible Cλ regions
Heavy Chain Gene Segments - Overview
Heavy chain V region is encoded by three separate gene segments:
- variable segments, V
- diversity segments, D
- small joining segments, J
In the germline DNA one D and one J are joined & the V gene segment joins the combined DJ to form a complete VH exon.
The H chain C-region gene is encoded by several exons
The C-region exons and the leader peptide (L) are spliced to the VH exon
The L sequence is cleaved after translation & the disulphide bonds linking the chains form
Heavy Chain Rearrangement & Synthesis
Germline contains multiple gene encoding for the C region – there are 9 constant region genes in humans, one for each Ig class or subclass.
µ or δ are transcribed first during B-cell development.
Primary transcript can be spliced in two different ways (alternative splicing) to give either a VDJ-µ or VDJ-δ mRNA, which when translated and combined with either λ or κ light chain can form IgM or IgD of identical specificity.
Transmembrane and Secreted Immunoglobulin
All classes of immunoglobulin are made in two forms:
- a transmembrane form (the B-cell receptor for antigen) & secreted, soluble antibody
- differ at the carboxy terminus of the heavy chain – transmembrane from has a hydrophobic anchor sequence, antibody has a hydrophilic sequence
Transmembrane chain: alternative splicing removes the SC and exons encoding the hydrophobic carboxy terminus are retained and incorporated into the mRNA when the introns are spliced out
Secreted antibody: SC hydrophilic carboxy terminus retained & sequences 3’ are discarded
Factors Affecting Antibody Diversity
Multiple V genes in the germline
VJ & VDJ combinatorial association
Random assortment of H and L chains
Junctional Diversity
- exact positions where V & J or V,D & J join are not constant
- imprecise recombination leads to deletions or changes in the amino acids in parts of the Ig hypervariable region
- -> affects the sequence of the antigen binding site and antigen specificity
- imprecision of joining of V, D and J segments - change the reading frame of the DNA sequence, do not translate into a useful protein -> non-productive rearrangement
Factors Affecting Antibody Diversity - Somatic Hypermutation
Single nucleotide substitutions
Occurs at a high rate in rearranged V regions of Heavy and Light chain genes
Low affinity antibodies are produced in a primary response to antigen
After secondary stimulation with the same antigen the antibodies’ affinity ↑ -> sequence has diverged from germline sequence
Regulation of Ig Gene Expression - Allelic Exclusion
Ig chains are encoded by only one set of genes, from either the maternal or paternal chromosome
- e.g. H chain from paternal chromosome genes & L chain (either κ or λ) from maternal chromosome genes
A productive rearrangement of V,D & J gene DNA on one chromosome giving a H-chain polypeptide, causes the other parental H-chain locus to stop rearranging
Similarly with L-chain loci, first with κchain and then with λ-chain genes
Productive rearrangement of V to J of either κ or λ genes causes the other to remain unarranged
- only one H-chain & one L-chain are expressed
- individual B cells synthesise antibody of only one specificity
B Cell Development
H-chains are rearranged first (Pro-B and Pre-B cells)
L-chains are rearranged later (Immature B cells)
If a developing B cells does not successfully rearrange its H or L chain it dies
The expression of a B-cell receptor is a requirement for survival
T Cell Receptor Structure and Development
T-cell receptor resembles a single arm (Fab fragment) of an Ig molecule:
- each chain has a variable region (V) and a constant region (C)
- V region binds the antigen
- sequence variability is clustered into regions of hypervariability → form the binding site for antigen
- called complementarity-determining regions (CDRs)
- V domains of the T-cell α-chain and the β-chain each have three CDR loops, i.e. CDR1, CDR2, CDR3
In T-cell development, gene rearrangement produces variability in the variable regions of the T-cell receptor - resembling what occurs in B cells to generate diversity in Igs
In T cell development, there is no equivalent of somatic hypermutation
T Cell Receptor Diversity - α-Chain β-Chain
α-chain:
- locus on chromosome 14
- many V & J gene segments
- one Cα gene (like an Ig light chain locus)
- → a Vα segment rearranges to join a J α segment creating a functional exon encoding the V domain (similar to Ig light chain)
β-chain:
- locus on chromosome 7
- many V & J gene segments
- two D gene segments
- two Cβ gene segments (like an Ig heavy chain locus)
- → rearrangement of a Vβ, a Dβ and a Jβ gene segments creates a functional exon encoding the V domain (similar to Ig heavy chain).
T Cell Receptor Diversity
After gene rearrangement exons are separated by introns, which may contain unrearranged gene segments
When the gene is transcribed the primary RNA transcript is spliced to remove the introns and is processed giving mRNA
Translation of the mRNA gives α and β chains
Newly synthesised α and β chains enter the endoplasmic reticulum where they pair to form the α:β T-cell receptor
T Cell Receptor Complex (CD3 Complex)
α:β T-cell receptor heterodimers can’t leave the endoplasmic reticulum unless associated with membrane proteins of the CD3 complex: CD3γ, CD3δ, CD3ε and a fourth chain ζ
CD3 proteins and the ζ chain associate with intracellular signalling molecules, and after antigen has been recognised, transmit signals
α and β chains have very short cytoplasmic domains - don’t signal
Two types of T cell receptor:
1. α:β T-cell receptor and
2. γ:δ T-cell receptor
All T cells express either α:β receptor or γ:δ receptor, but not both
γ:δ T Cell Receptor
The δ gene segments are within the α-chain locus
Successful rearrangement within the α-chain locus results in deletion and inactivation of the δ-chain locus
T-cell receptor genes make rearrangements that are productive & non-productive
- the first T-cell receptor gene rearrangements involve the γ and δ loci
- if a thymocyte makes productive γ & δ-chain rearrangements before a productive βchain rearrangement, it is committed to being a γ:δ T-cell
- then γ:δ heterodimer assembles with the CD3 signalling molecules and the γ:δ T cells leave the thymus
Development of α:β T Cells
> 90% of thymocytes that complete successful gene rearrangements commit to α:β lineage
If cells fail to make a successful gene rearrangement & don’t express a T-cell receptor they die by apoptosis in the thymus
Expression of both CD4 and CD8 allows immature T cells to use either co-receptor depending on whether its T-cell receptor is suited to recognising peptide antigens presented by self-MHC class I or self-MHC class II → increases the probability that the cell will complete its maturation
Positive and Negative Selection of the T Cell Repertoire
First stage of T-cell development:
- production of T-cell receptors regardless of antigenic specificity
Second stage:
- selection of those receptors that work well with the individuals MHC molecules to recognise pathogen-derived peptides.
- involves only α:β T cells,
- does not involve γ:δ T cells
- once γ- and δ-chain genes have been productively rearranged, thymic development is complete
Positive selection favours T cells that recognise peptides presented by a self MHC molecule
Negative selection eliminates potentially autoreactive cells - could be activated by peptides normally presented by MHC molecules on healthy cells
Positive Selection of the T Cell Repertoire
Positive selection occurs in the cortex of the thymus
Self-peptides from the normal breakdown of the body’s proteins assemble with MHC molecules
Self peptides are presented with both MHC class I and MHC class II
If a self peptide MHC complex is bound within 3 - 4 days after a functional T cell receptor is produced:
- positive signal and cell lives
- if no binding, no signal and cell dies
If the receptor binds self-peptide MHC class I complex:
- CD8 molecules are recruited & CD4 excluded
If the receptor binds self-peptide MHC class II complex:
- CD4 molecules are recruited & CD8 excluded
Negative Selection of the T Cell Repertoire
Negative selection deletes T cells whose antigen receptors bind too strongly to self-peptides complexed with MHC molecules
Such T cells are potentially auto-reactive:
- → tissue damage
- → autoimmune disease
Most important cells for –ve selection: bone marrow derived DCs, & macrophages
Only a small fraction of α:β T cell survive +ve and –ve selection and leave the thymus as mature naïve T cells
