5 Antibody diversity Flashcards
Amino acid isotopic variability in immunoglobulins
IgG antibody - heavy chain class (and subclass where applicable) is used, light chain (kappa or lambda)
Amino acid allotypic variability in immunoglobulins
allelic variations across populations - associated with heavy and light chains. Some have effect on overall physiochemical properties of antibody
example of allotypic variability in immunoglobulins
altering small amino acid changes that have effect on glycosylation patterns
Amino acid idiotypic variability in immunoglobulins
purely on the antigen binding area
identifying unique structure and sequence that makes a particular antibody (applies to TCR too) different from any other antibody, associated with variable of heavy and light chains. CDRs of variable regions.
Heavy and light Ig chains/T-cell receptor genes - germline sequences
Germline sequences are not functional
Heavy and light Ig chains/T-cell receptor genes - functional genes
functional genes are a result of somatic recombination - only occurs in B and T cells
Heavy and light Ig chains/T-cell receptor genes - germline genes
large cluster of gene segments in a multigene family
Heavy and light Ig chains/T-cell receptor genes - regions
- Variable region (V) segments (start at 5 prime) need a leader sequence upstream of all the variables
- Diversity (D) segments (Ig heavy & TCR beta chains – not light)
- Joining (J) segments
- Constant (C segments)
light chain rearrangement
random V & J segments recombine, make gene encoding VL domain
Specific recombinase system recognises (discrete signals within the gene) DNA recombination signal sequences (RSS) = joining, cut out the DNA in chromosome between
where are recombination signal sequences
present at the end of V and start of J regions
effect of recombination signal sequences
signalling for those pieces need to come together
how is diversity produced in B cells
Have coding joint – V and J segment joint in the chromosome DNA - totally imprecise, another way Ig can increase their diversity = 90% diversity in B cells when you make them
12/23 rule
only an Ig gene segment with a random 12 base spacer
RSS can be joined to an Ig gene segment with a 23 base spacer
effect of 12/23 rule on diversity region
When add diversity region – D region, still follows the same rule
12/23 light chain joins
light chain can only join V to J
12/23 heavy chain joins
Heavy chain can only join V to D and D to J
B cell development cell stages
Start with a stem cell, progress through series of stages: early pro-B cell, late pro-B cell, large pre-B cell (has a partial immunoglobin not fully functional), small pre-B cell then immature B cell
which chain is made first in B cell development
heavy then light
when is the B cell checked for self-reactivity
Once gets to having a mature antibody on its surface
what genes are expressed at different points throughout the B cell development process
RAG 1 and 2
when are RAG 1 and 2 expressed
only expressed during B cell and T cell development
expressed during B cell development of heavy chain then during the development of light chain
what are RAG 1 and 2 known as
proteins Recombination activating genes 1 &
what are RAG 1 and 2 essential for
somatic recombination
RAG 1 and 2 regulation
highly regulated, switched on and off at different stages
what happens if don’t have RAG 1 and 2
Without RAG 1 and 2 cannot make B or T cells – immune deficiency
what is Terminal deoxynucleotidyl transferase
(TDT) is a non-templated DNA polymerase which acts on the cleaved DNA ends and adds additional DNA bases
progression of rearrangement to form B cells - how is a signal formed to stop heavy chain rearrangement
Start with heavy
D-J rearrangement on both chromosomes at same time
After triggers late pro-B cell heavy chain gene rearrangement on only one chromosome (V to DJ region that’s already been made)
If make heavy chain and gets on surface of cell = signal to come down off cell surface and stop rearranging the heavy chain genes,
what happens if doesn’t make successful rearrangement
cell rearranges the second chromosome – if this one is non-functional too the cell will die
when is light chain rearrangement triggered
after heavy chain has been successfully rearranged and signal sent to stop
light chain rearrangement for B cells
kappa chain rearranged first on one chromosome (if successful and creates surface IgM, cell will no longer rearrange) if doesn’t will rearrange second kappa chain gene, if that fails will rearrange the two lambda – plenty of goes at making light chain gene
what is created at end of rearrangements
Immature B cell will either be IgM kappa or IgM lambda
why can light chains rearrange multiple times
Receptor editing can rescue B cells which have generated a self-reactive antibody – will try to rearrange to see if can make it not self-reactive
V-J configuration, can potentially have another go to rearrange until run out of V and J regions to join
when can’t light chain rearrange again
if use very first V region and very last J region first time round then don’t get a second chance
what is the joining of Coding Joint DNA like
imprecise
Joining of Coding Joint DNA for light chain
V-J
Joining of Coding Joint DNA for heavy chain
V-D and D-J
result of joining of Coding Joint DNA
joining can result in additional nucleotides added to the joint
what are the additional nucleotides in Joining of Coding Joint DNA
- Non-templated addition by Terminal deoxynucleotidyl transferase (TdT): Termed N region additions
- Palindromic additions by template directed fill in by DNA polymerases: Termed P-region additions
effect of adding more nucleotides in the joining of coding joint DNA
alter the potential peptide reading frame - As random they do not meet, some changes of removal ‘nibbles’ till they can come together
what occurs during N and P region addition
RSSs brought together
RAG complex generates DNA hairpin at coding ends
artemis: DNA-PK complex opens DNA hairpins generating palindromic P-nucleotides
N-nucleotide additions by Tdt
pairing of strands
unpaired nucleotides removed by exonuclease
gaps filled by DNA synthesis and ligation = coding joint
what causes variability in antibodies
Between D and J DNA is not encoded in chromosome
Allelic exclusion
Single T or B cell will only express product of a single allele for each of its relevant antigen receptor genes
B-cell will express a single specificity of IgH chain and a single specificity light chain
what causes mono-specificity
A single T-cell will express either a unique α and β, or a unique γ and δ TCR pair
Allelic exclusion leads to monospecificity of BCR
what is monspecificity
same antibody
if crossed animal with alpha and beta B cell what is expressed
heterozygous, has genetic material of both, only one – a or b is expressed on the surface of T cell
what does somatic hypermutation do
refines the immune response
Recognises the epitope and fine tuning it
where does somatic recombination occur
only in B cells and the secondary lymphoid organs
somatic recombination and mutations
- Targeted mutations in the DNA encoding the rearranged VJ or VDJ segment
- altered DNA sequence may encode different amino acids
- some mutations will improve binding to antigen.
- Occurs simultaneously with class switching
Somatic hypermutation results
small alterations in amino acid sequence within the Variable domains of immunoglobulins
Known details of somatic hypermutation
- Not random mutations - transitions >transversions
- especially serine codons AGC and AGT
- Activation induced cytidine deaminase (AID) is induced
Mechanisms for generation of diversity
- choice of multiple genes – multifaceted
- random recombination of gene segments
- V-J (light) and V-D-J (heavy)
- imprecise junction when they join, allows for P and N addition
- (N region addition, P region addition)
- Combination of heavy and light chain proteins
- somatic hypermutation
Refining the antibody o self-reactive
tend to die fairly rapidly – clonal deletion
Refining the antibody soluble self-molecule
can get out to periphery, they’re usually very unresponsive to any normal stimuli for B cells (anergic), don’t enter lymph nodes so die fairly rapidly
Refining the antibody no self-reaction
No self-reaction to self or soluble self = migrate to periphery and then become naïve B cell, when meet antigen = mature B cell
where does clonal deletion occur
All in the bone marrow
what does Isotopes (class) switching affect
the heavy chain genes only
Isotopes (class) switching - initial isotopes made
IgD and IgM are made (cell surface)
how are more isotopes produced
- Further class switching involves a second specific recombination system
- gives a switch to IgG, IgA, IgE
- occurs after antigen stimulation
how are IgM and IgD made
generated by RNA splicing – depends on RNA splicing whether end up with IgM or IgD
what enzymes are involved in isotope switching
activation-induced cytidine deaminase (AID)
what enzymes are involved in somatic recombination
activation-induced cytidine deaminase (AID)
