Antigen Receptor Development Flashcards
Describe the germline configuration of immunoglobulin gene families.
- there are 3 gene families: kappa light chain, lambda light chain, and heavy chain
- located on different chromosomes
- each has a variable region and constant region
What are the segments of the variable region for light chains?
V-segments are located at the 5’ end
J-joining segments are located near the 3’end
What are the segments of the variable region for heavy chains?
V-segments are located at the 5’end
D-diversity segments are located downstream
J-joining segments are located further downstream near the 3’ end
What do the J and D segments code for?
the 3rd CDR on the 3’ end of the antigen binding site
What is the order of gene rearrangement for heavy chains?
- D-J rearrangement
- V-DJ rearrangement
- VDJ gene will now be the only V region expressed in that cell
- alternative splicing => VDJ-C
What is the order of gene rearrangement for light chains?
- V-J rearrangement
- alternative splicing => VJ-C
- kappa or lambda light chain joins heavy chain => IgM
Describe B cell specificity.
A single B cell (and its progeny) will only express ONE antigen-binding specificity (1 Vh and 1 VL)
What is allelic exclusion?
In Ig-producing cells, only one allele is expressed to ensure antigen specificity
Describe plasma cell specificity.
Plasma cells can only make 1 kind of antibody:
1 heavy chain type
1 light chain type
=> B cells may violate this rule (express both IgM and IgD)
List the stages of B cell development.
- HSC
- pro-B cell (early, late)
- pre-B cell (large, small)
- immature B cell
- mature B cell
Describe gene rearrangement during the HSC stage of B Cell development.
all genes are in germline configuration
- no rearrangement occurs
Describe gene rearrangement during the pro-B cell stage of B Cell development.
- D-J rearrangement of the heavy chain (early)
- V-DJ rearrangement of the heavy chain (late)
Describe gene rearrangement during the pre-B cell stage of B Cell development.
- large: now that VDJ gene is produced, mu heavy chain is transiently expressed on the surface, associated with a surrogate light chain (pre-B receptor)
- small: V-J rearrangement of the light chain
What components make up the pre-B cell receptor?
mu heavy chain + surrogate light chain + Ig-alpha + Ig-beta
Which B cell developmental stage is the first time there is Ig expression?
pre-B cell large
Describe gene rearrangement during the immature B cell stage of B Cell development.
Now that the VJ gene is expressed, the newly produced kappa/lambda light chain will join the mu heavy chain on the surface => IgM on the surface
- no response to antigens
- critical control point for gene rearrangement (can no longer make another V region)
Describe gene rearrangement during the mature B cell stage of B cell development.
immature B cell enters the peripheral lymphoid tissue
begins to express IgD (with the same antigen specificity as IgM)
- responsive to antigens
What is the mechanism for Ig gene rearrangement?
- Recombination Signal Sequence (RSS) is located downstream of V, on both sides of D, and upstream of J.
- RAG1/RAG2 recognize the RSS.
- RAG loops out the intervening DNA between V/J or J/D
- RAG excises the intervening DNA => circularizes => BREC/TREC => deleted
- DNA Ligase ligates the ends
What is the RSS?
RSS is made of 7-9 bps separated by a 12 or 23 bp spacer
- highly conserved
What are BRECs/TRECs?
B-Cell Recombination Excision Circle
- excised intervening DNA gets circularized
- as cell proliferates, the BREC/TREC will get diluted and eventually deleted
What is the purpose of quantifying BRECs/TRECs?
to see if B cells and T cells are functioning and producing Ig and proliferating
How do you get transmembrane IgM vs secreted IgM?
alternative splicing of the primary RNA transcript leads to either a transmembrane exon added after the C region or a secretion exon added after the C region
How can one cell co-express IgM and IgD?
- primary RNA transcript contains all C-region genes
- alternative splicing leads to choosing either a Cmu or a Cdelta
- the VDJ region remains the same, thus conserving antigen specificity
What are the 3 mechanisms by which the body increases antibody diversity?
- Combinatorial Joining of VDJ segments
- Junctional Diversity by adding N sequences
- Combinations of H and L chain proteins
What is combinatorial joining of VDJ segments?
every B cell clone expresses a unique VDJ/VJ combination
facilitates increased diversity and antigen specificity
What is N-region addition (junctional diversity)?
- After RAG excises the intervening DNA, enzyme Tdt randomly adds nucleotides on the excised ends
- only occurs in the heavy chain
- on either end of the DJ segment
What is a consequence of N addition?
- nonfunctional antibody due to frameshift or stop/nonsense codon
What happens if a B cell receptor is non-functional?
apoptosis
What are the possible effects of an anti-self B cell receptor?
apoptosis, anergy, or receptor editing
What is anergy?
state of nonfunctionality of B cells that have anti-self receptors that have low autoreactivity
- decreases IgM expressed on the surface
- can bind to self proteins, but does not lead to a T cell activity
What is receptor editing?
If a B cell produces an anti-self receptor, when the Ig binds to a self-antigen a signal is sent to the cell to change the receptor
- RAG is activated to create a new genetic recombination (since there are still upstream Vs and downstream Js available)
- this only occurs on the light chain
- cell continues to mature
- if still anti-self, apoptosis
Describe the structure of T Cell Receptors.
2 different chains (alpha and beta)
2 domains each (variable and constant)
Which terminal corresponds to which domain of the TCR?
- N domain = variable
- C domain = constant
Alpha chains and Beta chains correspond to light or heavy?
alpha = light (VJ) beta = heavy (VDJ)
What is an isotype switch?
A B cell can be induced to change the heavy chain it expresses if there are still downstream C regions available
- occurs after B cell is activated and presents to T cells
What is the mechanism for an isotype switch?
- enzyme AID (activation-induce cytidine deaminase) recognizes switch regions upstream of C regions
- loop out, excise, ligate
- if there are downstream C regions available, you can create a new Ig isotype (with the same antigen specificity)
