B cell development II Flashcards
When does the light chain rearrangement start?
Pre-B cell stage
What is an immature B cell and where is it located?
What = contains a functional heavy and light chain which results in a IgM+ B cell
located = bone marrow
What is a transitional B cell and where is it located?
what = IgM+ B cell that leaves the bone marrow
located = secondary lymphoid organs
Why might junctional diversity sometimes cause problems
- introduction of stop codons -> truncated protein
- frameshift
- not able to form B sandwhich
- early stop codon
What is the ordered progression of BCR gene rearrangements?
- heavy chain before light chain
- kappa light chain before lamda (in mice, somewhat in humans)
Describe the progression of heavy chain gene rearrangement (describe what happens during productive and nonproductive outcomes)
- D to J
- occurs on both homologous chromosomes
- doesn’t lead to a lot of frameshifts -> almost always successful - V to DJ
- occurs on one homologous chromosome first
- 1/3 chance of being successful
- if the first rearrangement is productive (pre-BCR signaling inhibits rearrangement of the second chromosome and induces kappa rearrangement)
- if the first rearrangement is nonproductive, the second chromosome is tried
- if second rearrangement is productive -> development continues
- if second rearrangement is nonproductive -> cell death
Describe the progression of light chain gene rearrangement (describe what happens during productive and nonproductive outcomes)
- V to J
- starts at one allele of kappa gene (mouse)
- moves to lamda after two kappa chains tried
- if kappa 1 successful -> mu + kappa inhibit kappa 2
- if kappa 2 successful -> mu + kappa inhibit lamda
- if lamda 1 successful -> mu + lamda inhibit lamda 2
- if lamda 2 successful -> great success
- if lamda 2 unsuccessful -> death
What are some differences of the light chain rearrangement progressions compared to the heavy chain? Is light or heavy chain rearrangement the rate limiting step?
- light chains can make repeated rearrangements at each locus (can try the same allele again)
- time limited - essentially if a pre-B cell makes it through heavy chain rearrangement it will become an immature B cell
rate limiting step = heavy chain rearrangement
What is a heavy chain productive rearrangment and what are the effects?
productive rearrangement = heavy chain protein can pair with the surrogate light chain to form the pre-BCR
effects = temporally reduces expression of Rag-1/2 to limit access to other heavy chain locus (prevents rearrangement of other allele) -> allelic exclusion
What is a productive light chain rearrangement and what are the effects?
productive rearrangement = light chain pairs with the heavy chain and the BCR (IgM) is put on the surface
effects = ligand independent signaling produces survival signals and turns off Rag genes and limits access to other light chain loci -> prevents further light chain rearrangements
What is allelic exclusion and what does it ensure?
allelic exclusion = functional rearrangement of on heavy/light chain allele prevents the functional rearrangement of the other allele(s)
ensures that each mature B cell bears a BCR of one specificity (only one heavy chain protein and one light chain protein are expressed)
When testing for allelic exclusion experimentally, what was the question, observation and hypothesis?
Question: how can we show allelic exclusion experimentally
Observation: functional rearrangement of one heavy or light chain allele to turn off/prevent functional rearrangement of the other allele(s)
Hypothesis: expression of functionally rearranged heavy and light chain genes in B cell progenitors of a mouse should prevent the endogenous heavy and light chains from rearranging and expressing on the surface
What system was used to test allelic exclusion in mice?
create a transgenic mouse that expresses a BCR that recognizes HEL (anti-HEL)
Why was HEL used to study allelic exclusion in mice?
- well studied protein
- Ab.s that recognize HEL are available
- purified HEL is readily available
Describe the transgene used to test allelic exclusion in mice
- obtained genomic DNAs containing rearranged heavy and light chain genes that encode an anti-HEL Ab
- included heavy constant regions for IgM and IgD
- included endogenous regulatory elements (E(mu)) - randomly inserted into the genome of every cell
- endogenous IgH and IgL loci unaffected
What are allotypes?
minor sequence differences in heavy/light chain constant regions (alleles), doesn’t necessarily mean functionally different
What did the anti-HEL transgenic mouse vs WT results show, what was the conclusion, and what was this proof of?
- when looking for endogenous BCRs using the endogenous (b) allotype, only 1-6% of cells expressed the endogenous BCR in the transgenic in comparison to the control
- when looking for the transgenic BCR using the transgenic (a) allotype, over 90% of B cells expressed the transgenic BCR
conclusion = expression of reaaranged anti-HEL transgenes can inhibit endogenous Ig expression
proof of allelic exclusion
What are some features of transitional B cells?
- transitional B cells = immature B cells that are non self-reactive leave the bone marrow
- transition between immature and mature stages
- receive signals from cells in the spleen to continue development
- B cell activating factor (BAFF) required for survival and maturation
What are some characteristics of B cell activating factor (BAFF)
- secreted by cells in the spleen
- promotes survival and continued development
- also required for mature B cell survival
Describe the levels of IgM, IgD and BAFF receptor in the different stages of B cells: T1, T2 and mature B-2
T1:
- IgM = high
- IgD = very low
BAFF R = intermediate
T2:
- IgM = high
- IgD = intermediate
- BAFF R = high
Mature B-2:
- IgM = intermediate
- IgD = high
- BAFF R = high
What are some characteristics of mature B-2 cells?
- circulate amongst the secondary lymphoid organs
- express both IgM and IgD (more IgD than IgM)
Where are constant regions in the genome?
3’ to V(H)
How is expression of isotypes other than IgM and IgD mediated?
DNA recombination
How are IgM and IgD both transcribed?
- both mu and delta constant regions are transcribed as one primary RNA transcript
- mature IgM and IgD mRNA are generated depending on where transcript is cleaved and polyadenylated
Describe the alternative cleavage and polyadenylation where IgM is expressed
Immature cells only cleave at polyA site 2 and splice to the mu constant region exons
Describe the alternative cleavage and polyadenylation where mostly IgD and some IgM is expressed
Mature cells preferentially cleave at polyA site 4 and splice to the delta constant region, but can also use polyA site 2
