B cell development II

Question 1

When does the light chain rearrangement start?

Answer 1

Pre-B cell stage

Question 2

What is an immature B cell and where is it located?

Answer 2

What = contains a functional heavy and light chain which results in a IgM+ B cell

located = bone marrow

Question 3

What is a transitional B cell and where is it located?

Answer 3

what = IgM+ B cell that leaves the bone marrow

located = secondary lymphoid organs

Question 4

Why might junctional diversity sometimes cause problems

Answer 4

1. introduction of stop codons -> truncated protein
2. frameshift
   - not able to form B sandwhich
   - early stop codon

Question 5

What is the ordered progression of BCR gene rearrangements?

Answer 5

1. heavy chain before light chain
2. kappa light chain before lamda (in mice, somewhat in humans)

Question 6

Describe the progression of heavy chain gene rearrangement (describe what happens during productive and nonproductive outcomes)

Answer 6

1. D to J
   - occurs on both homologous chromosomes
   - doesn’t lead to a lot of frameshifts -> almost always successful
2. 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

Question 7

Describe the progression of light chain gene rearrangement (describe what happens during productive and nonproductive outcomes)

Answer 7

1. 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

Question 8

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?

Answer 8

1. light chains can make repeated rearrangements at each locus (can try the same allele again)
   - time limited
2. 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

Question 9

What is a heavy chain productive rearrangment and what are the effects?

Answer 9

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

Question 10

What is a productive light chain rearrangement and what are the effects?

Answer 10

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

Question 11

What is allelic exclusion and what does it ensure?

Answer 11

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)

Question 12

When testing for allelic exclusion experimentally, what was the question, observation and hypothesis?

Answer 12

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

Question 13

What system was used to test allelic exclusion in mice?

Answer 13

create a transgenic mouse that expresses a BCR that recognizes HEL (anti-HEL)

Question 14

Why was HEL used to study allelic exclusion in mice?

Answer 14

• well studied protein
• Ab.s that recognize HEL are available
• purified HEL is readily available

Question 15

Describe the transgene used to test allelic exclusion in mice

Answer 15

1. 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))
2. randomly inserted into the genome of every cell
3. endogenous IgH and IgL loci unaffected

Question 16

What are allotypes?

Answer 16

minor sequence differences in heavy/light chain constant regions (alleles), doesn’t necessarily mean functionally different

Question 17

What did the anti-HEL transgenic mouse vs WT results show, what was the conclusion, and what was this proof of?

Answer 17

• 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

Question 18

What are some features of transitional B cells?

Answer 18

• 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

Question 19

What are some characteristics of B cell activating factor (BAFF)

Answer 19

1. secreted by cells in the spleen
2. promotes survival and continued development
3. also required for mature B cell survival

Question 20

Describe the levels of IgM, IgD and BAFF receptor in the different stages of B cells: T1, T2 and mature B-2

Answer 20

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

Question 21

What are some characteristics of mature B-2 cells?

Answer 21

• circulate amongst the secondary lymphoid organs
• express both IgM and IgD (more IgD than IgM)

Question 22

Where are constant regions in the genome?

Answer 22

3’ to V(H)

Question 23

How is expression of isotypes other than IgM and IgD mediated?

Answer 23

DNA recombination

Question 24

How are IgM and IgD both transcribed?

Answer 24

• 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

Question 25

Describe the alternative cleavage and polyadenylation where IgM is expressed

Answer 25

Immature cells only cleave at polyA site 2 and splice to the mu constant region exons

Question 26

Describe the alternative cleavage and polyadenylation where mostly IgD and some IgM is expressed

Answer 26

Mature cells preferentially cleave at polyA site 4 and splice to the delta constant region, but can also use polyA site 2