B cell development I

Question 1

Where do B cells develop and where do they engage foreign Ag?

Answer 1

develop -> bone marrow
engage -> peripheral lymphoid organs

Question 2

How are different B cell stages be distinguished?

Answer 2

• rearrangement status of heavy and light chain genes
• cell surface markers: surface Ig (BCR), others (lamda5, VpreB, BAFFR)

Question 3

What are some defining features of a pro B cell?

Answer 3

• committed to becoming a B cell
• turns on B cell genes, including Rag-1/2, TFs for B cell genes
• turn off/repress genes important for other lineages
• starts to rearrange Ig heavy chain gene
• receive signals from bone marrow stromal cells that promote proliferation and further development

Question 4

What are some consequences of lacking Rag-1/2?

Answer 4

• B cells don’t get past the pro-B cell stage of development
• T cell development is also blocked at an early stage
• SCID phenotype

Question 5

What does SCID stand for?

Answer 5

Severe combined immunodeficiency

Question 6

What is SCIDs?

Answer 6

• defect in B and T cell function
• severely impaired adaptive immunity
• severely immunocompromised

Question 7

How can SCIDs be treated?

Answer 7

• live in a sterile environment (bubble boy)
• bone marrow transplant -> bone marrow from healthy person can fix the patient’s bone marrow
• gene therapy -> if we know the defect, introduction of a functional gene

Question 8

What makes mice a good model system to study human biology?

Answer 8

• mammal
• similar genetic diversity -> most human genes have analogous mouse genes
• similar physiology
• genetic abnormalities that cause disease are similar to mouse, including immunological disorders

Question 9

How was Rag deficiency studied in the mouse? Name the techniques and how the data was represented.

Answer 9

Generate mice with knock-out of Rag-1 gene; used flow cytrometry to identify cell populations; represented on a dot plot

Question 10

What are the functions of the green PMT, red PMT, side scatter, and forward scatter in flow cytrometry

Answer 10

green PMT = detect cells that have green Abs
red PMT = detect cells that have red Abs
side scatter = granularity (remove debris)
forward scatter = cell size (ensures single cell)

Question 11

What is the purpose of flow cytrometry?

Answer 11

• technique to measure the expression of specific proteins on the surface (or inside) of individual cells
• cells strained with fluorescently labeled Ab.s

Question 12

What are the two graphical representations of flow cytrometry data

Answer 12

1. histogram plot
2. dot plot

Question 13

When SCIDs was modeled in the mouse, what markers were used to identify mature T cells and what markers were used to identify mature B cells

Answer 13

T cells:
- CD4
- CD8

B cells:
-B220 (B cell phosphotase; CD45R)
- IgM and IgD (IgD is a marker of a mature B cell)

Question 14

When SCIDs was modeled in the mouse, where were the cells isolated from and why?

Answer 14

where: spleen
why: this is where a large population of mature B and T cells are found

Question 15

When SCIDs was modeled in the mouse, and Rag-1 was knocked-out, what was the major finding?

Answer 15

No mature B and T cells in the spleen

Question 16

What are some defining features of a pre-B cell?

Answer 16

• characterized by rearranged heavy chain
• two sub-types (early/large; late/small)
• heavy chain expressed and pairs with surrogate light chain to for the pre-B cell R (pre-BCR)
• several rounds of proliferation at this stage -> daughter cells will have the same heavy chain, but will go on to have different light chains
• transcription of heavy chain is linked to VDJ recombination

Question 17

What are some features of the IgH locus?

Answer 17

1. contains promoters upstream of each V segment
   - binds RNAP II
   - sites where ts is initiated
2. enhancer elements in intron between variable and constant regions (E(mu)) and 3’ of alpha constant region (3’(alpha)E)
   - bind TFs
3. IgH gene is in inactive chromatin in non-B cells, including early hematopoietic progenitors

Question 18

What are some features of the IgH locus in early pro-B cells?

Answer 18

1. chromatin becomes accessible
2. B cell specific gene regulatory proteins are expressed and bind to enhancer elements
   - e.g. Oct-2
3. not much transcription from V segment promoters
   - too far from enhancers
   - however VDJ recombination is underway

Question 19

What are some features of the IgH locus in pre-B cells?

Answer 19

1. rearrangements bring enhancers closer to V segment promoters
2. efficient transcription of heavy chain gene

Question 20

What are some features of the pre-BCR?

Answer 20

1. rearranged heavy chain pairs with surrogate light chain
2. surrogate light chain is the same on all pre B cells = VpreB (like V(L)) + lamda5 (like C(L)))
3. surrogate light chain expressed by all pre-B cells
4. VpreB has extra C-term acidic residues and lamda5 has extra N-term basic residues
5. allows multiple pre-BCRS to self-aggregate (no ligand needed)
6. self-aggregation of pre-BCR initiates signaling via BTK = critical development checkpoint)

Question 21

What are some features of XLA?

Answer 21

• almost all patients are male
• patients are hemizygous for btk mutant allele
• females are asymptomatic carriers

Question 22

How is XLA treated?

Answer 22

1. current therapies directed at reintroducing Ab.s rather than correcting defect in B cells
   - patients receive weekly IgG injections from many blood donors which restores humoral immunity
2. patients are treated more liberally with antibiotics