B cell development

Question 1

What is required for pluripotent HSCs to commit to becoming a B cell?

Answer 1

• Pluripotent HSCs express CD34 and CD43
• Those that stay in the bone marrow express transcription factor Pax5 → differentiation into **pro-B **cells
  
  • ​Pax5 is expressed throughout the life of a B cell b/c it is needed for the transcription of many B cell genes, including Ig genes
  • If Pax5 is not expressed, CLP will become a monocyte (or later a NK cell), not a B cell.

Question 2

What are the first steps needed to generate a B cell receptor (BCR)?

Answer 2

• Rearrangement of Ig-µ heavy chain gene locus
  
  • There are two Ig-µ gene loci (one for each chromosome) → cell has two chances to execute productive VDJ recombination
  • If VDJ fails to be productive on either chromosome → cell apoptoses
  • VDJ productive/produces a heavy chain that can bind the surrogate light chain → pre-B cell
• The generation of a µ heavy chain that is able to form a pre-B cell is the first of two B cell development checkpoints

Question 3

What is allelic exclusion?

Answer 3

• If **VDJ **recombination of the Ig µ heavy chain is productive with the first chromosome, subsequent rearrangement is stopped by the elimination of RAG recombinase proteins
• This limits synthesis of the µ heavy chain to only one of the two loci and is called allelic exclusion

Question 4

What steps are needed to generate a pre-B cell?

Answer 4

• µ heavy chain must demonstrate capacity to bind a surrogate light chain, made up of the proteins VpreB and λ5
  
  • VpreB = proxy for light chain variable region
  • λ5 = proxy for light chain constant region
• Newly synthesized µ heavy chain bound to VpreB and λ5 translocates to the cell surface along with transmb proteins Igβ and Igα → forms pre-B cell receptor → cell is now a pre-B cell

Question 5

How is light chain synthesis completed?

Answer 5

• Pre-B cell undergoes **proliferation **→ generates a large population of pre-B cells with the same µ heavy chain **already **paired to a surrogate light chain (VpreB and λ5
• **RAG **gene expression is turned on → light chain undergoes VJ recombination
  
  • There are four light chain loci: one κ and one λ on each chromosome
  • Recombination begins with the κ gene one chromosome
    
    • If doesn’t work, more to κ gene on second chromosome, then λ gene

Question 6

How is a functional IgM receptor generated?

Answer 6

• IgM formed in the ER when a light chain is paired with a µ heavy chain
• IgM, Igβ, and Igα move to the cell membrane → assembly Mature BCR → cessation of light chain rearrangment
• If the cell does not produce a functional light chain → cell apoptosis
  
  • ​∴ capacity to make a functional IgM receptor = **second **B cell development checkpoint

Question 7

B cell negative selection

Answer 7

• Although B cells now have mature IgM receptors, they cannot leave th BM, since most of them are still self-reactive and must undergo negative selection first before leaving the BM
• BM stromal cells express self-antigen → B cell monovalent and multivalent (many epitopes) sampling of self-antigens
  
  • **Multivalent **negative selection happens entirely in the BM
    
    • ​B cells that bind multivalent antigens undergo receptor editing
  • Monovalent negative selection begins in the BM, but continues in the periphery
    
    • ​B cells that bind self antigen → anergy

Question 8

Receptor Editing

Answer 8

• Before apoptosis occurs during negative selection, B cells that have recognized multivalent self-antigens have the chance to “redeem themselves”
• They do this through a process called receptor editing in which the autoreactive B cell:
  
  • reduces the level of surface IgM (turns off autoreactivity)
  • upregulates RAG expression and
  • begins a new rearrangement of the light chain locus → excision of the previously expressed rearrangement → production and testing of a new rearrangment
• Process can happen again and again until the cell either:
  
  • finds a receptor that lacks autoreactivity → continued B cell development - OR -
  • runs out of recombination possibilities → apoptosis via clonal deletion

Question 9

What are the relative levels of IgD and IgM on mature BCRs?

Answer 9

• B cells that are not self-reactive must make both IgD and IgM before they leave the BM → heavy chain mRNA splicing to produce both heavy chains
• When a B cell leaves the BM it is not yet fully mature so has more IgM on its surface than IgD.
• Once a B cell is fully mature it will have more IgD than IgM on its surface.
  
  • This is due to interactions with antigens on follicular DCs

Question 10

What is the fate of B cells that are self-reactive against monovalent antigens?

Answer 10

• B cells that bind these antigens are allowed to leave the BM, but they are in a state of anergy and have a short life span of 1-5 days
• They produce very little IgM on the cell surface, ∴ they are not activated when their IgD BCR binds the self antigen

Question 11

How is B cell maturation completed?

Answer 11

• Completed in the **periphery **(2° lymphoid organs)
• BM-derived B cells interact with follicular dendritic cells (FDCs; not real DCs) via two proteins on the B cell surface:
  
  • Lymphotoxin (Similar to TNF-α)
  • BAFF (B cell activating in the TNF family)
• Interaction → stimulation of B cell to mature → surface IgD expression > surface IgM expression
• B cell then reenters circulation as a mature naive B cell that can be activated by non-self antigen