B-cell Maturation and Activation (Part 1)

Question 1

B-cells, antibody, and B-cell receptor (BCR)

Answer 1

Antibody/BCR is formed by heavy chain (IgH) and light (IgL) chains
Each B cell has one specificity
BCR is the membrane-bound antibody assocIated with signaling chains.
Antibody/BCR has mutiple isotypes/classes (constant regions).

Question 2

What is a B-cell?
(Bursa of Fabricius)

Answer 2

B-cells make antibodies
In mice and humans, B-cells come from bone marrow.

Question 3

The Genomic Distribution of Immunoglobulin Genes

Answer 3

IgL (Light Chain): Including Ig(kappa) and Ig(lambda) - two separate genes.
There is one heavy chain gene (IgH) but two light chain genes (IgL)
Only ONE ALLELE of each will be used in each B cell.

Question 4

The Life of a B-cell

Answer 4

Most B-cells develop in the bone marrow (mouse/human)
Continue to develop even in adults (unlike T-cells)
Differentiate from HSC (hematopoietic stem cells) to CLP (common lymphoid precursor)
Dependent on bone marrow stromal cells.

Question 5

Probability of in-frame arrangement

Answer 5

Question 6

B-cell bone marrow development

Answer 6

B-cell development can be divided into multiple stages.
Antibody genes are rearranged during ProB (IgH) and PreB (IgL)

Question 7

Rearrangement of B-cell receptor (BCR)

Answer 7

V(D)J recombination
Require the recombinases RAG1/RAG2
Two alleles of IgH:
* DJ recombines on both alleles (Early Pro-B)
* V recombine on one of the alleles (Late Pro-B)
* Will use another IgH allele if the 1st VDJ recombination failed

Question 8

Step 1
Rearrangement IgH (Heavy Chain)

Answer 8

**Allelic Exclusion: **
Only ONE allele of IgH is expressed in one cell
Successful rearrangement will prevent further rearrangement by:
1. Downregulation of RAG expression
1. RAG degradation
1. Epigenetic regulation

Allelic exclusion limits one B cell to only express one allele of IgH and one allele of IgL

Question 9

How does the cell know if it has a good IgH rearrangement?

Answer 9

PreBCR Checkpoint
The function of PreBCR is to detect the successful IgH rearrangement
Ensures that only B-cells with functional HEAVY CHAIN proceed in development
Failed rearrengment -> no IgH -> no preBCR -> die

Question 10

Step 2
Rearrangement IgL (Light Chain)

Answer 10

Allelic and isotypic exclusion:
* Only ONE ALLELE IgL is expressed in one cell
* Successful rearrangment -> BCR -> STOP subsequent arrengement.
* Either Igk (Chr6) or Ig(Gamma) (Chr16) arrange 1st.

Igk : Ig(gamma) ratio
Mouse 95%:5%
Human 65%:35&
Cat 5%:95%

Question 11

Antibody Diversity
Combinatorial Diversity

Answer 11

Combinatorial Diversity: Random combination of VDJ of IgH and IgL

Question 12

Antibody Diversity
Junctional Diversity

Answer 12

Junctional Diversity: DNA sequence introduced at joint during V(D)J recombination

P-nucleotides (palindromic) - mediated by Artemis and DNA-PKcs
N- Nucleotides - mediated by TdT (Terminal Deoxynucleotidyl Transferase). TdT adds random nucleotides to 3’.

Question 13

Step 3
Maturation-self tolerance (bone marrow)

Answer 13

Diverse BCRs = potential self-reactivity
Process that ensure B cells can recognize foreign antigens while avoiding reactivity to the body’s own tissue.
If a BCR is self-active, receptor editing or other mechanism promote tolerance, reducing the likelihood of autoimune responses.

Question 14

Step 3
Receptor editing

Answer 14

Allows self-reactive B cells to rearrange their light chain genes and potentially generate a BCR that does not recognize self-antigens.
If succesfull, the B-cell can mature and exit the bone marrow, it undergoes apoptosis.

Question 15

Step 4
Maturation-self tolerance (spleen)

Answer 15

Two sites (BM and spleen) for maturation increase the exposure of B cells to different self-antigens

Question 16

Mature Follicular B cells

Answer 16

B cells enter the lymph node or spleen at the B cell zone (primary lypmhoid follicie).
Also called follicular B cells (B2 B cells)

Question 17

Step 4
Maturation (spleen/lymph nodes)

Answer 17

Transitional B cells require access to B cells follicles to mature and survive
Transitional B cells differentiate into mature/follicular B cells (or marginal zone B cells; later)
B-cell activating factor (BAFF) - important for B cell survival
Without BCR –> half-life of new B cells is 3-6 days

Question 18

Antibody and B cell receptor (BCR)

Answer 18

**BCR is the membrane-bound antibody associated with signaling chains **
Antibody (immunoglobulin) on the cell membrane
Associate with other signaling chains (Ig alpha and Ig beta) and co-receptors.
BCR crosslinking will trigger a signaling cascade and activate B cells.

Question 19

How do B cells express both IgM and IgD at the same time?

Answer 19

By alternate splicing.
Zfp318: suppress transcriptional termination and polyadenylation after Cu.
Alternatively Spliced from VDJ to C(delta)

Question 20

Alternative Splicing

Answer 20

Expression of transmembrane and secreted Ig is also controlled by alternative splicing

Question 21

BCR Signaling Pathway

Answer 21

1. Cross-linking of membrane Ig by antigen
2. Tyrosine phosphorylation events
3. Biochemical intermediates
4. Active enzymes
5. Transcription Factors

Question 22

Summary 1.1

Answer 22

B-cell develop in bone marrow
B-cells rearrange IgH and IgL (Ig-kappa and Ig-lambda)
PreBCR checkpoint to ensure successfull IgH rearrangement
Allelic exclusion ensures singel specificity
Multiple mechanism to remove self-reactive B cells

Question 23

Summary 1.2

Answer 23

Each B cell has one specificity (allelic exclusion)
BCR is the membrane-bound associated with signaling chains (Ig alpha and Ig beta).
Aleternative splicing regulates: membrane-bound/secreted forms og Ig and expression of IgM vs IgD.