Immuno Test 3 - B Cell

Summary of B cell life/what happens in bone marrow/periphery

1. Bone marrow: B-cell development

B cells rearrange their immunoglobulin (Ig) genes without needing antigen to generate a unique B-cell receptor (BCR).

They pass the heavy-chain and light-chain checkpoints to produce a functional BCR.

2. Bone marrow: central tolerance

Immature B cells are tested for binding to self-antigens.

• Strong self-reactivity: receptor editing may occur; if self-reactivity persists, the cell may die or become anergic.

• No dangerous self-reactivity: the cell can survive and leave the bone marrow.

3. Periphery: maturation and surveillance

B cells travel to peripheral lymphoid organs, such as the spleen and lymph nodes, and mature into naïve B cells.

They circulate while waiting to encounter their specific antigen.

B cell development is dependent on….

bone marrow stromal cells

stages of b cell development

stem cell - heavy and light genes are germline (uncombined DNA)

lymphoid progenitor cell (the cell binds to the bone marrow stromal cell)

early pro b cell (the cell expresses kit, which interacts with SCF (stem cell factor) presented by the bone marrow cell)

late pro b cell (the cell expresses the IL-7 receptor, and the stromal cell expresses IL-7 signaling the cell to proliferate and proceed to the next phase)

pre b cell (The cell continues to receive IL-7 signals while attached to the stromal cell, Heavy-chain gene rearrangement completes during this stage)

immature b cell (The cell detaches from the bone marrow stromal cell, Light-chain gene rearrangement is completed, leading to full expression of functional surface IgM antibodies. At this point, the immature B cell is ready to leave the bone marrow)

VDJ rearrangement at stages of b cell development

early pro b cell

• heavy chain - DJ rearranging, done first bc is antibody foundation and takes longer

late pro b cell

• heavy chain - VDJ rearranging

large pre b cell - The cell successfully makes a heavy chain (specifically the mu chain) and displays it temporarily on the cell surface as part of a "pre-B-cell receptor" to check that it works, though most of it is still kept inside the cell.

• heavy chain - complete

small pre b cell - The heavy chain is made and stored inside the cell (ER, pulled back from cell surface), but it is not yet expressed on the cell surface because the light chain has not been made yet.

• heavy chain - complete

• light chain - VJ rearranging

immature b cell - Both heavy and light chains are fully assembled, creating functional IgM antibodies that are displayed directly on the cell surface for the first time.

• heavy chain - complete

• light chain - complete

mature b cell - The cell co-expresses both IgM and IgD antibodies on its surface by using alternative RNA splicing on the heavy-chain gene transcripts, making it fully mature and ready to encounter antigens.

• heavy chain - complete

• light chain - complete

Each B cell has 2 copies of ________, 2 copies of _________ and

2 copies of ___________-

Heavy chain

kappa light chain

lambda light chain

early and late pro b cell h chain gene rearrangement

what genetic material is available - Each diploid B cell carries two copies (alleles) of the…

heavy-chain locus, (kappa) light-chain locus, (lambda) light-chain locus

Early Pro-B Cell: D–J Rearrangement

Diversity (D) and Joining (J) gene segments are joined together on both chromosomes simultaneously → then it can progress onto the next stage if successful

Late Pro-B Cell: V–DJ Rearrangement (First Chromosome)

VDJ is attempted

• if successful - a survival signal is sent. The cell stops rearranging heavy-chain genes (allelic exclusion) and transitions into a pre-B cell

• if it fails - the cell tries again on the second chromosome, if it fails again the cell dies

Late Pro-B Cell: V–DJ Rearrangement (Second Chromosome)

If successful, the cell receives a survival signal and successfully moves on to become a pre-B cell, if not and a signal is not received the cell dies

If a successful heavy chain (VDJ-IgM) has been developed, can/how does it associate with a light chain to become a complete antigen-specific receptor?

1

Successful heavy-chain rearrangement (VDJ-IgM)

The developing B cell successfully rearranges its immunoglobulin heavy-chain gene and produces a μ heavy chain.

2

Association with the surrogate light chain

The μ heavy chain pairs with a surrogate light chain made of VpreB and λ5 (lambda 5). This forms the pre-B-cell receptor (pre-BCR).

This tests whether the heavy chain can pair properly and produce a functional signaling complex.

3

What if the heavy chain cannot pair?

• If the first heavy-chain rearrangement fails, the cell can attempt rearrangement on the other heavy-chain chromosome.

• If a productive heavy chain still cannot be made, the cell generally undergoes apoptosis.

Important: A heavy chain that successfully pairs with the surrogate light chain sends signals that inhibit further heavy-chain rearrangement.

4

Pre-BCR signaling

The pre-BCR associates with the signaling proteins Igα and Igβ (CD79a and CD79b). These transmit signals into the cell, promoting survival, proliferation, and progression to the next developmental stage.

The pre-BCR is expressed on the cell surface, even though it contains a surrogate rather than a conventional light chain.

5

Clonal expansion

The successful pre-B cell divides repeatedly, producing many daughter cells. Your estimate of around 100 cells is a reasonable rough illustration, although the exact number varies.

6

Light-chain rearrangement begins

After proliferation, the cells stop dividing, and RAG1 and RAG2 are re-expressed. The cells begin rearranging their actual light-chain genes, usually κ (kappa) first, followed by λ (lambda) if necessary.

7

Formation of a complete B-cell receptor

A successfully rearranged conventional light chain pairs with the μ heavy chain, replacing the surrogate light chain. The resulting membrane-bound immunoglobulin M (IgM), associated with Igα/Igβ, forms the B-cell receptor (BCR).

What would the phenotype of a λ5 deficient individual be?

• Status of B cell receptor rearrangement

  • 
    

• B cell development arrest

  • at large b cell phase

• Susceptibility

  • reoccurring infections (ones dependent on b cell reaction)

• Treatment

  • give them antibodies, bad cases stem cell transplants

How to restrict the number of heavy chain rearrangements and why?

Allelic exclusion: not well understood

All B-cell receptors on the cell surface are identical because of Allelic Exclusion (Homogeneous Receptors)

If they were all different (no allelic exclusion) there would be fewer total receptors participating in binding the antigen, and the overall interaction strength would be diluted, leading to inefficient recognition and signal transduction

Successful generation of functional B-cell antigen receptors

A LOT of “unreadable” nucleotide sequences could /do result during somatic recombination

• Random selection of VDJ, VJ

• Junctional diversity (P and N nucleotide insertion/deletion)

Immune system tries several times to create fully readable nucleotide sequences

• Can use both homologous chromosomes for heavy chain

• Can use all four chromosomes for light chain (two for kappa and two for lambda)

• Only “successful” B-cells will live; unsuccessful cells will die

Result should be (and usually is) immature B-cells with functional IgM isotype antigen-specific receptors

Allelic exclusion

• Uses only one heavy chain

• Uses only one light chain

Because heavy (H) chains use up all their D gene segments in an attempt to make an H chain…

they get only one chance per locus for a productive rearrangement. However, in light chains multiple attempts at gene rearrangements are possible

The maximum number of attempts in light chains is equal to the number V

gene segments or J gene segments, which ever is fewer

Rearranges until there is a functional Ig (heavy and light) or until it runs out of V or J segments and dies

Light chain rearrangement

Occurs independently

Large pre-B cell replicate (a single heavy chain specificity → 100 progeny cells (small pre-B cells)

Light chain rearrangement more efficient (no TdT expression)

Successful generation of functional B-cell antigen receptors - what are the checkpoints?

Heavy-chain checkpoint — Pro-B cell

ight-chain checkpoint — Small pre-B cell

• RAG1 and RAG2 are re-expressed.

• Light-chain V-J rearrangement occurs, usually κ first and then λ if needed.

• The conventional light chain must pair with the μ heavy chain to form a complete IgM BCR.

• The receptor must be expressed at the cell surface and signal appropriately.

If unsuccessful: The cell can make additional light-chain rearrangement attempts; if it cannot generate a functional receptor, it generally undergoes apoptosis.

Self-reactivity checkpoint — Immature B cell

Rearrangement is complete, what’s next?

Next checkpoint

• Are receptors reactive to self antigens?

• 75% are self reactive → 25%can leave the bone marrow and go into the peripheral

• Most self antigens are expressed in bone marrow

Trigger

• Surface IgM on an immature B cell encounters and binds to soluble univalent self-antigens

Cellular Response

• The signal does not induce activation or cell death (apoptosis) directly; instead, it signals the cell to downregulate IgM expression and express IgD

Outcome

• The anergic B cell is allowed to leave the bone marrow into peripheral circulation, but it has a vastly reduced lifespan and dies quickly

What is the final decision point after an autoreactive B cell has undergone receptor editing (rearranging its light chain genes to create a new receptor specificity)?

The B cell expresses a newly edited receptor with a new specificity

Persistent Self-Reactivity

• Condition: The newly edited receptor still binds to multivalent self-antigens.

• Outcome: The cell receives a signal leading to apoptosis (programmed cell death). This eliminates autoreactive cells to prevent autoimmune reactions.

Successful Receptor Rescue

• Condition: The new receptor no longer recognizes self-antigens.

• Outcome: The immature B cell passes central tolerance screening, leaves the bone marrow, migrates to peripheral tissues, and matures.

Why would an autoreactive B cell undergo receptor editing (rearranging its light chain genes to create a new receptor specificity)?

Multivalent Self-Reactivity

• Condition: Surface IgM binds strongly to self-antigens expressed on bone marrow cells.

• Outcome: The immature B cell is retained in the bone marrow. Instead of being immediately released, it is held back to undergo receptor editing (or apoptosis if editing fails).

If it does not bind to self-antigens on bone marrow cells, it is released into the bloodstream. In circulation and secondary lymphoid organs, it begins expressing both IgD and IgM

If self-reactive can edit its receptor - how?

Rearranges a second light chain - primary rescue pathway for self-reactive immature B cells in the bone marrow

1. Self-Antigen Detection

• Trigger: The surface IgM on an immature B cell binds to multivalent self-antigens expressed on a bone marrow stromal cell.

• Response: Instead of immediately dying, the cell receives a signal to halt development and reactivate its gene rearrangement machinery.

2. Light-Chain Gene Rearrangement

• Re-initiating Recombination: The immature B cell continues to express RAG proteins to rearrange unused light-chain gene segments (V and J) to make a new light chain

3. Selection Outcomes

• it works

• try again if it fails until you run out of attempts then it dies

Elimination/inhibition of self-specific B-cells

If immature B-cell is specific for a monovalent antigen in the bone marrow (often on

soluble, non cell-surface molecules) why does that matter

Will generate IgD version of the receptor (and downregulate igm)

Will leave the bone marrow

But will be “anergic” (non-reactive)

Will NOT respond (even if it encounters the self antigen)

Lives for only 1-5 days

gets rid of antibodies that cross react with self

Mature naïve B cells

Most B cells are clonally deleted

Due to bad VDJ rearrangement

Due to strong self-reactivity

Lack of receptor editing rescue

Central tolerance

Those that escape that might be self reactive are

anergic

Die due to peripheral tolerance mechanisms

want to know if new cells are productive

do they maintain their structure and do they react to self (bad)

IL-7 and FLT3

both bind to CLP (common lymphoid progenitor)

These transcription factors turn on B-cell-specific genes (such as CD19, VpreB, lambda5, and the recombination machinery RAG-1/RAG-2), committing the CLP to become a pro-B cell.