B Cell Development

Mature B Cell (Event, Expression)

*EVENT*: -Alternative mRNA splicing of heavy chain transcripts leads to production of IgD EXPRESSION: Express both IgM and IgD and *possess the same antigen specificity* These are *Naive* B Cells.

Heavy Chain Gene Rearrangements

*Heavy-chain rearranges before light-chain* D-J can occur in both heavy alleles V-DJ occurs one allele at a time; if unproductive, progress to 2nd allele

What genes control the proteins responsible for Ig gene rearrangement?

*R*ecombinase *A*ctivating *G*enes; RAG-1 and RAG-2 *T*erminal *d*eoxynucleotidyl *T*ransferase (TdT)

Rules for B Cell Rearrangements

*Rearrangements can be made on both homologous chromosomes* -Only 1 will eventually be expressed -Allelic Exclusion occurs at both heavy- & light-chain loci *Only 1 heavy chain locus & 1 light chain locus will rearrange at any given time* *Heavy chains have TWO CHANCES to rearrange before B cell death occurs* -2 alleles *Light chains have FOUR CHANCES to rearrange before B cell death occurs* -2 κ chain and 2 λ chain alleles

VpreB

*V*preB substitutes for a light chain *v*ariable region Resembles the variable light chain but has an extra amino terminal region.

Summary of B Cell Development

1. Heavy Chain Locus rearranges. *If successful*: 2. Pre-B Cell Receptor is made 3. Heavy Chain gene rearrangement ceases 4. Resulting Pre-B Cell proliferates 5. Rearrangement of Light Chain Locus begins. *If successful*: 6. Complete Immunoglobulin B Cell Receptor is formed 7. Gene rearrangement ceases 8. B Cell development continues → antigen-specific phase

Stages of B Cell Development

1. Stem Cell 2. Early Pro-B Cell 3. Late Pro-B Cell 4. Large Pre-B Cell 5. Small Pre-B Cell 6. Immature B Cell 7. Mature B Cell

Immunoglobulin Synthesis Steps

1. V(D)J rearrange to form the V exon 2. C-region is encoded by 1+ exons 3. Rearranged V exon is spliced to C-region exon 4. mRNA is translated 5. Leader sequence is removed 6. Immunoglobulin protein is either membrane-expressed or secreted

Productive Rearrangement

1/3 chance of a correct reading frame Rearrangements that result in a functional Ig chain are *productive*

How many pro-B cells make productive rearrangements?

50%

How many pre-B cells make productive rearrangements?

85%

How many B lineage cells succeed?

<50%

How many B cells are generated by the bone marrow per day?

> 10¹⁰

Phase 4: Proliferation/Differentiation

Activated B cells proliferate and differentiate into effector forms (plasma cells & memory B cells).

Extra Nucleotides during rearrangement?

Added during heptamer-nonamer joining (*junctional diversity* -*P Nucleotides*: added on as a result of hairpin cleavage -*N Nucleotides*: random nucleotides added on by TdT

Terminal deoxynucleotidyl Transferase (TdT)

Adds random nucleotides to cut ends of DNA Important for junctional diversity, but not essential for recombination ON in Pro-B Cells, adding mostly N nucleotides in heavy chain -It is a Pro-B Cell marker that is lost as they develop OFF in Pre-B Cells

Regulation of B Cell Rearrangements

As each complete receptor-chain gene is generated, the protein it encodes is expressed as a part of a receptor, which signals the cell to progress to the next step. *Pro-B Cell*: No µ protein; gene rearrangement is active *Pre-B Cell*: -µ heavy chain is synthesized & assembles with light surrogate chain -Pre-B cell receptor is transported to surface → heavy chain rearrangement stops -Cells proliferate & begin light chain rearrangement *Immature B Cell*: Functional light chain assembles with µ for functional expression on surface → light chain rearrangement stops

Just-in-time Mechanism

Body posses a minimal # of needed B-cell templates w/ certain antibody specificity. -Expand only needed B-cells that's rapid and based on memory

Major Site of Antibody Production

Bone Marrow

Immature B Cell Development

Can develop without direct stromal cell influence but does require growth factors & cytokines

Severe Combined Immunodeficiency (SCID)

Caused by RAG mutations

Mutations in Btk

Causes B cell deficiency, seen in Pro-B Cell Stage, called X-Linked Agammaglobulinemia (XLA). Patients with XLA get recurrent infections from common extracellular bacteria. -No mature B cells

Ligand Binding

Cross-linking of the Pre-B Cell Receptor by the stromal-cell ligand may stop V-DJ heavy chain rearrangement and induce V-J light chain rearrangement.

2 Potential Problems in B Cell Development

Developing specific antibodies against self-antigens Lymphocyte expansion is conducive to tumor formation *B cell development requires multiple levels of control*

Requirement for Pro-B to Pre-B Cell Development

Direct stromal contact (Supplies IL-7)

Importance of Stages of B Cell Development

Each stage is marked by successive changes in the rearrangement & expression of immunoglobulin genes, and represents a *checkpoint* to control gene rearrangement.

B Cell Development & Bone Marrow Location

Early pro-B cells are located near the endosteum & move toward the center of the marrow cavity as they mature.

Bruton's tyrosine kinase (Btk) KNOW

Encoded on X Chromosome & essential for B cell maturation.

B Cell Immunoglobulin Loci

Every B cell has 2 copies of each of the immunoglobulin loci: -Heavy-Chain Locus -κ Light-Chain Locus -λ Light-Chain Locus

Immature B Cell (Formation, 3 Events)

Formation signified by a fully functional IgM receptor on the surface: *EVENTS*: -Light chain rearrangement is complete -Light chains have assembled with µ chain to form IgM -Fully functional IgM present on surface *Up to this point*: Development is independent of specific Ag, posses autoreactive B cells

Small Pre-B Cell (Formation, Capacity for Self-Renewal, 4 Events)

Formed after large Pre-B Cell has gone through 5-6 rounds of cell division and after presumptive ligand-pre-B cell receptor interaction (???) DO NOT divide. *EVENTS*: -Majority of Pre-B Cell Receptors are internalized -Surrogate Light Chain synthesis ceases -Ig Heavy Chain rearrangement ceases -Rearrangement of variable light chain begins (V-J rearranging)

Surrogate Light Chain Development

Formed by 2 proteins, *λ5* and *VpreB*, encoded separate from Ig. Expression on surface with complete heavy chain is *essential for progression from pro-B cell to pre-B cell*

Immunoglobulin Genes in Malignant B Cells (Monoclonal)

Genes ARE rearranged and the tumor represents a single rearrangement. V and C regions are in the same fragment (?)

Immunoglobulin Genes in Non-Lymphoid Cells

Genes NOT rearranged V and C regions are in distinct regions (?)

How is B Cell development controlled? (Big Picture)

Genes/proteins control immunoglobulin gene rearrangement. These genes/proteins are developmentally controlled.

Early Pro-B Cell (Rearrangements, Surface Ig)

H-Chain Genes: D to J rearranging L-Chain Genes: Germline Surface Ig: Absent

Late Pro-B Cell (H Chain, L Chain, Surface Ig)

H-Chain Genes: V to DJ rearranging L-Chain Genes: Germline Surface Ig: Absent

Stem Cell

Heavy Chain Genes: Germline Light Chain Genes: Germline Surface Ig: absent.

Repeated Light-Chain Locus Rearrangement

If initial rearrangement is unproductive, a 2nd one can occur between: -Any Vκ on the 5' side of the 1st joining -Any Jκ on the 3' side of the 1st joining The intervening DNA containing the unproductive 1st joining will be excised. There can be as many as 5 successive attempts on 1 light-chain gene. -If all J regions are used up, cell death is induced.

Signal Transduction Proteins

Igα and Igβ Bruton's Tyrosine Kinase (Btk)

Immunoglobulin Genes in Mature B Cells (Polyclonal)

Immunoglobulin genes ARE rearranged Each B cell has a different immunoglobulin gene rearrangement (appears as a smear on a southern blot)

Summary of if initial-light chain rearrangement is unsuccessful

L-chain rearrangement continues until productive rearrangement made. -If all J regions used up --> DEATH

Phase 2: Selection (Location, Function)

LOCATION: Bone Marrow FUNCTION: Elimination of self-reactive B cell lineages -Few anergic (lack of immune response to an antigen) cells may be seen in circulation

Phase 1: Maturation (Location, Requirements)

LOCATION: Bone Marrow REQUIREMENTS: Stromal Cells

Ligation of Immature B Cell IgM

Leads to cell death

Plasma Cells (Location, Function)

Migrate to particular sites in secondary lymphoid tissues. -produce antibodies (where?)

Interleukin 7 (IL-7)

Necessary for proliferation & Pre-B Cell Development Receptor (on the Pre-B Cell): IL-7 Receptor

Pro-B Cell (Precursor, Capacity for Self-Renewal, Events)

PRECURSOR: Derived directly from pluripotent stem cells CAPACITY FOR SELF-RENEWAL: Limited; produce more pro-B cells EVENTS: Rearrangement of heavy chain genes, which results in a "functional" µ heavy chain that is *not surface expressed*

4 Phases of B Cell Development

Phase 1: Maturation Phase 2: Selection (elimination of self-reactive) Phase 3: Migration/Activation Phase 4: Proliferation/Differentiation

Light Chain Gene Rearrangements

Pre-B cells divide 5-6x before light-chain rearrangement Rearrangements take place 1 light-chain locus at a time κ locus usually rearranges before λ locus *Rearrangement continues until a successful light-chain is produced* -If all J regions are used up, cell death is induced.

What are the earliest identifiable cells of B cell lineage?

Pro-B Cells

Large Pre-B Cell (Event)

Production of a *functional µ heavy chain*, which is transiently expressed on the surface to form part of the *Pre-B Cell Receptor*

What is the survival of a developing B cell dependent on?

Productive rearrangement of the light & heavy chain genes. Not all rearrangements result in a suitable reading frame to be translated into an immunoglobulin chain. -DNA is cut & spliced during recombination

Stem Cell Factor (SCF)

Promotes Pro-B Cell Development Receptor (on the Pro-B Cell): c-Kit

Memory B Cells

Recirculate throughout secondary lymphoid tissues

Non-Lymphoid Stromal Cells

Required for B cell development in bone marrow. 2 Functions: -Make specific cell-surface contacts with developing B cells through adhesion molecules on stromal cells and ligands on B cells. -Provide growth factors that stimulate lymphocyte differentiation & proliferation

Igα and Igβ

Signal Transduction Proteins Disulfide-linked transmembrane proteins that are necessary for transport and stable membrane expression of Ig. Associate with Ig in ER and are essential for internalization of Ig signal. Cease expression when B cell becomes a plasma cell.

Growth Factors secreted by Stromal Cells

Stem Cell Factor Interleukin 7

Phase 3: Migration/Activation (Locations)

Surviving mature B cells leave bone marrow circulate between bloodstream and lymphatics. Activation occurs in secondary lymphoid tissues.

Transcription Factors Involved in Regulating B Cell Development

There are several. Mutations result in B cell-specific deficiencies.

RAG-1 and RAG-2

They recognize recombination signal sequences, specifically heptamer-nonamer, and make dsDNA breaks. Function temporarily ceases during proliferation.

Pre-B Cell Receptor

µ chain combines with 2 proteins that mimic Ig light chains, called *Surrogate Light Chains*, *VpreB* and *λ5*. Possesses some signaling capacity.

λ5

λ5 substitutes for a light chain constant region