B Cell DevelopmentAssignment: Chapter 4 (Skip Figs. 4.6, 4.12)

Notes

1. Tens of billions of B cells generated each day in the bone marrow; only ½ survive

2. Bone marrow: primary lymphoid tissue

3. Development means the cell surface expression of a unique B Cell Receptor (BCR), which is an Ig molecule (monomeric IgM and IgD)

Learning Objectives for Lecture 11

1. Discover how lymphoid stem cells become B cells destined to make antibodies

2. Understand the importance of Ig gene rearrangement

3. Appreciate mechanisms leading to B-cell leukemias

Stage 1: Immunoglobulin (Ig) Gene Rearrangement (Absence of Antigen)

Notes1. Pre-B-cell Receptor: rearranged HC, surrogate LC, Ig, Ig initiates cell division resulting in 30-

70 small pre-B cell clones (all have same heavy chain, but with potential to have different light chains)

2. Signal from pre-B-receptors halts HC gene re-arrangement & sLC synthesis; cell proliferation to yield lots of small pre-B-cells; cell division stops and light chain gene rearrangement begins

3. Immature B cells selected for tolerance (prevents autoimmunity)

4. Tolerant immature and mature B cells enter periphery (immature cells mature in the spleen)

5. Naïve B cells (never seen antigen) circulate looking for foreign microbes

RAG proteins responsible for gene recombination

Ig and Ig are Signaling Subunits of the B Cell Receptor (BCR; surface Ig molecule)

Notes

1. The Ig molecule (either pre-BCR or BCR) can not travel to the surface of the B cell without Ig and Ig

2. The pre-BCR and BCR consist of an Ig molecule plus Igand Ig3. Ig and Ig genes turned on at the pro-B-cells stage and remain on

until cell becomes and antibody secreting plasma cell

4. Ig and Ig send signals when receptors are engaged (bound antigen)

Bone Marrow Stromal Cells Direct B Cell Development

Notes

1. Adhesions molecules: CAMS, VLAs, VCAMs2. Signaling molecules: Kit (receptor); SCF (membrane bound growth factor), IL-7

Productive Gene Rearrangement is Key to Developing B Cell Survival

Notes1. Two copies of heavy chain and light chain loci (one on each chromosome;

remember there are 2 light chain loci and )2. Most DJ rearrangements are successful (D can translate three reading frames)3. VDJ rearrangement is consecutive; 50% success rate (remember: chain)4. Unproductive gene rearrangement results in apoptosis (ordered cell death)

Light Chain has Several Chances to Rearrange

Notes

1. Large pre-B-cells undergo cell division before becoming resting small B cells; LC rearrangement

2. Starts with and goes until all possibilities have been tried

3. LC rearrangement, 85% successful

4. Overall success of Ig gene rearrangement is less than 50%

Ending Gene Rearrangement

Notes

1. Once productive gene rearrangement has occurred, need to prevent further gene rearrangement. Why?

2. The ligation (binding) of a rearranged immunoglobulin gene at the cell surface sends a signal to shut down gene rearrangement. How is preventing gene rearrangement accomplished?

3. B cell diversity: generation of different heavy chain; each paired with a different light chain

Combinatorial DiversityLC: : 40 V x 5J=200; : 30V x4J=120 320 TotalHC: 40V x25D x6J = 6000LC + HC 320 x 6000 = 1.9 x 106 possible combinations

Junctional Diversity

Random insertion of N and P nucleotides at VJ, DV, VDJ junctions

Total Possible Different Ig Receptor CombinationsCombinatorial plus junctional = 1011 estimated possibilities

Regulating B Cell Development

1. Genes essential for gene recombination are turned on at selective stages of B cell development

2. Genes encoding RAG;

-turned on in Early pro-B cell and late pro-B cell (HC rearrangement)

-turned off in Large pre-B cell (to allow proliferation)

-turned back on in Small-pre-B cell (LC rearrangement)

3. Terminal deoxynucleotidyl transferase (TdT)

-responsible for functional diversity (N nucleotides)

-turned on in pro-B cells, silent in small pre-B cells

4. Genes encoding Ig and Ig

-turned on in pro-B cells and remain on

5. Burton’s tyrosine kinase (Btk)

-signaling molecule whose deficiency prevent B cell development

X-linked Agammaglobulinemia

1. Ig and Ig signal to a signaling molecule: BTK

2. Btk needed to signal B cell to develop

3. Btk is located on X-chromosome

4. Patients lacking Btk (mostly boys) have B cell development blocked at the pre-B-cell stage and therefore have no circulating antibodies

5. Suffer from X-linked Agammaglobulinemia

6. Recurring infections: Haemophilus influenzae; Streptococcus pneumoniae, Streptococcus pyrogenes; Staphylococcus aureus

7. Treatment: antibiotics and infusion of antibodies

Formation of B Cell Tumors (Leukemias & Lymphomas)

Notes

1. High transcriptional and splicing activity during B cell gene rearrangement

2. Mistakes made that can result in deregulated cell growth leading to leukemia

3. Ig gene segment is mistakenly joined to a gene regulating cell growth

-translocation: gene on one chromosome joined to a gene on a different chromosome

-B cell tumors: Burkitt’s lymphoma; Ig gene segment mistakenly fused to a gene called MYC that regulates the cell cycle; along with additional mutation leads to Burkitt’s lymphoma

CD5+ B Cells (B-1 Cells)

1. Arise early in embryonic development

2. Express CD5 on surface

3. No surface IgD; restricted BCRs; Abs to bacterial polysaccharides

4. Predominate in pleural and peritoneal cavities

5. Capacity for self-renewal

6. Most B cell tumors causing chronic lymphocytic leukemia (CLL) are transformed B-1 cells (express CD5 on surface)

7. Treatment: bone marrow transplant

Summary1. B cell originate from lymphoid progenitor stem cells and

development in the bone marrow thoughout life

2. Consecutive gene rearrangements of Ig genes results in the expression of a unique BCR (Ig molecule with H and L chains)

3. Several loci (2 HC; 4LC) to counter unproductive rearrangements

4. HC rearranges first and this must be productive to continue

-forms pre-BCR (rearranged HC and surrogate LC); ligation on cell surface halts HC gene rearrangement

5. LC rearrangement following proliferation of large pre-B cells

-4 loci; several attempts at each loci (85% success rate)

-productive light chain rearrangement halts further rearrangement

6. B cell repertoire is diverse (1011)

7. Mistakes cause B-cell leukemias and lymphomas