B Cell Development

Assignment: Chapter 4 (Skip Figs. 4.6, 4.12) 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 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)

Stage

1: Immunoglobulin (Ig) Gene Rearrangement

(Absence of Antigen) RAG proteins responsible for gene recombination Notes 1. Pre-B-cell Receptor: rearranged HC, surrogate LC, Ig chains) a , Ig b; initiates cell division resulting in 30 70 small pre-B cell clones (all have same heavy chain, but with potential to have different light 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

Ig

a

and Ig

b

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 a and Ig b 2. The pre-BCR and BCR consist of an Ig molecule plus Ig a and Ig b 3. Ig a and Ig B genes turned on at the pro-B-cells stage and remain on until cell becomes and antibody secreting plasma cell 4. Ig a and Ig b send signals when receptors are engaged (bound antigen)

Bone Marrow Stromal Cells Direct B Cell Development

Notes 1. Adhesions molecules: CAMS, VLAs, VCAMs 2. Signaling molecules: Kit (receptor); SCF (membrane bound growth factor), IL-7

Productive Gene Rearrangement is Key to Developing B Cell Survival

Notes 1. Two copies of heavy chain and light chain loci (one on each chromosome; remember there are 2 light chain loci k and l ) 2. Most DJ rearrangements are successful (D can translate three reading frames) 3. VDJ rearrangement is consecutive; 50% success rate (remember: m 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 k 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 Diversity LC: k : 40 V x 5J=200; l : 30V x4J=120 320 Total HC: 40V x25D x6J = 6000 LC + HC 320 x 6000 = 1.9 x 10 6 possible combinations Junctional Diversity Random insertion of N and P nucleotides at VJ, DV, VDJ junctions Total Possible Different Ig Receptor Combinations Combinatorial plus junctional = 10 11 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 a and Ig b -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 a and Ig b 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

Summary

1. 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. m HC rearranges first and this must be productive to continue -forms pre-BCR (rearranged m 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 (10 11 ) 7. Mistakes cause B-cell leukemias and lymphomas