Lymphocyte Development Part I: B cell Development

6 Stages of B Cell Development

1. Repertoire assembly
2. Negative selection
- take place in bone marrow
- shift from Antigen independent to Antigen dependent occurs
3. Positive selection
4. Searching for infection
5. Finding infection
6. Attacking Infection
- in periphery
- all antigen

1. Repetoire assembly
2. Negative Selection

1. Generation of diverse and clonally expressed B cell receptors in the bone marrow
2. Alteration, elimination, or inactivation of B-cell receptors that bind to components of the human body

Positive Selection

promotion of a fraction of immature B cells to become mature B cells in the secondary lymphoid tissues
- still naive/virgins at this stage - haven't seen their antigens yet

Searching for infection

- re-circulation of mature B cells b/w lymph, blood, and secondary lymphoid tissues

Finding infection

- activation and clonal expansion of B cells by pathogen-derived antigens in secondary lymphoid tissues

Attacking infection

- differentiation to antibody-secreting plasma cells and memory B cells in secondary lymphoid tissue

B cell Destinations

- originate and develop in Bone marrow= primary lymphoid tissue
--> circulation
--> secondary lymphoid tissue (spleen, lymph nodes, Payer's patches in GI tract)

B Cell Development Names

1. Pluripotent hematopoietic stem cell
2. Common lymphoid progenitor (can go either B cell or T cell lineage route)
3. B-cell precursor
4. pro B cell

B Cell Development and Cell Markers

B Cell Development Is Associated with Changes in Cell Markers
- can ID the different stages by what is being expressed by the cells
- CD34 = pluripotent hematopoietic stem cell marker, used clinically for stem cell transplantation
- B cell precursor chara

Stages of B Cell Development

- are defined by the status of Ig gene
- each step is characterized by a specific gene rearrangement
Stem Cell stage
- no rearrangements
Pro B cell stage
- rearrangement of heavy chains
Pre B cell stage
- rearrangement of light chains
Immature B cells
- h

Development in the Bone Marrow

- the process is contact-dependent (B cells with bone marrow stromal cells)
- B cells contact with many bone marrow stromal cells (promiscuity)
- as a consequnce of interaction b/w ligand and receptors, B cell starts to develop
Key players:
- Cell Adhesio

Ig Gene Rearrangement

- starts in the Pro-B cell stage with heavy (H) chain rearrangement
- Productive rearrangements result in ability to generate functional protein.
- Unproductive rearrangements result from introduction of stop codons during joining --> so not every pro-B c

Productive Heavy Chain Rearrangement Leads to...

- Expression of Pre-B cell receptor:
- mu heavy chain, surrogate light chain (consisting of VpreB and lambda5)
- gets expressed on membranes on the B cell along w/ signaling molecules (Igbeta and Igalpha) --> the signal it generates is important for B cel

Expression of Pre-BCR

- affects gene arrangement!
Importance of Heavy Chain expression with surrogate light chain:
1. Stops heavy chain rearrangement
** so once a heavy chain is productively rearranged you can do no more heavy rearrangements
2. Starts light chain rearrangement

First Consequence of Ordered Rearrangement

- allotypes distinguish Ig
- In homozygous parents, every B cell expresses the same Igh allotype
- pure a allotypes (Iga/a); pure b allotypes (Igb/b) - mated these bunnies
--> mixed baby bunnies Igha/b
- outside of babies' B cells express only one type of

Productive Light Chain Rearrangement

- leads to expression of IgM on the cell surface, rearrangement then stops
- The hallmark of the Pre-B Cell stage is light chain rearrangement
- Only one light chain can be expressed (Light Chain isotype exclusion)
- An immature B cell expresses only mIgM

The Advantage of Allelic and Light Chain Isotype Exclusion

- B cell is monospecific!
- allelic exclusion yields homogenous B cell receptors w/ high-affinity binding
- w/o allelic exlcusion, heterogenous B cell receptors are yilded with low-affinity binding
- so B cell is is more easily activated

B cell survival

- helped by multiple rearrangements of light chains
- The opportunity to undergo multiple rearrangements increases the likelihood of forming a BCR

Can Heavy chains undergo multiple rearrangements?

NO
- to make a heavy chain: have V region (far left), D's (middle left) J's (middle right) and C's (far right)
- first, D rearranges with a J
- all downstream D's are eliminated by this (b/c DNA in b/w is lost)
- V then rearranges with the D-J
- when you

B Cell Development Overview

First Checkpoint

- selects for functional heavy chains
- if pre-B-cell receptor continues on
- no pre-B- cell receptor --. apoptosis
- summary: Allelic exclusion and initiation of light chain rearrangement

Second Checkpoint

- selects for functional light chains
- B cell receptor (includes light chain rearrangement and complete Ig) continues on
- no B cell receptor --> apoptosis
- this checkpoint stops all Ig gene rearrangement

Expression of proteins involved in rearrangement

- this is developmentally controlled
1. RAG-1 an RAG-2
- lympohid-specific recombinase
- when you bring gene segments together you need these (glue)
- during first check point these get momentarily turned off
2. TdT
- adds some nucleotides after genes are

Summary of Ig Rearrangements

1. Early pro-B cell = H chain rearrangememt
- D-J rearrangements on both chromosomes -->
2. Late pro-B cell = H chain rearrangement
- V-DJ rearrangement on first chromosome -->
- V-DJ rearrangement on 2nd chromosome
- apoptosis
3. Pre B cell = L chain gen

B cell Tumors

- caused by aberrant rearrangements
- there are specific recognition sequences that allow segments to come together correctly
- cryptic recogintion sequences occur in other genes
- so possible to have a rearrangement that juxtaposes Ig genes with other ge

Classes of B cells

1. B-2 B cells (what we are usually talk about)
2. B-1 B cells - develop first (reason for number)

Properties of B-1 B cells

First identified based on CD5 expression
Subsequently defined on basis of high IgM, low IgD expression
Not clear if they represent a separate B cell lineage or come from precursor that could generate either a B-1 or a conventional B cell
** Recognize comm

Comparison of B1-B cells and B2-B cells

memory allows us to use vaccines
B1 - limited repertoire

Stromal cells produce _______, which is a secreted B-cell development growth
factor influencing the progression of B cells from the late pro-B-cell to the pre-B-cell stage:
a. stem-cell factor
b. Kit
c. IL-7
d. VLA-4
e. VCAM-1

C

Which of the following characterizes the B-1 cells that develop prenatally?
a. They lack N nucleotides.
b. They possess polyspecificity for bacterial polysaccharide antigens.
They arise early in embryonic development preceding the development of
the major

E

A defect in which of the following proteins blocks B-cell development at the pre-B-cell stage,
resulting in almost no circulating antibodies in individuals with this defect?
a. IL-7 receptor
b. terminal deoxynucleotidyltransferase (TdT)
c. Pax-5
d. Bruton

D
IL-7 receptor is earlier in the process; would prevent even making a pre-B cell

Self-reactive vs Non-self reactive B cell fates

- if IgM on immature B cell is non-self reactive, leaves bone marrow and expresses both IgM and IgD, exits into the blood
- if IgM on immature B cells bind multivalent (multiple epitopes) self antigen --> retained in bone marrow and get a second chance
-

Soluble Self-Antigens

- IgM of immature B cells bind soluble univalent self antigen
- B cell is signaled to make IgD and to become unresponsive to antigen
-

B cells that Survived

- circulate within lymphoid tissue
- enters lymph node into T cell zone via HEV
- tries to get specifically into a primary follicle - need this for survival
- Primary Follicle: populated by follicular dendritic cells which both retain antigen and give a n

B Cell Maturation Completion

- occurs in the follicle
- B cells have to fight to get into the follicle
- once in follicle, it gets the maturation signal via follicular dendritic cells and cytokines
- then mature B cell can finally exit and look for antigen

Fates of Antigen Stimulated B Cells

- come back to the lymphoid (but remember, it's mature this time)
- can either go back into the follicle --> part of germinal center (isotype switching here and affinity maturation and memory cell development)
- OR go to bone marrow and become a plasma ce

Tumor Counterparts of Normal Cells

1. Acute lymphoblastic leukemia (ALL)
2. Pre B-cell leukemia
3. Mantle cell lymphoma
4. Chronic lymphocytic leukemia
5. Follicular center cell lymphoma; Burkitt's lymphoma
6. Hodgkin's lymphoma
7. Waldenstrom's macroglobulinemia
8. Multiple Myeloma

Acute lymphoblastic leukemia (ALL)

Normal cell equivalent and stage in development:
lymphoid progenitor
Location:
bone marrow and blood
Status of Ig V genes:
unmutated

Pre B-cell leukemia

Pre-B cell (w/ pre B cell receptor)
- bone marrow and blood
- unmutated

Mantle cell lymphoma

Resting, naive B cell
- periphery
- unmutated

Chronic lymphocytic leukemia

activated or memory B cell
periphery
usually unmutated

Follicular center cell lymphoma; Burkitt's lymphoma

mature, memory B cell; resembles germinal center B cell
periphery
mutated, intraclonal variability

Hodgkin's lymphoma

germinal center B cell
periphery
mutated +/- intraclonal variability

Waldenstrom's macroglobulinemia

IgM-secreting B cell
periphery
Mutated, no variability w/i clone

Multiple Myeloma

Plasma cells; various isotypes
Bone marrow
Mutated, no variability w/i clone

Review of Stages of B Cell Development

- bone marrow - primarily antigen independent

Tom Jones is a 1 year old boy who presents with 1 day of fever, cough and
dyspnea. The mother recorded a fever of 102.3 F and notes increased
irritability, rhinorrhea, frequent ear pulling, diarrhea and vomiting. The
cough began yesterday and has progress

1. Use CD19; add fluorescence to CD19 on a blood sample; could also look for IgD
2. Btk - probably wouldn't make it through pre-B cell development (needed for transition from pro to pre)
IL-7 Receptor deficiencies - no B cells
3. No memory B cells; can't

Ingrid Alshouse was a 55 year old housewife when she complained of excessive fatigue. A blood sample revealed mild anemia and increased sedimentation rate. Sedimentation rate is increased when fibrinogen or IgG is elevated. Both IgG and IgA were elevated.

1. late stage plasma cell; myeloma
2. a specific Ig rearrangement; if all came from one cell they would all have the same rearrangement
3. tumor in bone marrow could be affecting further B cell development
4. how do you get a variable region associated wi