Immunology Chapter 6

The body carries an incomplete inventory of B cells, one that expands and contracts its individual clones according to the current need

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How many B cells are generated everday?

60 billion

B cell development divides into

6 phases

Phase 1

Repertoire assembly

generation of diverse and clonally expressed BCRs in the bone marrow

Phase 2

Negative Selection

Alternation, elimination, or inactivation of BCRs that bind to components of the human body

Phase 3

Positive Selection

Promotion of a fraction of immature B cells to become mature B cells in the secondary lymphoid tissues

Phase 4

Searching for selection

Recirculation of mature B cells between lymph, blood and secondary lymphoid tissues

Phase 5

Finding Infection

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

Phase 6

Attacking infection

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

The first 3 phases correspond to

development in the bone marrow

The last 3 phases correspond to

development in the secondary lymphoid tissues

Negative selection

prevents the emergence of mature B cells bearing receptors that bind to normal constituents of the body because they could potentially attack health tissue and cause autoimmune disease

Positive selection

immature B cells compete for access to a limited number of follicles in the secondary lymphoid tissues where their maturation is completed

Central Tolerance source

Negative Selection

Peripheral Tolerance Source

Positive Selection

How many BCRs in IgG

2 binding sites, 6 CDRs = 12 BCRs

How many BCRs in IgM

10 binding sites * 6 CDRs = 60 BCRs

Pro-B cells

earliest identifiable cells of the B cell lineage. Progenitor B cells that retrain some capacity for self renewal and divide to produce additional pro-B cells as well as cells that proceed to a further stage of development

Early pro-B-cell

early stage of differentiation where the rearrangement of heavy chain genes and light chain gene rearrangement occurs

What gene rearrangement occurs at the early pro-B-Cell stage?

the joining of the Dh gene segment to the Jh gene segment

What gene rearrangement occurs at the late pro-B-Cell stage?

the joining of a Vh gene segment to the rearranged DJH to give VDJH

What marks the point where a developing B cell ceases to be a pro B cell and becomes a pre B cell?

The expression of the mew (u) chain

What does the heavy chain of a late pro B cell encode?

the membrane associated form of the u chain

Pre-B cells represent two stages in B-cell development:

the immature large pre-B cells and the mature small pre-B cells

What must a large pre-B cell must demonstrate in order to rearrange a light chain gene?

It must demonstrate that its mew (u) heavy chain has the capacity to bind a light chain and form a viable cell surface receptor.

How does the large pre-B cell take the test to rearrange a light chain gene?

The large pre-B cell synthesizes a surrogate light chain, which is able to assemble with high-quality mew (u) chains and Iga and Igb to form a pre BCR.

When the pre BCR synthesized, what happens?

The pre BCR delivers signals for the large pre-B cell to proliferate and reactivate the recombination machinery for light chain gene rearrangement which then causes the Large pre b-cell to give rise to a clone of small pre-b cells.

What happens if the large pre B cell fails to assemble a pre BCR?

no signals are transduces and the large pre B cell dies by apoptosis

What can cause a large pre b cell to fail to form a pre BCR?

the presence of N and P nucleotides that perturb the folding of the heavy chain or its interaction with the surrogate light chain

Where does the rearrangement of light chain genes occur?

in the small pre-b cells

What does the surrogate light chain consist of? What does each part surrogate for?

VpreB = Vl

lambda 5= Cl

Order of light chain rearrangement

kappa, then lambda if kappa fails

If K light chain rearrange successfully,

then gene rearrangements are stopped and B cell makes a kappa light chain

If K light chain genes fail to make a viable K chain,

then the lambda light chain genes process to rearrange. If successful, then light chain is synthesized and light chain gene rearrangement is halted.

Why are the light chains and mew (u) heavy chains translocated to the ER

they are assembled with IgA and IgB to form the BCR

After correct assembly of the heavy and light chains,

the resulting IgM associated with Iga and Igb to form the BCR which is then transported to the cell surface.

With surface expression of a BCR,

the small pre-B cell becomes an immature B cell

Stromal cells

network of nonlymphoid cells that provide specialized micro-enivronments for B cells at various stages of maturation.

Stromal Cell Function

1. make specific contacts with the developing B cells through the interaction of adhesion molecules with their ligands

2. produce growth factors that act on the attached B cells

Secondary lymphoid tissue

spleen, peyers patch, lymph node

In early stages of B cell development, how are the B cells dependent on bone marrow stromal cells?

Stem cells and early pro-B cells use the integrin VLA-4 to bind to the adhesion molecule VCAM-1 (vascular cell adhesion molecule 1) on stromal cells. This and interactions between other cell-adhesion molecules (CAMs) promote the binding of Kit on the B cell to stem-cell factor on the stromal cell. Activation of Kit causes the B cell to proliferate. B cells at a later stage of maturation require interleukin-7 (IL-7) to stimulate their growth and proliferation.

What induces the synthesis of all the proteins needed for immunoglobulin genes (RAG1 and RAG2) to be rearranged?

E2A and EBF

What do RAG1 and RAG2, E2A and EBF induce the expression of?

Pax-5

Pax-5 function

transcription factor that switches on genes for the cohort of proteins expressed only in B cells, including IgA IgB, and CD19

CD19

cel surface protein of B cells and a component of the B cell coreceptor

nonproductive rearrangements

gene arrangements that do not translate to give a functional heavy chain sequence

productive rearrangements

rearrangements that maintain a proper heavy chain reading frame

What causes nonproductive and productive rearrangements?

N and P nucleotide additions

Gene rearrangement is initiated at one heavy chain loci to avoid 2 heavy chains from being made

Treu

What does the first gene rearrangement of the heavy chain entail? Is it usually successful?

The first rearrangement joins a D gene segment to a J gene segment, producing a DJ segment.

THis is usually successful because all 3 reading frames of the small D gene segments give rise to a functional protein sequence.

What does the second gene rearrangement of the heavy chain entail? What happens if it fails?

The second rearrangement joins a V gene segment to the DJgene segment to form a VDJ gene segment.

Only 1 out of the 3 possible reading frames produces a V region

In the B cells where heavy chain gene rearrangement is successful….

transcription of the rearranged gene increases and the IgM heavy chain (The MEW u chain) is made

If the B cells make an unsuccessful heavy chain rearrangement at the first locus? What happens? What if they fail again?

They have a second attempt at the second gene locus. If they fail again then the cell undergoes apoptosis

Survival Signals

A general feature of the lymphocyte development is for apoptosis to be the default pathway unless a positive signal for survival and further differentiation is received

How does formation of the pre BCR prevent the pre B Cell from making a second form of mew (u) chain and light chain

the pre BCR sends signals to:

1. terminate the transcription of the RAG genes

2. induce the degradation of RAG proteins

3. cause the chromatin of the heavy chain locus to be rearranged into a state that prevents gene rearrangement

Allelic exclusion

a cell expresses only one of its two copies of a gene (ex when the pre BCR shuts down the production of RAG transcription to prevent the formation of another mew and light chain).

Although individual B cells express only one allele, the maternal and paternal alleles are expressed equally within the B-cell population.

True

What is the possible outcome if RAG 1 and 2 aren’t shut down after a productive rearrangement in a B cell?

you lose avidity

Large pre B cells reactivate RAG genes, Why?

They need to make a proper light chain.

How does light chain gene rearrangement proceed?

Rearrangements take place one light chain locus at a time with the Kappa locus being rearranged before the lambda locus.

What effect does having a single event of a V-J joining have on diversity? What advantage does this have?

It reduces the somatic diversity achieved by combining different gene segments, it has the advantage that several attempts can be made to rearrange the same light chain gene by using V and J gene segments not involved in previous rearrangements.

Why is several rearrangements not possible at the heavy chain locus?

THe first rearrangements that join V, D , and J togther excise all other D segments

Where does the functional BCR send the signal to shut own light chain rearrangemnt?

After the mew chain and light chain is assembled in the ER, the BCR forms a membrane bound IGM and associates with IgA and IgB and moves to the cell surface where it sends the signal to shut downlight chain rearrangement.

Isotypic exclusion

ensures that the light chain produced by an individual B cell is either κ or λ, but not both

1st checkpoint; where does it act?

assess the quality of the heavy chains; acts in a pro- B cell after it has rearranged a mew chain gene and is making mew chain protein; assesses the capacity to bind to a surrogate light chain and assemeble a function pre BCR

2nd checkpoint

asseses the quality of the light chains; operates in a pre B cell when it has rearranged a light chain gene and is making light chain protein which is delivered to the ER.

What is the role of Igα and Igβ for a B cell receptor? When would you expect to see them expressed? When would they be turned off (not expressed/suppressed)?

role of Iga and Igb are responsible for transducing signals, halting gene rearrangements, establishing allelic and isotypic exclusion.

They are never turned off. expressed since pro-b cell

What causes tumor cells?

Sometimes normal processes go awry when V cells cut, splice and mutate their Ig genes which causes the mutations to form a tumor cell

For B-cell tumors, such disruption is often associated with an

aberrant immunoglobulin-gene rearrangement that joins an immunoglobulin gene to a gene on a different chromosome

Translocations

Events that fuse part of one chromosome with another. Tumors are formed when B cell genes are fused with genes in control of cell growth

Proto-oncogenes

Genes that can cause cancer when their function or expression is perturbed

Oncogenes

Viral genes responsible for transformation

How does Burkitt’s Lymphoma arise and what two chromosomes are affected?

Translocations between chromosome 14 (Ig gene) and chromosome 8 (MYC).

What is the normal role of the MYC gene and how is it involved in Burkitt’s lymphoma?

The normal role of MYC gene is regulating cell cycle but abnormal expression as a result of translocation causes increased growth

What are the 2 proto-oncogenes

MYC and BLC-2

BLC-2 normal function

protective against premature apoptosis in B cells

B-1 cells

A minority of human B cells that arise early in embryonic development differs from other B cells by their expression of CD5

B-2 Cells

the majority type of B cell. It undergoes somatic hypermutation and affinity maturation, doe snot express the CD5 glycoprotien and makes antibody of narrow specificity

What is meant by self-renewal? Which of the two demonstrates this?

B-1 Cells; With time, B-1 cells are no longer produced by the bone marrow, and so the population of B-1 cells in adults is maintained by proliferation of the existing B-1 cells at sites in the peripheral circulation. This process of self-renewal is dependent on the cytokine IL-10.

Which produces memory?

B-2

What major immunoglobulin isotypes are secreted for each?

B-1 =IgM

B-2 = IgM and IgG

Immature B Cells immunoglobulin isotype

IgM

Mature B cell immunoglobulin isotype

IgM and IgD

Self antigens

Immature B cells that express surface IgM embrace a wide range of antigenic specificities and this initial repertoire includes many CRS that recognize healthy human tissue

Self reactive (autoreactive B cells)

B Cells with the potential to respond to self antigens

To prevent the emergence of mature B cells that are self reactive,

the BCRs of immature B cells are programed to generate negative intracellular signals when they bind to antigen which induce the B cell to either die by apoptosis or inactivate

As a consequence, the resulting population of mature B cells in a healthy individual cannot respond to self antigens and is therefore said to be

self tolerant

If the cell surface IgM of an immature B cell binds a multivalent self antigen in the bone marrow,

this signals the B cell to reduce the amount of IgM onits surface and to maintain the expression of the RAG complex. This permits the B cell to continue rearranging its light chain loci.

Receptor editing

Occurs when self reactivity is still retained with the second light chain, further light chain gene rearrangements are tried. This process of assessing compatibility of receptors produced from successive gene rearrangements.

Anergy

elf-reactive B cells that bind to these monovalent self antigens are not signaled to continue light-chain gene rearrangement nor are they signaled to die by apoptosis. Instead, they become inactivated and unresponsive to their specific antigens. This state of developmental arrest is called ( ).

Clonal deletion

The selective elimination of self-reactive receptor specificities from the B cell Repertoire

Is Anergy typically in reference to immature B cells or mature B cells? What ends up happening to anergic B cells?

immature B cells

Anergic cells enter peripheral circulation and die in 1-5 days

Central Tolerance

the type of immunological tolerance where it is induced in the primary lymphoid organ (bone marrow)

three mechanisms for immature B cells that react to self antigens in the bone marrow

1. receptor editing

2. apoptosis

3. anergic

this tolerance is central tolerance

Peripheral Tolerance

self-reactive immature B cells outside the bone marrow either die by apoptosis or are rendered anergic when they engage self antigens. Tolerance induced to antigens outside the bone marrow

The final stage of maturation begins when

Immature B cell enters a secondary lymphoid tissue

CCL21

chemokine that is secreted by stromal cells and dendritic cells in secondary lymphoid tissues and attracts leukocytes and dendritic cells to the tissue

CCR21 & CCL19

receptor on leukocytes for chemokines CCL19 and CCL21

Primary lymphoid follicles

A B Cell area in a secondary lymphoid tissue in the absence of an immune system response. It contains resting B lymphocytes