B-cell development, activation and differentiation

Question 1

B-cell development begins with a hematopoietic stem cell (HSC) that develop into a CLP, what does CLP stand for?

Answer 1

CLP - common lymphoid progenitor, which all lymphocytes develop from.

Question 2

Wherer does development of B cells primarily occur?

Answer 2

B cell development occur in the bone marrow and is influenced by various niches established by stromal cells.

Question 3

What three things define the different stages of B cell development?

Answer 3

Stages of B-cell development are defined by:
- the presence of sets of cell-surface markers (which include cytokine and chemokine receptors and adhesion molecules),
- expression of specific transcription regulators
- the rearrangement status of immunoglobulin genes.

Question 4

What controls the different stages of B cell development?

Answer 4

The stages of B-cell development are controlled by networks of transcription factors and by epigenetic changes that influence the expression of key genes. The cells become increasingly committed to becoming B lymphocytes.

Question 5

What are the most common type of B cells?

Answer 5

The most common type of B cell is the B-2 B cell, which follow the “classical” developmental pathway. There are subtypes of B cells that have a differing development pathway.

Question 6

Briefly describe the pathway from a HSC to a cell committed to B cell development.

Answer 6

HSCs develop into the ELP (early lymphoid progenitors, which is defined by the expression of RAG1/2 in prerparation for receptor rearrangement. A subset of ELPs migrate to the thymus and serve as T-cell progenitors while other ELPs remain in the bone marrow as B-cell progenitors. As the levels of the IL-7R increase, expression of c-kit and Sca-1 proteins decreases, and the ELP develops into a common lymphoid progenitor (CLP). The CLPs then get in contact with stromal cells secreting the chemokine CXCL12 where they develop into the first stage committed to the B cell lineage.

Question 7

At what stage does the CLP cells get commited to the B cell lineage?

Answer 7

When it has become a pre-pro-B cell.

With the acquisition of the B cell specific marker B220 (CD45R), and the expression of increasing levels of the transcription factor EBF1 (Early B-cell factor 1), the developing common lymphoid progenitor enters the pre-pro-B-cell stage.

Question 8

Name the different stages of B cell development in order.

Answer 8

CLP –> pre-pro-B cell –> Pro-B cell –> Pre-B cell –> Immature B cell –> periphery –> spleen –> transitional B cells –> mature B cells.

Question 9

What happens during the pre-pro-B cell stage?

Answer 9

• Accessibility to the BCR/Ig heavy chain locus is increased
• Activation of B-lineage genes and inhibition of expression of T cell commitment factors, further contributing to the commitment to the B cell lineage.
• Pre-pro-B cells remain in contact with CXCL12-secreting stromal cells in the bone marrow.

Question 10

What happens during the pro-B cell stage?

Answer 10

In the progenitor B cell, pro-B cell stage:

• early: D-to-J recombination is completed and the cell
  begins to prepare for V -to-DJ joining, which requires the TF PAX5.
• late: PAX5 expression –> initiation of V-DJ rearrangement, when finalized, the heavy chain is completed by the onset of early pre-B cell stage, capable of expressing µ heavy-chain protein.

Question 11

What happens during the pre-B cell stage?

Answer 11

During the early pre-cell stage, the genes for the surrogate light chain (SLC) is expressed and two SLCs pair with two µ heavy chains, forming the pre-B-cell receptor (pre-BCR). A functional pre-BCR is the first checkpoint, aka the pre-B-cell checkpoint. A functional pre-BCR leads to proliferation. Cells that have not formed a functional µ heavy chain, one that can associate with both another µ heavy chain and surrogate light chains to form a pre-BCR that transmits signals, undergo apoptosis.

The proliferating large pre-B cells form a pool of daughter cells all encoding for the same heavy chain (small pre-B cell stage) that will eventually go through individual rearrangement of the light chain, but before that, the heavy chain locus is closed off by chromatin remodeling and surrogate light-chain gene transcription is terminated by a negative feedback round of signaling through the pre-B-cell receptor.

The small pre-B cells undergo light chain rearrangement (random if kappa or lambda starts in humans). This is the second checkpoint, aka the immature B-cell (second) checkpoint. If light chain rearrangement successful a BCR is formed. If not the cell goes into apoptosis. Once a light-chain gene rearrangement has been successfully completed, the IgM B-cell receptor is expressed on the cell surface and signals the cell (apparently spontaneously, without ligand binding or self-aggregation) to terminate any further light-chain gene rearrangements. The cell has now entered the immature B cell stage.

Question 12

During the pre-B cell stage, two checkpoints take place. Explain what their purpose are in short.

Answer 12

1st checkpoint - the pre-B-cell checkpoint: Check if heavy chain rearrangement was successful –> pre-BCR formed. If not –> apoptosis.

2nd checkpoint - the immature B-cell checkpoint: check if the light chain rearrangement was successful. If light chain rearrangement successful –> BCR is formed. If not –> apoptosis.

Note: most pre-B cells that have successfully rearranged their heavy chains will express the complete BCR (membrane IgM/mIgM), and go on to form immature B cells.

Question 13

After a successful in 1st checkpoint, the formation of the pre-BCR play a very important role in the development of B cells. What role?

Answer 13

Pre-B cell signaling induces:
- downregulation of RAG1/2 and loss of TdT activity, which ensures that no further heavy chain recombination is possible - ensuring allelic exclusion, i.e only one heavy chain allele expressed per B cell. The chromatin of the unrearranged heavy-chain locus undergoes physical changes that block further rearrangement.
- Proliferation followed by light chain rearrangement

Question 14

What happens during the immature B cell stage in the bone marrow?

Answer 14

Immature B cells only express IgM, not IgD and continue to express B220 and CD19.

During the immature B cell stage the BCR must be tested for self-reactivity. Immature B cells are very sensitive to apoptosis, and are tested against self-antigens in the bone marrow on stromal cells. There is only negative selection of immature B cells, those that don’t react strongly to self antigens present in the bone marrow go out in the periphery while B cells with autoreactive receptors undergo one of three fates (Collectively referred to as central tolerance):
– Clonal deletion in bone marrow (apoptosis)
– Receptor editing in bone marrow: altering the specificity of the BCR by reactivation of their RAG genes, re-editing the light chain, if not self reactive after that, it moves on out into periphery.

Note: anergy can occur in the periphery.

Question 15

When immature B cells have been shown to be non-self reactive, they go out into the periphery, where do they complete their maturation and how do they get there?

Answer 15

Immature B cells express the S1P receptor, which recognizes the lipid chemoattractant sphingosine 1-phosphate (S1P) in the blood and migrate to the spleen to complete their development.

Question 16

Explain the process of immature B cell maturation to T2 B cells in the spleen.

Answer 16

When the immature B cells leave the bone marrow, they are still functionally immature. When they enter the spleen they are referred to as T1 B cells (Transitional B cells). These cells undergo autoreactive screening against soluble self-antigens. If they react strongly to any self antigen, they undergo apoptosis. The non-autoreactive T1 cells are negatively selected and differentiate into T2 B cells.

The majority of T1 B cells die in this negative selection process, around 55-75% which sounds harsh but T2 B cells are resistant to antigen-induced apoptosis, so better safe than sorry.

There is also a third subpopulation of T3 cells that are unresponsive to self antigens, these become anergic and die.

Question 17

How does the T2 B cells mature into B-2 B cells (follicular B cells)?

Answer 17

T1 cells that have completed the selection process start to differentiate into T2 B cells that are able to enter the follicles. In the follicle, the T2 B cells receive a signal that upregulates the expession of the BAFF receptor, and in order to mature, the T2 B cells require BAFF-signaling, so If the cell doesn’t express the BAFF-R it will die (small fraction). The BAFF signalling promotes survival of transitional B cells by inducing the synthesis of anti-apoptotic factors such as Bcl-2, Bcl-xL, and Mcl-1. These cells also upregulate their expression of IgD. Then they have completed their development into fully mature, recirculating conventional (B-2) B lymphocytes that can migrate to the lymphoid follicles in the lymph nodes.

Note: Some T2 B cells instead enter the marginal zone and become marginal zone B cells.

Question 18

What are the four key properties of B2 B cells?

Answer 18

The key features of B-2 B cells (follicular B cells) are:

• They express high levels of IgM/IgD on their surfaces
• They recirculate between blood and lymphoid organs

– They respond to antigens by producing antibodies but reqire T-cell help to do it.

• They have a half-life of approximately 4.5 months in the periphery (if they get a sufficient supply of (B-cell activating factor, BAFF), memory cells can be even more long lived.

Question 19

Compare marginal zone (MZ) B cells and B1-B cells to the conventional B-2 B cells in terms of function, location, and development.

Answer 19

The MZ B cells are more similar to the B-2 B cells than the B-1 B cells. The MZ B cells originate the same way and mature through the same process. The only differences are that the MZ B cells occupy the marginal zone of the spleen while the follicular B cells migrate to other secondary lymphoid organs. MZ B cells appear to be specialized for recognizing blood-borne antigens and are capable of responding to both protein and carbohydrate antigens. MZ B cells have high levels of mIgM and low mIgD (the opposite of B-2 B cells) and mostly secrete IgM compated to B2 cells that secrete mostly IgG.

B-1a B cells are very different from B-2 B cells. Location wise, they occupy the pleural (around the lungs) and peritoneal (around the abdomen) cavities. They develop from existing B-1 cells (derivatives from embryonic development) - not from HSCs. They are also functionally distinct, they have a very limited receptor repertoire that recognize conserved self-carbohydrate and lipid antigens, such as phosphatidylcholine (PtC) exposed on aged erythrocytes and apoptotic cells, so they are specialized to clear dead cells and debris. Furthermore, they don’t require T cell activation to secrete antibodies (mainly IgM like MZ B cells), so they provide an early response extracellular pathogens.

Question 20

Compare the development of T cells and B cells, what is similar? What differs?

Answer 20

Similarities:
- Both B and T cells go though a negative selection process when screening for autoreactivity (although the processes are different)
- Most cells (>90%) are lost before reaching the periphery.
- Sequential rearrangement of antigen receptor genes
- Signalling from the completed receptor is necessary for continued survival (positive selection), although these processes differ too.

Differences:
- Mainly, B cells don’t mature in the thymus (or a structure analogous to it. They do fully mature in the spleen generally but it’s not necessary as it is for T cells in the thymus.
- There negative selection of T cells involves MHC and expression of peripheral tissue antigens in the primary lymphoid organs, These processes are not involved for B cell development.

Overall, the results of these developmental processes usually are mature B and T cells with appropriate antigen receptor repertoires that will protect us from infections and avoid undesirable autoreactivity.

Question 21

Explain the basics of the clonal selection hypothesis.

Answer 21

The clonal selection theory provides the basis of how millions of cells/antibodies perfect for combating the pathogen can be produced from an extremely low number of cells. Upon activation of a lymphocyte, that cell receives proliferation signals to clonally expand to produce many effector cells specific to the antigen. This allows for having big diversity with little energy spent that can be invested only when needed to make more.

Question 22

There are two types of B cell responses, which?

Answer 22

T-cell dependent and T-cell independent B cell responses.

Question 23

What determines what type of B cell response will occur?

Answer 23

They type of antigen determines which type of B cell response will take place.

• T-dependent (TD) antigens trigger the TD response, mediated by B-2 B cells (follicular B cells) and requires the help of CD4+ T cells.
• Multivalent/polymerized antigens trigger the T-independent (TI) responses, which do not require T-cell help.

Question 24

There are two types of TI antigens, which?

Answer 24

• TI-1 Ag bind to B cells through PRRs and mIgs. The TI-1 antigens are mitogenic and (at high antigen concentrations) elicit a polyclonal, antibody-secreting response.
• TI-2 Ag cross-link large numbers of BCRs. They are highly multivalent and are capable of delivering an acticvation signal in the absence of T cell help. T1-2 antigens do not bind to innate immune receptors

Note: Both types of T-independent responses are enhanced by interactions with other cell types, including T cells, macrophages, and monocytes, and neutrophils.

Question 25

Give an example of a TI-1 antigen and how it activates B cells.

Answer 25

One example of a TI-1 antigen is LPS. LPS from gram-negative organisms binds to B cells via both membrane-bound immunoglobulin (mIg) and TLR4, resulting in signaling from both receptors, which is enough to activate the B cell.

Question 26

Give an example of a TI-2 antigen and how it activates B cells.

Answer 26

TI-2 antigens have highly repetitive structures, such as polysaccharides found on gram-positive bacteria such as pneumococcus. These repetetive structures are frequently bound by the complement component C3d which is recognized by the coreceptor CD21 on B cells. Cross-linking of both mIg and CD21 receptors on B cells lead to an activation signal. Cross-linking of between 12 and 16 Ig receptors by TI-2 antigens has been shown to be sufficient to deliver an activating signal.

Question 27

Three signals are necessary for TD B cell activation, which?

Answer 27

Signal 1: The B cell binds antigen via its Ig receptors/BCR. Signal 2: An activated T cell binds to the B cell both through its antigen receptor and via a separate interaction between CD40 on the B cell and CD40L (CD154) on the activated T cell. Signal 3: Upon binding to the B cell, the T cell releases activating cytokines.

Question 28

Upon T cell dependent activation of a B cell, it can respond in three different ways, which?

Answer 28

Following T-dependent stimulation with antigen, B cells may: - Differentiate into antibody-producing plasma cells in the primary focus - Or differentiate into early, low-affinity memory cells. - Alternatively, they may enter the follicles to participate in the germinal center reaction.

Question 29

Describe the TD B cell response from infection to B cell antigen recognition.

Answer 29

Upon infection, the antigen gets concentrated in various peripheral lymphoid tissues. From there, the antigen is drained by the lymphatic system into the lymph nodes. The antigen can enter the lymph nodes alone, bound to antigen transported cells or complement coupled. In the lymph nodes, antigens enter through one of two routes, depending on their size. - Some low-molecular-weight (<70 kDa) antigens enter the lymph nodes via a leaky network of conduits that are sampled by the follicular B cells. - Higher molecular weight antigens are taken up first by Fc or complement receptors on subcapsular sinus macrophages or by similar receptors on B cells, dendritic cells, and circulating macrophages, and subsequently passed on to the B cells.

Question 30

What happens when the B cells are presented with antigen in the follicles?

Answer 30

Once the B cells have bound the antigen, the BCRs that are normally in nanoclusters starts to aggregate to form microclusters of BCRs and associated coreceptors and signaling molecules. This triggers the B cell membrane starts to spread over the target membrane and then contract, resulting in the BCRs cluster and to move into lipid rafts in the membrane which brings the ITAMs of the Igα and Igβ into close proximity of a tyrosine kinase, Lyn, that phosphorylates the ITAMs and initiates the BCR signaling cascade. An immunological synapse forms which sustains the signal. The signal magnitude depends on the strength and duration of antigen binding and by additional interactions between the BCR and coreceptors like CD21, CD40 and BAFF-R. The signal induces the "signalsome" which ultimately lead to activation of genes for B cell survival, proliferation, differentiation and importantly motility of the B cells. The immunological synapse also facilitates internalization of the antigen (e.g. by BCR-mediated endocytosis), which is loaded onto MHC II molecules for subsequent antigen presentation to T cells. BCR mediated signalling also causes increased expression of costimulatory molecules CD80, CD86, and CD40 on the B-cell surface, also needed for antigen presentation to T cells. B cells are highly motile and respond well to chemoattractant signals. Chemokine signalling causes the antigen exposed B cells migrate closer to the T cell zones, where they present the antigen to T cells. Once contact with an antigen-specific, activated T cell is made they interact through the TCR-MHC antigen (signal !), costimulatory receptor CD40 on the B cell and ligand CD40L on the activated T cell (signal 2) & T-cell CD28 with B-cell CD80 and CD86 and the T cell secretes cytokines such as IL-4 and IL-21 (signal 3) - which lead to activation of the B cell and induces differentiation. Individual B cells may differentiate into plasma cells, early memory cells, or cells that enter the germinal center.

Question 31

What determines the fate of the activated B cells?

Answer 31

Different combinations and concentrations of cytokines determine the transcriptional changes in the activated B cells and different transcription factor combination determines the fate of the activated B cells. - Pax-5 and Bcl-6 (eg by IL-21 and IL-6), along with low levels of IRF-4, favor the generation of proliferating, germinal center cells. - The expression of BLIMP-1 and of high levels of IRF-4 support the generation of antibody-secreting plasma cells Since these are opposing roles, some transcription factors that lead to GC formation also suppress differentiation into plasma cells, eg Pax-5.

Question 32

Explain the process of formation of plasma cells.

Answer 32

Following activation of B cells having received the specific signals to differentiate into plasma cells, they differentiate into plasmablasts that migrate to the medullary chords in the lymph nodes or to the border between the white and red pulp of the spleen and form extrafollicular primary foci (singular primary focus). Here they complete their differentiation into plasma cells and secrete large numbers of IgM and IgG that can neutralize or opsonize antigen by the first 5 to 6 days. The plasma cells are thus responsible for the early humoral response, with antibodies that are specific to the antigen being produced fast. The size of the primary foci peaks at around 7-8 days, and then most die by apoptosis by day 14. The antibodies produced by plasma cells have not been honed through somatic hypermutation to have higher affinity to the antigen, this takes place in germinal centers which produces antibodies involved in the late humoral response.

Question 33

What is the difference between plasmablasts and plasma cells?

Answer 33

Plasmablasts are cells that have begun to secrete antibodies but have not yet terminally differentiated into plasma cells (still express BCRs on their surface and have the capacity to proliferate). Plasma cells are fully differentiated and can no longer divide (little to no surface Ig), their sole purpose is to produce and secrete large amounts of antibodies. (Compared to naive B cells that bear cell surface IgM and do not secrete antibodies.)

Question 34

Activated B cells that go into the lymph node follicles initiate a germinal center response. What cells are included?

Answer 34

Although GCs consist primarily of rapidly dividing B cells, they also contain follicular dendritic cells (FDCs), T follicular helper (Tfh ) cells, and macrophages.

Question 35

What are the three key events occurring in the germinal center?

Answer 35

The function of germinal centers is to generate a very good and specific response to the pathogen. The key events in the GC are: – Proliferation – Somatic hypermutation – Ig class switching (also outside GC)

Question 36

What is somatic hypermutation (SHM)? What are the five key properties of SHM?

Answer 36

Somatic HyperMutation (SHM) produces individual point mutations in Ig heavy- and light-chain rearrangements – SHM occurs following antigen contact – Affects only variable regions – Requires T cell engagement (CD40-CD40L interactions) – Mutations increase over time and with repeated exposures – Followed by affinity selection result in increased affinity for Ag over time

Question 37

The GC consists of two compartments, which and what cells occupy each zone?

Answer 37

Two compartments: – Dark zone—densely packed with rapidly proliferating B cells (centroblasts) – Light zone— less rapidly proliferating B cells (centrocytes), interspersed with a network of FDCs (that keep them alive) and Th cells (to activate).

Question 38

Go through germinal center formation step-by-step.

Answer 38

In the germinal centers, B cells undergo a period of intense proliferation (centroblasts) in the dark zone, during which they are also subject to somatic hypermutation (SHM) facilitated by AID and error prone DNA polymerases with a mutation rate of ~1/1000 nucleotides compared to the 1/100 000 that is the "background" mutation rate. The B cells then move to the light zone where they interact with Tfh cells and FDCs and their mutated receptors are "tested" towards the antigen, to see if they have higher affinity than the parental B cell. Those B cells with higher affinity will bind higher levels of antigen on their surfaces and will spend more time in interaction with Tfh cells and outcomplete the lower affinity siblings and be selected and are allowed to undergo class switching and from there they either exit the GC or recycle back to the DZ again for more rounds of SHM. The cycling back can occur several times. Cells in which the mutations lead to less or no affinity to the antigen will die by apoptosis after no or very low interaction with Tfh cells. The B cells that exit the GC undergo class switching and differentiate into plasma cells that produce high affinity antibodies to combat the pathogen or become memory T cells.

Question 39

How does AID work in somatic hypermutation, briefly?

Answer 39

AID or Activation-induced cytidine deaminase mediates somatic hypermutation. It targets genes that are under active transcription and deaminates a cytidine base to uridine, creating a uridine-guanosine (U-G) mismatch that can be resolved through several DNA repair mechanisms - creating opportunity for a mutation of the variable Ig region. Mutational hot spots are sequence motifs far more likely to be targeted. Higher concentration of mutational target sequences within the complementarity determining sites (CDRs) of the Ig variable regions than in framework sequences. AID also work in class switch recombination, but its a completely independent process.

Question 40

What is the purpose of class switch recombination?

Answer 40

Class switch recombination is the process in which the Fc region of an Ig receptor is changed to another class, resulting in different effector functions.

Question 41

Explain the process of class switch recombination.

Answer 41

Naive B cells express IgM and IgD, in order to secrete another class of Ig they need to undergo class switch recombination, which is only allowed in activated B cells (mostly in the GC) and requires co-stimulation via CD40 (so it requires T cells to happen). The Ig heavy chain locus lies downstream of the variable region, and contain the genes for the constant regions IgM and IgD first, then IgG, IgE and IgA. Before each constant Ig gene, there is a switch region that consists of simple, short, G rich repeats in tandem (20–80 bp long) that are different for the different classes. AID targets these switch regions, depending on the cytokine signal received by the B cell. Example: The cytokine signal that promote class switching to IgA is IL-5, so when a B cell in the GC received signals from the CD40 and IL-5, aid will bind to the switch region before the IgA constant segment (acceptor switch region), and induce a DSB. It will also target the initial switch site (before IgM/IgD (donor switch site) and induce a DSB. The two switch regions will them be ligated together by the NHEJ machinery, and the sequence in the middle will be excised (excision circle). Since DNA is excised, this process is irreversible, but in theory, a cell that has class switched to for example IgG could class switch to IgA since IgA is downstream of IgG and therefor is not included in the excision circle.

Question 42

Which class is switched to upon IL-4 stimulation?

Answer 42

IgE (or IgG1)

Question 43

Which class is switched to upon TGF-beta stimulation?

Answer 43

IgA (or IgG2b)

Question 44

Which class is switched to upon IFN-gamma stimulation?

Answer 44

IgG3 or IgG2a.

Question 45

B cells that emerge from the GC can differentiate into memory cells, compare memory B cells with naive B cells and name three key differences.

Answer 45

Memory B cells respond with antigen secretion to antigen challenge much quicker that naive B cells (1-3 days compared to 4-7 days). Also the peak response is much earlier for memory B cells, at around 3-5 days compared to 7-10 days for naive B cells. The magnitude of the response is generally 10-1000 times higher for memory B cells. They have secrete high affinity antibodies compared to the initial low affinity antibodies secreted by newly activated B cells. They are very long lived, up to the life span of the host while naive B cells survive for days/weeks max). Memory B cells har highly T celll dependent while naive B cells can be activated independently of T cells (TI activation). They also have different surface marker and chemokine receptor expression and express anti-apoptotic molecules.

Question 46

Early and late memory cells differ in one aspect, which?

Answer 46

First memory cells to appear during the primary response are IgM+ memory B cells while IgG+ memory B cells appear later, have mutated and affinity selected receptors.

Question 47

Where do long lived plasma cells (LLPCs) reside?

Answer 47

LLPCs reside in the bone marrow, where stromal cells and eosinophils provide the necessary components for sustained survival.

Question 48

Most newly generated B cells are lost at the end of the primary immune response, how?

Answer 48

We don't know! After Ag is cleared, most of the effector cells are no longer required and the exact mechanism behind apoptotic clearance isn’t fully characterized - questions remain: - Is the trigger simply lack of Ag, or a more active switch? - What roles do residual Ag, FDCs, and T-cell signaling play in survival/death of B cells after primary responses? - How are memory cells selected for survival instead of death induction?

Question 49

Which model organism is mostly used to study TI B cell responses?

Answer 49

Nude mice, which have a mutation in Foxn1 that results in alopecia and the absence of a thymus (athymia), leading to no mature T cells. They can only response to TI antigens.

Question 50

Which regulatory mechanisms are there for B cells?

Answer 50

All the regulatory mechanisms are negative (as the activation pathways are already so regulated). - CD22 and FcγRIIb receptor shut down BCR signalling. - CD5 also negatively regulates BCR (and TCR signalling). - B-10 B cells act as negative regulators by secreting IL-10 that shuts down inflammatory responses by T cells and APCs.

Question 51

How are B cells regulated by CD22?

Answer 51

Shutting down BCR signaling may be necessary to stop proliferation when it is no longer required. This is done by CD22: a cell-surface receptor that contains Immunoreceptor tyrosine-based inhibitory motifs (ITIMs) which are similar to itams but mediate inhibitory functions instead of activating. The ITIM domain is phosphorylated upon activation, allowing association of SHP-1, a phosphatase that strips activating phosphate groups from BCR signaling molecules. This enables CD22 to act as a negative regulator of the B-cell immune response.