B Cell Development Flashcards
____________ are the “traditional” B cells we think about when we discuss antigen activation and antibody production
B-2 Follicular B cells
B-2 Follicular B cells are referred to as follicular because….
They are the only subset that can enter splenic and lymphoid node follicles due to the high levels of IgD on their surface
The other recognized subsets are B-1 B cells and B-2 Marginal zone B cells which may have a small amount of _______ but do not ______________________
IgD; recirculate in/out of the follicles
B-1 B cells and B-2 marginal zone B cells are enriched with ___________________ and respond to _____________________________.
BCRs that recognize self-antigens and respond to T-independent antigens rather than T-dependent ones
Which subset of B cells has the most diversity of BCRs?
Follicular B cells
B cell development is intimately tied to ___________________.
The rearrangement of H and L chains
B cell development occurs in _________________ and is dependent on _____________________________________________.
The Bone marrow;
Dependent on bone marrow stromal cells and soluble factors (such as cytokines and growth factors) produced by these stromal cells
What is meant by the statement that “B cell development is independent of antigen”?
The initial, antigen-independent phase of B cell development generates mature, immune competent B cells that can bind to a unique antigen
- survival of the developing B cells in the initial stages depends only on successful H & L chain rearrangement
- after B cell is fully matured, it enters circulation “looking” for its cognate antigen (antigen dependent stage)
What happens to a B cell if Cognate antigen is not encountered?
Within a few weeks (approximately 2-8 weeks), the B cell dies by apoptosis
What happens to a B cell if cognate antigen is encountered?
If antigen is encountered, the B cell is activated —> clonal proliferation —> differentiation into plasma cell (antibody production) or memory cell
Pre-pro-B cell (undifferentiated leukocyte stage)
Several key proteins are expressed at this stage
- CD45R is a B cell lineage-specific molecule that marks the cell specifically as a cell for B cell lineage (as opposed to another lymphocyte- T cell or NK cell)
- EBF-1 (early B cell factor 1) is expressed at greater levels
- E2A (immunoglobulin enhancers-binding factor)
- EBF-1 + E2A promote accessibility of the Dh-Jh locus for upcoming recombination
Pro-B cell
- PAX5 transcription factor (necessary for Vh-Dh-Jh rearrangement) is expressed, and this prevents expression of non-B-cell lineage factors (ie T cell lineage genes)
- RAG-1 and RAG-2 (recombination-activating genes) enzymes are upregulated to initiate VDJ recombination
- TdT enzyme is initially expressed at this stage so N-nucleotide addition can occur between the genes
- CD19 is expressed- a B cell specific marker (in mature B cells, it can interact with other cell surface proteins to help activate the B cell)
- Transcription of Ig-alpha and Ig-beta (NOT SAME AS IgA)
- Successful rearrangement of the Ig μ chain indicates the end of the pro-B cell stage
RAG enzymes function
The RAG enzymes first orchestrate the Dh-Jh rearrangement step, followed by VhDhJh rearrangement to complete the V region of the H chain
- if either step in the rearrangement process is not successful, the cell dies by apoptosis
Pre-B cell- steps
Large Pre-B cell (early)
Small (resting) pre-B cell (late)
Large Pre-B cell (early)
Synthesis of the μ chain protein
Expression fo the μ chain on the surface with surrogate light chains λ5/14.1 and V preB, both of which are linked to the μ chain through S-S bonds
The μ chain, λ5/14.1, V preB, Igα and Igβ form what is called the pre-B cell receptor complex (pre-BCR complex)
Expression of the pre-BCR complex on the surface does 4 things:
It ensures that VDJ rearrangement (ie H chain recombination) on the other allele stops (i.e. allelic exclusion)
- downregulates RAG and TdT
Small (resting) pre-B cell (late)
Pre-BCR signals contribute to providing access of the L chain locus so VJ rearrangement of the L chain genes can occur
Immature B cells (in the bone marrow)
L chains pair with the μ chain, forming a complete BCR complex (IgM + Igα + Igβ) on the surface
Defining characteristic on an immature B cell is ___________________.
A fully assembled IgM BCR complex on the surface (IgM + Igα & Igβ)
Immature B cell (in bone marrow)- Negative Selection
Cells are now capable of interacting with antigen via the IgM BCR complex
Negative Selection:
Those that do NOT recognize self-antigen proceed to the spleen!
Those that DO recognize self antigen at this step have 3 possible fates:
1) apoptosis
2) anergy - lacks of reactin to foreign substances
3) Receptor editing
Receptor editing
Rearrangement of other L chain alleles to try and make a BCR that does not recognize self-antigen
- If successful, the cell goes to the spleen!
- If unsuccessful, the cell undergoes apoptosis
Splenic Immature B cell
Functional B cell with an IgM BCR goes to the spleen
More primary RNA is made (with both μ and δ genes) and alternatively spliced to produce IgD mRNA
Negative selection: (another round) occurs as the immature B cell percolates through the T cell zone of the spleen
- surviving immature B cells now express approximately 10 x more IgD BCRs than IgM allowing them to enter the splenic follicles
Positive selection: once in the splenic follicles, the immature B cells receive a signal to increase expression of BAFF-R
- if cells don’t receive the signal, or don’t upregulate BAFF-R, they will undergo apoptosis
Naive/Mature B cell (Follicular B cells) - name surface molecules and general function
The defining characteristic of mature, but naive, follicular B-2 B cell is the presence of both IgM and IgD on the surface
- After this point, changes to the BCR will occur in the secondary lymphoid organs and will be antigen-dependent
Ie Isotype switching and affinity maturation
Naive/mature B cells upregulate additional important surface molecules upon leaving the spleen:
CR1- interacts with C3b opsonin
CR2- CD21 which interacts with C3d
CD40- to interact with the Th cell
L-selectin- allows entry of B cells into specific tissues
The defining characteristic of mature, but naive, follicular B-2 B cell is ….
The presence of both IgM and IgD on the surface
Antigen-dependent development
If cognate antigen is encountered, the cell becomes activate and differentiates into either
- plasma cell- secretes ab’s
- memory cell- recirculates
If cognate antigen is not encountered within 2-8 weeks, the cell undergoes apoptosis
Antigen-dependent development:
Upon antigen recognition a B cell will…. (3)
- Make copies of itself (clonal expansion)
- Differentiate into plasma cells or memory cells
- Undergo SHM/affinity maturation and possibly isotype switching
B-1 B cells
Have very little IgD on the surface
They locate primarily to the peritoneal and pleural cavities (as they can’t enter lymphoid follicles)
They mainly produce IgM antibodies (they don’t interact with Th cells in the follicles which helps with isotype switching)
Mainly recognize microbial carbohydrate epitopes which results in T-independent response
Marginal Zone B-2 B cells
Have very little IgD on the surface
They locate primarily to the splenic marginal zone (as they can’t enter lymphoid follicles)
They mainly produce IgM antibodies (they don’t interact with Th cells in the follicles which helps with isotype switching)
Mainly activate in a T-independent manner
Antigen delivery to B-2 follicular B cells: where are the B-2 follicular B cells activated?
Secondary lymphoid organs
Antigen delivery to B-2 follicular B cells: What are the mechanisms by which this delivery of antigens to B cells can occur?
Small, soluble antigens enter the subcapsular sinus (afferent lymphatics) in lymph
- Smaller antigens are carried to the follicle via conduits
- Larger antigens are captured by subcapsular macrophages and brought to the follicle
Even Larger antigens or antigen-antibody complexes enter the lymph node and are captured by medullary dendritic cells (ie in the medulla)
Antigen delivery to B-2 follicular B cells: Are these antigens already processed when they are delivered to the B cells?
No. In all cases, the antigen is captured and left whole for BCR recognition
B cell activation signals: What comprises the BCR complex?
A membrane bound antibody (IgM + Igα +
Igβ or IgD + Igα + Igβ in a naïve, mature B cell)
What key roles does the BCR play in the activation of a naive B cell?
1) binding of antigen to the BCR
- when this occurs, the cytoplasmic tails of Igα and Igβ become phosphorylated, initiating the 1st signal needed to activate a naive B cell
2) Internalizing the antigen
- B cells internalize protein antigen and process them for presentation to T cells
Engagement of the BCR is rarely enough to activate a naive B cell- additional receptor engagement and signals are usually required- what are they and what do they do?
For T dependent antigens (ie proteins) signals from CD4 T helper cells are needed, but the threshold level of antigen can be lowered when other receptors/signals are involved
For T independent antigens (ie polysaccharides), engagement of additional receptors such as PRRs (ex TLR) or complement receptors are often needed to fully activate a B cell when T cells are not involved
CD21/CR2
Follicular B cells express small amounts of CD21 (aka CR2) whereas marginal zone B cells express high levels of this receptor
When complement is activated, C3 is cleaved to create C3a & C3b which binds to the pathogen. In certain circumstances, C3b is further cleaved into C3c & C3d.
It is C3d which remains attached to the pathogen that interacts with CD21 on the B cells.
When CD21 binds to C3d on the pathogen surface, this enhances the strength of the signal provided by Igα & Igβ of the BCR complex
TLR
B cells also express many different TLRs which can bind to microbial products and help with the activation of B cells
When a TLR is bound to a PAMP, the signal sent via the TLR enhances the signal sent via Igα & Igβ when the BCR is engaged by antigen.
- allows B cell to be activated at a lower threshold of antigen
Different types of antigens and how they activate B cell responses
Polysaccharide antigens contain multiple/repeating identical epitopes
B cell activation by TD antigens:
Full activation of the native B cell requires 2 signals:
1) Recognition of cognate antigen via the mIg and the 1st signal sent via Igα and Igβ- this creates a primed B cell
2) Once linked recognition occurs, CD40 (on the primed B cell) binds to CD40L (on the Th cell), providing the 2nd signal
B cell activation by TD antigens:
What does it mean when a B cell is primed?
Primed B cell internalizes, processes and expresses antigen on MHC II for T cells
Primed B cell migrates to the boundary of the follicle where it can interact with T cells in the T cell zone
B cell activation by TD antigens:
List the steps of B cell activation with T-dependent antigens, where it occurs, important cells, molecules, enzymes, etc in the process
1) Recognition of cognate antigen via the mIG and 1st signal sent via Ig-alpha and Ig-beta. This creates a primed B cell
2) Primed B cell internalizes, process, and expresses antigen on MHC II for T cells
3) During this time, DC’s have processed Ag and presented it to CD4 T cells in the T cell zone
4) The activated CD4 T cell differentiates into Thelper cell and upregulates CD40L
5) The Th cell migrates toward the follicle to interact with the primed B cell
6) Primed B cell presents antigen to the Thelper cell to ensure they recognize the same pathogen = linked recognition
- 2nd activation signal: interaction of CD40 on the B cell with CD40L on the CD4 Th cell fully activates the B cell
7) The Th cell secretes cytokines to provide “instruction” to the now activated, B cell
- SHM, isotype switching, differentiation- plasma/memory
8) The fully activated B cell and some Th cells (Tfh) move back into the follicle for B cell clonal proliferation
- this forms a germinal center
T-dependent activation: Isotype switching and Somatic Hypermutation (SHM) depends on ?
Isotype switching (aka: class switch recombination or CSR) and SHM are dependent on the enzyme activation-induced cytosine deaminase (AID)
A mutation in AID can result in a lack of isotype switching —> hyper IgM syndrome
What is the difference between a primary and secondary follicle?
Primary follicle- before immune stimulation
Secondary follicle = Germinal center- follicle after the B cell has undergone activation
What is a germinal center, where is it, and what happens there?
Within the follicle
Within the germinal center, the clones of the activated B cell will undergo affinity maturation and possibly, isotype switching if needed
- Tfh secrete cytokines to direct B cell activities
In addition, differentiation of the clones into either plasma or memory B Ellis
What type of T helper cells are in the germinal center and what is their role?
Tfh (T follicular helper) cells
Secrete cytokines which helps direct B cell activities
Describe the two different types of plasma cells.
Germinal center plasma cells have 2 possible fates:
1) Remain in the follicle to secrete class-switched antibodies for a few days (before undergoing apoptosis)
2) a small percentage migrate to the bone marrow to become long-lived plasma cells
What happens to the fully activated B cells that migrate back into the follicle?
The majority of the activated B cells move back into the follicle and form a germinal center (secondary follicle)
1) Proliferate (clonal expansion)
2) Undergo SHM and affinity maturation
3) May undergo isotype switching
4) Differentiate into plasma cells or memory cells
What is the role of the follicular dendritic cells for these cells?
Cells with their altered BCR receptors “check” their newly formed (mutated) receptors on antigens held by FDC’s
- those with higher affinity receptors competitively bind to antigen on the FDCs which then provide survival signals for these cells. Those with lower affinity receptors cannot compete and do not receive the survival signals
What is somatic hypermutation and why is it important?
Point mutations in the variable region of L and H chains aiding in affinity maturation ie increased affinity for antigen
Important because it is the ONLY mechanism of generating diversity AFTER antigen exposure
Where do plasma cells go to secrete antibodies?
Move to the medullary cords (LN) or red pulp (spleen) to secrete low affinity antibodies for 5-10 days
Germinal center plasma cells - remain in the follicle and secrete class-switched ab’s before undergoing apoptosis. Some of these will go to bone marrow to become long-lived plasma cells
Where do memory B cells “go” after a naive cell has been activated and differentiated into a memory B cell?
- Re-enter circulation to search for cognate antigen
- OR remain in lymph node/spleen
What happens when a memory B cell encounters cognate antigen again? Where will this encounter occur?
- Becomes activated
- Migrates to follicle to undergo the same changes as any activated B cell (SHM and affinity maturation, isotype switching, proliferation)
How is a memory B cell activated (what does it take to activate a memory B cell vs a naive B cell)?
- Must simply encounter antigen (1st signal ONLY)
- Does not require T cell help or interaction like a naive B cell would
How does an activated memory B cell know which isotype to make when its clones become plasma cells?
Model the receptors of the precursor B cell
?????
What types of antigens can activate B cells without T cell help (ie what kind of antigens are T-independent antigens)?
Marginal zone B-2 cells respond to polysaccharide antigens in the blood
- these responses initiated in the spleen
Antigens in the mucosa or the peritoneum activate the B-1 B cells
B cell activation by T-independent antigens results in … (5)
1) No memory B cells
2) Little isotype switching
3) Lower titer of antibody achieved
4) No affinity maturation- ie ab doesn’t improve
5) Subsequent challenge does not improve affinity or antibody titer
Why are these antigens T-independent?
- Activate B cells and stimulate antibody production without the involvement of CD4 Thelper cells ie not presented to T cells via MHC molecules
- High epitope density (cross links BCRs) + IFN-gamma (produced by cells involved in the innate response) provide a strong enough signal
What role does C3d play in reducing B cell dependence on T cells for activation?
- TI-2 antigen can be naturally bound by C3 complement products (C3d)
- C3d can then be bound by CD21 on B cell surface
- Antigen is then bound by both BCR and CD21, lowering threshold for activation (bypasses need for Thelper)
Two categories of T-independent antigens
TI-1 antigens (mitogens)- activate mature and immature B cells of any antigen specificity
TI-2 antigens- activate only mature B cells that are specific for that antigen (antigen-specific activation)
- high epitope density —> cross-linking of the BCRs
Where do these types of responses normally occur?
1) Blood- Polysaccharide antigens activate Marginal zone B-2 cells
2) Mucosa or Peritoneum- antigens activate B-1 B cells
What is the 2nd signal that is required for full activation of B cells to a T-independent antigen?
2nd signal comes from the IFN-gamma cytokine produced by innate immune cells responding to the infection
What is a mitogen? What kinds of things are mitogens?
- Can activate B cells through receptors that are not the BCR (ex. TLR)
- Tend to be components of a bacterial cell wall (ex. LPS)
What types of cells can be activated by a mitogen?
All B-cells (both mature and immature)
- Mitogen receptors are present and identical on all B cells (ex. TLR)
What is polyclonal activation? How does it differ from regular B cell activation?
Polyclonal activation —> at higher concentrations, mitogens can activate B cells regardless of antigenic specificity
- At higher concentrations, the TI-1 Ag can activate solely through receptors that are not the BCR ie TLR —> polyclonal activation
- At lower concentrations, the TI-1 Ag must also bind the BCR and therefore only activates Ag-specific B cells
What type of antibodies do you get from polyclonal activation?
Non-specific antibodies
Name some TI-2 antigens
Usually encapsulated polysaccharides but can be repetitive protein antigens such as flagellin
What is a TI-2 antigen? What are characteristics of a TI-2 antigen?
- Activate B cells using the BCR
- High epitope density causes extensive cross-linking of the BCR’s
What types of cells are activated by TI-2 antigens?
ONLY antigen-specific B cells (mature B cells)
What is the name of the immunodeficiency in which the individual cannot respond to TI-2 antigens?
Wiskott- Aldrich Syndrome
- highly susceptible to infection by encapsulated bacteria
B cell- CD4 T cell interactions:
Full activation of B cells requires two signal for T-dependent antigens- what are they?
1st signal: the appropriate antigen-specific T cell encounters the antigen on the primed B cell, the 2 cells interact for several hours (immunologic synapse)
- drives changes in the B cell: proliferation, SHM, differentiation, isotype switching, upregulation of CD80/86, downregulation of ligands holding the B cells in the germinal center (ex. L-selectin)
2nd (co-stimulatory signal for B cells): CD40 (on B cell) interacts with CD40L (on Thelper cell)
- required for affinity maturation, isotype switching, and differentiation of B cells into memory cells
What functions occur as a result of this interaction?
1) 1st signal: Increases expression of MHC II, CD80, CD86, on the B cell
2) 2nd signal: Required for isotype switching, production of memory cells, and affinity maturation
On what type of cell can CD40 be found?
B cells
On what type of cell can CD40L be found?
Thelper cells (CD4 and occasionally CD8)
What happens if one of these molecules is mutated or non-functional? (What is the result if this interaction fails to occur)
Hyper-IgM syndrome
- Undergo very little isotype switching
- mostly IgM B cells
What isotype is found on naive, mature B-2 follicular B cell?
IgM and IgD
What isotype is found on memory B cell?
Only one isotype
- can be be IgM, IgG, IgA or IgE (NOT IgD!!)
Describe how isotype switching occurs in B cells:
Movement of already rearranged Vh (VDJ) genes next to a new Ch gene (changes antibody class/function but NOT antigen specificity)
- each Ch gene (except δ) has a switch region (non-coding region in front) that regulates this process
- intervening DNA between Cμ (1st Ch gene) and the new Ch gene will be looped out and deleted
How does a B cell know which isotype to make?
Subsequent clones will not be able to make isotypes for Ch genes that were previously in front of new Ch gene (the other C# were deleted and their isotypes wont be made for it or future clones)
Describe hyper-IgM syndrome
Hyper-IgM syndrome can be caused by a number of things, but a mutation in CD40L is one of them and this form of the immunodeficiency is generally X-linked so the specific disease is called X-linked hyper-IgM syndrome
Mutation in the enzymes Activation-Induced Cytidine Deaminase (AID) can also result in a lack of isotype switching,also resulting in hyper-IgM syndrome
The inability to respond TI-2 (polysaccharide) antigens results in an immunodeficiency referred to as Wiskott-Aldrich Syndrome.
The exact mechanism of how this results in failure to activate T cells with an eventual decline in T lymphocytes, has not been elucidated
The significance of this defect in immune protection is that most human bacterial pathogens have a polysaccharide capsule and humoral immunity is the main mechanism of protection against these types of infection
In which stage of B cell development does antigen specificity become fixed?
Pre B cell stage
Where does B cell development occur?
• Bone marrow
What is the role of Igα and Igβ in the developing B cell?
• Ig alpha and beta- send 1st signal of B activation and are a part of the BCR complex
◦ Immature B cell- IgM BCR complex consists of IgM + Igalpha + IgBeta on the surface. At this stage, activates PI-3 kinase that sends a signal to keep the cell alive. Cells are now capable of interacting with Ag via the IgM BCR complex, but do not proliferate in response to antigen creating a primed B cell.
What is the role of Igα and Igβ in a mature B cell that has fully developed and left the spleen?
• Igalpha and IgBeta transmit a signal to the nucleus when the B cell binds antigen
How can you tell the difference between a mature B cell that is naïve and one that has experienced its antigen?
• They undergo antigen-dependent development
◦ Activated B cells undergo affinity maturation of their H and L chain CDRs
◦ Activated B cells undergo isotype switching to produce IgG, IgA, or IgE
What B cell activities occur in the germinal center?
• Affinity maturation and isotype switching
What role do Follicular Dendritic Cells pay in B cell maturation?
• secrete cytokines to provide “instructions” (SHM, isotype switching, memory vs plasma cell) to the activated B cells
Which surface molecule(s) provide the 1st signal for B cell activation and when does this occur?
• Ig alpha and beta due to recognition of cognate Ag by B cell
Which surface molecule(s) provide the 2nd signal for B cell activation and when does this occur, and where does this occur?
• CD40-CD40L interaction between B cell (CD40) and Th cell (CD40-L)
Which two (2) molecules on the surface of the B cell and T cell must interact to initiate B cell isotype switching and memory cell development?
• CD40-CD40L
Why do memory B cells not develop in response to T-indep antigens?
• Because no affinity maturation ??
Which genes are replaced during isotype switching?
Ch genes
Which genes are replaced during affinity maturation?
?????
Which genes are altered or mutated during affinity maturation?
• Same VJ and VDJ genes, but point mutations are introduced during clonal proliferation
Compare and contrast T-dep and T-indep antigens.
T ind antigens don’t have:
- No memory B cells
- Very little isotype switching
- Lower titer of antibody is achieved
- No affinity maturation - ab’s will not improve
- Subsequent challenge does not improve ab or titer
