B Lymphocytes & Humoral Immunity - Detailed Notes
B Lymphocytes & Humoral Immunity
Learning Objectives
Understand the BCR complex structure and its role in B cell activation.
Describe the differences between T-dependent (TD) and T-independent (TI) antigens.
Know the functional significance and location of Follicular (B-2) B cells, Marginal zone (MZ) B cells, and B-1 B cells.
Understand initial B cell activation involves BCR cross-linking.
Describe the 3 main ways the BCR can be cross-linked by antigen.
Differentiate between TD and TI responses in B cells.
Understand B cell activation by TD antigens:
First and second signals.
Involvement of TH cells (contact and cytokines).
Understand B cell activation by TI antigens:
Signals provided for activation.
Understand the development of natural antibodies against TI antigens.
Describe the following processes, their significance, and which antigens allow them to occur:
Affinity maturation.
Isotype switching.
Differentiation into long-lived plasma cells and memory cells.
Describe how the humoral immune response is regulated (turned off).
Compare and contrast a primary with a secondary humoral immune response.
Understand X-linked agammaglobulinemia (XLA) and hyper-IgM syndrome concerning:
The specific defect.
How the disease is characterized.
B Cells & Adaptive Immunity
B cells are part of the adaptive immunity, working in conjunction with innate immunity.
Innate immunity provides the first line of defense:
Block entry, lysis, interferons: block and eliminate pathogens
Adaptive Immunity: Neutralization (toxin)
Defensins, lysozyme: Some Bacteria
Acute inflammation is managed by polymorphonuclear neutrophils (PMNs), while chronic inflammation involves mast cell activation.
Complement activation and antibody production enhance adherence and opsonization.
T cells provide help, and natural killer (NK) cells induce cytotoxicity.
B cells present specific antigens and differentiate into plasma cells.
Humoral Immune Response
Host defenses mediated by antibodies in plasma, lymph, and tissue fluids.
Effector functions of B lymphocytes.
Principal protective immune response against extracellular bacteria and foreign macromolecules.
Antibodies eliminate bacteria and neutralize toxins.
Deficiencies in B cells or antibody production increase susceptibility to recurrent bacterial infections.
Overview: B Cell Development
Three stages (similar to T cell development):
Maturation:
Begins with progenitor B cells expressing CD19.
Generation of mature, immunocompetent B cells.
Occurs in bone marrow.
Involves immunoglobulin-gene rearrangements.
Involves negative selection of self-reactive B cells.
Activation:
Interaction with antigen.
Occurs in secondary lymphatic tissue.
Differentiation:
Plasma cells or memory B cells.
Class switching.
Affinity maturation.
Maturation: BCRs & Somatic Recombination
BCRs are membrane-bound antibodies with the same extracellular structure but with a transmembrane domain.
Many B cells are needed as each B cell produces a BCR / antibody molecule with a single specificity.
Variable domains of BCRs are made up of 2 – 3 gene segments (V, J or V, D, J).
Different specificities for antigen result from:
Combinatorial diversity: combining V, D, and J domains to make the heavy and light chains (key enzyme RAG).
Junctional diversity: variations in the joining of gene segments (key enzyme TdT).
Maturation: Sequence of Events in BCR Formation
Heavy chain rearranged first:
Pro-B (progenitor B) cells have rearranged D and J to form DJ.
Pre-B (precursor B) cells have rearranged V and DJ to form VDJ.
Rearranged heavy chain is expressed on the surface of Pre-B cells with surrogate light chains.
Maturation: Pre-BCR
Rearranged μ heavy chain along with surrogate (temporary) light chains and Igα/Igβ are expressed.
Surrogate light chains are structurally homologous to κ and λ light chains but are invariant.
Expression of the pre-BCR complex allows for:
Continued survival and proliferation of pre-B cells (and apoptosis of those that do not).
Prevents the second μ heavy chain allele from undergoing recombination (if the first was successful).
Stimulates recombination of the light chain (remember there are two alleles - κ is always tried first, if not successful, tried second).
Inhibits synthesis of surrogate light chains
The cytoplasmic tail of the BCR is short and cannot signal (as was the case for the TCR). Igα & Igβ are analogous to CD3 + chains on the T cell
Immunoglobulin Gene Rearrangement
Immature B cells express H and L chains as mIgM. RAG (recombination enzymes) are key.
Cells which do not successfully rearrange their light chain will undergo apoptosis.
Maturation: Negative Selection of Self-Reactive Cells
A total of 4 light chain alleles are available for recombination (2 κ and 2λ alleles).
Immature B cells that express mIgM that bind with high avidity to self-antigen are deleted via apoptosis.
To avoid negative selection, some self-reactive immature B cells can be “rescued” by undergoing light chain editing = rearranging a different light chain allele to try and create a non-self-reactive BCR.
If successful, the immature B cell will escape negative selection.
Maturation: Alternative RNA Splicing
Once immature B cells pass negative selection, the cells alternatively splice heavy-chain primary RNA transcripts so that both IgM and IgD are created and expressed on the surface (this step is specific for B cells).
Expression of both IgM and IgD on the surface of the B cell defines this cell as mature.
This occurs after the B cell produced in the bone marrow has passed through the spleen.
Summary of B Cell Maturation
Generation of mature B cells occurs in stages that are defined by certain proteins expressed on the cell surface.
Progenitor B cell (Pro-B cell):
Initial expression of a common B cell marker, CD19
Precursor B cell (Pre-B cell):
Immunoglobulin heavy chain gene has successfully rearranged and is expressed on the surface with surrogate (temporary) light chain
Immature B cell:
Heavy and light chain gene have both successfully rearranged
Expression of membrane bound IgM (mIgM)
Negative selection
Exported from bone marrow to peripheral lymphoid organs
Mature B cell:
Expression of membrane bound IgM and IgD (mIgM & mIgD)
Summary: B vs. T Cell Development
Stage | Common to Lymphocytes | B Cells | T Cells |
|---|---|---|---|
Maturation | Generation of mature, immunocompetent cells | Involves gene rearrangements; negative selection of self-reactive cells; occurs in the Bone marrow (and spleen); Cell Types: Follicular, Marginal Zone, & B1B cells | Occurs in the Thymus; Cell types: CD4+ TH subtypes & CD8 cells CTL |
Activation | Occurs in secondary lymphatic tissue | Requires interaction with antigen (1st signal); Second signal required | |
1st signal: Antigen-BCR: Variety of antigens including protein (linear and conformational epitopes) & non-protein (carbohydrate, lipid) | 1st signal: Antigen-TCR Peptide antigens processed by APCs and presented | ||
2nd signal: VARIES: Leads to differential activation and different effector cells | 2nd signal: B7 – CD28 | ||
Differentiation | Effector & Memory Lymphocytes | Plasma cells In Follicular B cells: Affinity maturation, Class switching, Memory cells | TH, CTL Memory cells |
Activation of Mature, Naive B Cells
Activation requires antigen interaction with B cell in the peripheral (2o) lymphoid organs.
Spleen - blood-born antigens
Lymph nodes, MALT, & tonsils - tissue antigens
Without antigen, naive B cells will die within months by apoptosis.
Subsets of B Cell
Two signals required for B cell activation:
1st signal always involves antigen binding to the BCR.
2nd signal varies.
The type of antigen recognized by the BCR determines the nature of the 2nd signal, and the B cell response differs.
The 3 distinct subsets of B cells tend to:
bind different antigens,
activate with different signals, and
have different degrees of differentiation in response to antigen.
There are 3 distinct subsets of B cells classified as follows:
Follicular B cells:
In the follicles of the lymph node.
Respond primarily to T-dependent antigens presented via class II MHC on TH cells.
Marginal Zone (MZ) B cells:
In the marginal zone of the spleen.
Respond primarily to T-independent antigens (e.g., polysaccharide antigens or encapsulated bacteria).
First line of defense against blood-born pathogens.
B-1 B cells:
In the peritoneal and pleural cavities and mucosal areas.
Respond primarily to T-independent antigens (e.g., polysaccharide antigens or encapsulated bacteria).
First line of defense against pathogens in these locations.
Located in different peripheral lymph organs.
Specialize in recognizing either TD or TI antigens.
Follicular B Cell Migration
Mature, naïve follicular B cells migrate throughout the secondary lymphatic tissues in search of antigen.
As these B cells enter, they reside in & circulate through the follicles within the secondary lymphoid tissues
Follicular B cells are attracted to the follicles by CXCL13 = a B cell-specific chemokine.
CXCL13 is only produced in the follicle by follicular dendritic cells (fDCs).
If unactivated, B cells will exit following the S1P concentration gradient (similar to T cells).
Follicular B Cell Antigens
Antigens enter the secondary lymphoid organs from the lymph/blood, by crossing epithelial barriers, or carried in by dendritic cells.
Macrophages and dendritic cells present processed protein antigens in class II MHC molecules to TH.
Results in activated TH cells which can provide 2nd signal.
Soluble antigens enter the lymphatics and can be delivered to the follicles to directly bind to the BCR expressed by follicular B cells
Binding of soluble antigen binding to BCR results in 1st signal in three ways
Initiation of Follicular B Cell Activation: First Signal
Follicular B cells are initially activated upon antigen binding to the BCR.
As in T cells, there is a need to aggregate receptors in order to initiate signal transduction pathways needed to induce B cell proliferation.
Depending on the antigen, there are three ways receptor aggregation can occur:
Antigen binding and cross-linking BCRs.
Antigen cross-linking a BCR with the CR2/CD21 coreceptor.
Antigen cross-linking a BCR with a PRR.
The CR2/CD21 coreceptor also includes CD19 and TAPA-1 (CD81).
Complement (C3d) coated antigen crosslinks the BCR and the CR2/CD21 coreceptor.
Epitopes on the antigen can bind and signal the B cell through both the BCR and through PRRs such as toll-like receptors.
Events Occurring After First Activation Signal
Upon antigen stimulation, signal transduction pathways are initiated, generating active transcription factors that regulate the expression of various genes.
Results in:
Expression of CD69 (retention signal)
Synthesis of proteins to promote survival and proliferation
Expression of B7 and chemokine receptors
The follicular B cell is “preparing” for interaction with a helper T cell.
Follicular B Cell Migration Out of Follicle
Follicular B cells activated by the first signal migrate to the outer edge of the follicle into the T cell zone.
Now, the B cell can interact with an activated TH cell, or the B cell can activate antigen-specific TH cells if unactivated previously (remember, B cells are APCs!).
B cells will receive their second activation signal from TH cells.
Follicular B Cell Activation: 2nd Signal
The second signal for follicular B cell activation comes from both the interaction of the B cell with the TH cell and any cytokines produced by the TH cell:
CD40 - CD40L (CD154) interaction
TH cytokines: the specific cytokines vary depending on the TH subset
B cells can activate naïve TH cells first
Summary of B Cell Activation
T cell dependent (TD) antigens:
Proteins (or substances covalently bound to proteins) binding to BCR generates the 1st signal.
The second signal requires direct contact with T helper cells.
CD40-CD40L interaction + various cytokines
Some cells immediately differentiate into short-lived plasma cells secreting IgM, but TD activation of B cells also leads to:
Extensive class switching
Affinity maturation
Memory cell generation
Also involves cytokines secreted by TH cells
Follicular B Cell Differentiation
Immediately following activation, some B cells differentiate into antibody-producing plasma cells.
Location is outside of follicle (= extrafollicular focus)
Short-lived plasma cells that produce mainly IgM
Some activated B cells migrate back into the follicle (germinal center) and stimulate the differentiation of previously-activated helper T cells to become follicular helper T cells (TFH).
Germinal Center Reactions
Follicular B cells interact with fDCs and TFH cells.
These B cells will undergo affinity maturation:
Produce antibodies with the same specificity but a higher affinity for antigen (tested with the help of follicular dendritic cells).
Requires TFH cell help
These B cells will also undergo isotype (class) switching:
Switch from making IgM to another isotype
Requires TFH cell help
Some of these B cells will then develop into memory cells
Affinity Maturation
Affinity maturation is the process that leads to increased affinity of antibodies for a particular antigen.
The goal is NOT to change the specificity of the antibody but to improve the binding affinity of the antibody to the antigen.
Affinity maturation occurs by somatic hypermutation of the variable domains of both heavy and light chains.
These mutations are point mutations and occur at an extremely high rate!
Affinity Maturation
These point mutations are initiated by the enzyme AID = activation-induced deaminase, which is expressed in follicular B cells upon receiving the second activation signal from TFH cells.
Clinical Application: Vaccines
Many vaccines approved in the U.S. are inactivated, fractional (toxoids or subunits), or are recombinant vaccines that are very good at stimulating B cells, resulting in the development of antibodies and memory B cells.
These vaccines do not replicate inside of host cells.
Good inducers of B cells and result in high antibody titers and memory B cell production.
Most of these vaccines are composed of proteins or polysaccharides covalently bound to proteins.
Result After Affinity Maturation
These mutations are occurring in the proliferating B cells in the follicle, resulting in the generation of numerous follicular B cells, each having different mutations in the variable domains of the heavy and light chains.
Some of these B cells may now have an antibody with a higher binding affinity for the original antigen.
Some of these B cells may now have an antibody with a lower binding affinity for the original antigen.
Some of these B cells may now have an antibody that does not bind to the original antigen at all.
Therefore, these B cells that have undergone affinity maturation need to be “tested” to ensure that their BCR (mIg) can still bind to the original antigen with higher affinity.
These affinity-matured B cells undergo a second round of selection. This is the job of the follicular dendritic cells (fDCs).
Follicular Dendritic Cells (fDCs)
After somatic hypermutation, the follicular B cells interact with follicular dendritic cells (fDCs) displaying the antigen.
This process occurs in the light zone of the germinal center.
fDCs are not APCs but display antigen using FcRs and complement receptors.
Selection of High-Affinity Follicular B Cells
Follicular B cells expressing the highest affinity BCRs can competitively bind to antigen displayed by fDCs.
BCRs that do not have high affinity or can no longer bind antigen undergo apoptosis.
Only those B cells within the germinal center that can bind to antigen are selected to survive.
These activated B cells have a BCR with a higher affinity for antigen than they did before activation.
Results After Selection
After selection, the surviving follicular B cells also interact with TFH cells and can undergo class switching (isotype switching) prior to differentiation into plasma cells and memory B cells.
Class switching results in follicular B cells being able to produce a different isotype of antibody with the same antigen specificity!
Heavy Chain Class Switching
1st signal
2nd signal
In the absence of T cell involvement, follicular B cells secrete IgM.
With T cell involvement, the follicular B cell can switch which isotype is made, depending on the cytokine secreted:
IFN-: IgG
IL-4: IgE
TGF-: IgA
Isotype switching is directed by TFH cell contact and by cytokines.
Heavy Chain Locus
Recall: the mature B cell recombined the VDJ region during development to create variability in BCRs expressed by B cells.
Downstream of the VDJ segments are the various constant gene segments encoding for the constant domains of the mu, delta, gamma, alpha, and epsilon heavy chains.
The C constant region is closest to the VDJ region, which is why IgM is produced first in a primary immune response!
Production of other isotypes (other than IgD) requires recombining the VDJ region with a different constant region.
Isotype (Class) Switching
The CD40-CD40L interaction induces the production of AID = activation-induced deaminase, which also initiates class-switch recombination.
The VDJ region recombines with a downstream constant (CH) region, and the intervening DNA is deleted.
The recombination events occur at the switch (S) regions, located in the introns at the 5’ end of each CH gene segment.
These regions contain targeting sites for AID.
AID induces dsDNA breaks in the switch regions
Isotype (Class) Switching
Which downstream switch region recombines with Sε is dependent on the cytokines.
For example: IL-4 causes the Sε and S switch regions to recombine with the deletion of all intervening DNA, resulting in the Cε region being adjacent to the VDJ region.
This results in IgE synthesis.
Clinical Application: Hyper-IgM Syndrome
Mutations in the CD40L gene result in a disease called hyper-IgM syndrome, which is characterized by defects in antibody production, isotype switching, affinity maturation, and memory B cell generation.
Patients have extremely high levels of IgM in their serum with very low levels of other isotypes.
Patients also have defects in Cell-Mediated Immunity due to the CD40-CD40L interaction being important in the activation of macrophages too.
Patients are susceptible to many types of infections.
CD40L binding to CD40 is critical for all the above processes to occur!
Differentiation of Follicular B Cells
After affinity maturation and isotype switching occur in the germinal center, follicular B cells with high-affinity BCRs will differentiate into long-lived plasma cells and memory B cells.
Long-lived plasma cells reside in the bone marrow and produce antibodies for months to years after the antigen is no longer present.
Memory B cells enter the recirculating lymphocyte pool.
Effector Functions of Plasma Cells
Long-lived plasma cells migrate to the bone marrow and continue to secrete antibodies for months-years.
Depending on the antibody isotype, the antibodies participate in the elimination of extracellular microbes and their secreted products (such as toxins).
Opsonization: IgG
ADCC: IgG and IgE
Complement activation: IgM and IgG
Neutralization: All Igs
Mast cell degranulation: IgE
Regulation of the Humoral Immune Response
As in T cells, there needs to be a way to turn off B cell activation and proliferation. One mechanism is through antibody feedback.
As the immune response develops, many antigen-antibody complexes form.
Antigen-antibody complexes can bind to FcRs on the B cell and cross-link the FcR with the BCR, resulting in the activation of inhibitory signaling pathways to prevent further B cell activation.
Memory B Cells
A fraction of activated follicular B cells do not differentiate into plasma cells but instead become memory B cells.
Populations of memory cells, each specific for a given antigen, circulate in the blood for months to years.
Ready to respond rapidly if antigen is re-encountered.
Memory B cells express a membrane-bound BCR that will be of the same isotype as generated in the primary response.
Example: If the follicular B cell class-switched to IgG during the primary immune response, then the memory B cell will express IgG as a BCR.
There are more IgG-expressing memory B cells than other isotypes.
Memory B cells have a BCR, can become activated in the same manner, and will differentiate into plasma cells that secrete antibodies with a higher affinity for antigen.
Primary vs. Secondary Follicular B Cell Responses
Review: Primary immune response:
Activation of naïve B cells
Synthesize IgM first (short-lived plasma cells), affinity mature and class-switch to synthesize IgG, IgE, or IgA (long-lived plasma cells)
Memory B cells are produced
Secondary immune response:
Faster activation of memory cells
Synthesize IgG (or others); no IgM made
Affinity maturation occurs creating higher affinity antibodies
More memory B cells are produced.
Review: Subsets of B Cell
There are 3 distinct subsets of B cells classified as follows:
Follicular B cells:
In the follicles of the lymph node
Respond primarily to T-dependent antigens presented via class II MHC on TH cells
Marginal Zone (MZ) B cells:
In the marginal zone of the spleen
Respond primarily to T-independent antigens (e.g., polysaccharide antigens or encapsulated bacteria)
First line of defense against blood born pathogens
B-1 B cells:
In the peritoneal and pleural cavities and mucosal areas
Respond primarily to T-independent antigens (e.g., polysaccharide antigens or encapsulated bacteria)
First line of defense against pathogens in these locations
Located in different peripheral lymph organs
Specialize in recognizing either TD or TI antigens
B Cell Activation: Two Signal Hypothesis
Similar to T-cells, B cell activation requires 2 signals:
First signal = binding (or recognition) of antigen by the BCR complex
Unlike T cells, the specific second signal varies and depends on the nature of the antigen
Shown below protein antigen with Helper T (TH) cells and cytokines which are providing second signal but this is limited to protein antigens
For non-protein antigens, what does the 2nd signal look like?
TI Antigens & B Cell Activation
T cell independent (TI) antigens:
Nonprotein antigen + BCR = 1st signal (polysaccharide, lipid, nucleic acid)
Second signal varies but involves co-receptor
No direct contact with TH cells but may bind cytokines produced by TH cells
Two types of TI antigens:
TI-1 (top figure): non-protein binding to a Toll-like receptor (TLR).
CR2 (complement receptor for C3d) also used for C3d coated antigens
TI-2 (bottom figure): non-protein, multi-valent antigen → extensive BCR cross-linking
No additional receptor involved
MZ & B-1 B Cell Activation
Upon receiving first and second signals by a TI antigen, B cell activation occurs, resulting in survival and proliferation of these B cells by activating signal transduction pathways
As there is no T cell input, these B cells differentiate into short-lived plasma cells and secrete IgM antibodies with relatively low affinity.
Allows for a rapid, initial antibody response to bacterial polysaccharides.
Clinical Application: XLA
Mutations in Bruton’s tyrosine kinase (Btk) result in a disease known as X-linked agammaglobulinemia (XLA).
Btk plays a crucial role in the ability of B cells to synthesize and express a BCR.
The specific block is in the differentiation of pre-B cells into immature B cells within the bone marrow.
Pre-B cells will undergo apoptosis.
Disease is characterized by a lack of B cells, a lack of Igs in serum, and no germinal centers in lymphoid tissues.
B Cell Activation: Summary
Activation of B cell subsets occurs differently:
Follicular B cells respond primarily to TD (protein) antigens and result in:
Their ability to process and present antigens to TH cells in order to activate and/or stimulate TH cells to provide signals to the B cell
Differentiation into subpopulations of plasma cells, some of which are short-lived while others are long-lived
Some B cells will undergo isotype switching
Some B cells will undergo affinity maturation
Development of memory B cells
Marginal zone B cells and B-1 B cells respond primarily to TI (non-protein) antigens and result in:
Differentiation into plasma cells that are generally short-lived
Very limited class-switching may occur, but usually just IgM is produced
Very limited memory B cell production occurs, but often no memory
Lecture Summary
B cell maturation
Somatic recombination of immunoglobulin genes
Heavy chain rearrangement & testing with surrogate chain
Light chain rearrangement, testing, and editing
Expression of IgM and IgD BCR
Overview of B Cell Activation
Types of BCR antigens (T-dependent vs. T-independent)
Subsets of B cells (Follicular, Marginal Zone, B1B)
Activation of Follicular B cells
Migration of naïve Follicular B cells
1st signal provided by antigen binding with BCR by cross-linking with
Other BCRs
Complement receptors
PRRs
Results of 1st signal:
CD69, B7, proliferation signals, cytokine & chemokine receptor expression
Migration out of follicle to receive 2nd signal: interaction with TH
Differentiation of Follicular B cells after TH activation
Generation of short-lived plasma cells producing IgM
Migration back into follicle for Germinal center reactions (TFH)
Affinity maturation: Improves affinity (contributes to the efficacy of vaccines)
Testing matured Ab for ability to bind antigen
Class switching to match the type of antigen
Cytokines and subsets of TH
Differentiation into plasma cells expressing antibody isotypes
Memory cell differentiation
Review of effector functions of plasma cells
How do antibodies promote immunity?
Regulating humoral immune response
How B cells activation is down regulated (antibody feedback)
TI antigens (lipids & carbohydrates)
Marginal zone B cells
Marginal zone of the spleen
Specialize in blood born pathogens
B1B cells
MALT, pleural & peritoneal cavities
Lipid & polysaccharide antigens
Sample Question
Why are TI antigens unable to involve helper T cells during the process of B cell activation?
A. TI antigens are not displayed in MHC molecules
B. TI antigens are too large to bind to the TCR
C. TI antigens are processed internally and can only stimulate CTLs
D. All of the above
Sample Question
Why do B cells start expressing B7 (CD80/86) when they are responding to protein antigens?
A. B cells need to bind CD28 on macrophages to receive their second signal.
B. B cells are APCs and can activate helper T cells.
C. B cells need B7 in order to proliferate.
D. B cells use B7 to inhibit the proliferation of T cells.