B Cell Activation and Immune Response

• B cell activation differs from T cell activation, and can be initiated by either cell-bound or soluble peptide antigens.
• B cells use various methods to recognize antigens and activate an immune response.
• B cell activation strategy depends on whether the antigen is cell-bound or soluble.
• B cells largely require T cell activation, but can be T cell independent if antigens are soluble

B Cell Activation Signals

• Activation of B cells requires two distinct sets of signaling events.
• Signaling events are generated by different pathways in response to T cell independent or dependent antigens.

Diversified B Cell Activation

• The human immune system employs a diversified approach to B cell activation to combat the diversity of pathogenic antigens.
• B cells can initiate an immune response without T cells, and have a broader antigen recognition repertoire.
• Depending on the antigen, B cell activation can occur independent of T cells, falling into two groups: TI-1 and TI-2.
• Most B cell activation is supported by T cell contact.
• Both activation pathways require a two-step initiation.

Thymus-Dependent (TD) Antigens

• Structure: Protein-based, requiring processing and presentation to T-helper (Th) cells on MHC class II molecules.
• Humoral Response: Requires Th cell help for B-cell activation, resulting in isotype switching, affinity maturation via somatic hypermutation, and memory B cell generation.

Thymus-Independent (TI) Antigens

• TI-1 Antigens:
• Structure: Polyclonal activators like lipopolysaccharides (LPS), which bind non-specifically to Toll-like receptors (TLRs) or other pattern recognition receptors (PRRs).
• Activates B cells independent of antigen specificity and induces a polyreactive response, often producing low-affinity IgM.
• TI-1 antigens do not involve isotype switching, affinity maturation, or memory cell formation.

Polyclonal Activators

• Polyclonal activators stimulate multiple B cells or T cells regardless of their antigen specificity and bypass normal antigen-specific activation pathways.
• Examples include: Lipopolysaccharides (LPS), interact with Toll-like receptor 4 (TLR4).
• LPS are polyclonal activators because they nonspecifically stimulate a wide range of B cells regardless of their antigen specificity.

TI-2 Antigens:

• Structure: Highly repetitive, multivalent structures like polysaccharides.
• Directly cross-link B-cell receptors (BCRs) on the surface of specific B cells.
• Humoral Response: Primarily produces IgM antibodies, can induce limited isotype switching in the presence of cytokines, but does not typically generate memory B cells.

Competence Signal from Antigen Binding to mIg

• Binding of antigen to membrane-bound immunoglobulin (mIg) on B cells provides the first signal (Signal 1) for activation.
• Effective Antigens:
• Protein-based TD antigens: Require T cell help.
• Repetitive TI-2 antigens: Cross-link multiple mIg molecules.
• Signal Transduction Pathway:
• Antigen and mIg interaction clusters BCRs.
• Activation of signaling molecules (e.g., Igα/Igβ) transduces the competence signal, priming the B cell for further activation.
• TD antigens rely on Th cell help, while TI antigens directly activate B cells through BCR or PRR engagement.
• The antigen type dictates the humoral response and the presence of long-term immunity.

T Cell-Independent Activation

• T cell independent activation one (TI-1) occurs via antigens that are polyclonal activators.
• TI-1 Polyclonal activators bind to surface structures other than BCRs, such as lipopolysaccharides binding to TLR4.
• T cell independent activation two (TI-2) activates B cells via antigens containing repetitive epitopes that are often polysaccharides with repetitive epitopes
• Complement component three can bind to an antigen that is bound to the BCR, CD21 then binds to the antigen bound C3 component and activates the B cells.
• Repetitive epitopes can also cross link multiple PCRs that induce B cell activation.

B Cells as Antigen-Presenting Cells (APCs)

• B cells can act as APCs.
• The interaction between MHC class II complex and the TCR/CD3 complex acts as a second signal for B cell activation.
• The first signal is the interaction between BCR and antigen epitope.
• Co-stimulation molecules anchor cell interaction and provide additional B cell stimulation.
• Co-stimulation molecules are the same as previously covered during signal transduction.

Signal Transduction in Naive B Cells

• Naive B cells transduce signals induced by cross-linkage of mIg
• Activation of naive B cells begins with the recognition of antigens by the B cell receptor (BCR), which is the membrane-bound immunoglobulin (Ig) on the surface of B cells.
• After the cross-linking of receptors on the B -cell surface with antigen, the material is endocytosed and processed via the exogenous pathway to generate an MHC class II:peptide complex, which interacts with the TCR of naïve T cells.
• Cross-linking of membrane Ig by antigens initiates a series of signal transduction events that lead to B cell activation and differentiation.

B Cell Receptor (BCR) Signaling:

• Cross-linking of membrane Ig activates the intracellular domains of the Ig molecules.
• The intracellular domains of Ig contain immunoreceptor tyrosine-based activation motifs (ITAMs).

Syk Kinase Activation:

• The phosphorylation of ITAMs leads to the recruitment and activation of the Syk kinase (spleen tyrosine kinase).
• Syk plays a central role in transducing signals downstream of the BCR.

Phospholipase Cγ2 (PLCγ2) Activation:

• Syk activation leads to the phosphorylation and activation of PLCγ2.
• Activated PLCγ2 catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).

Calcium Signaling:

• IP3 triggers the release of calcium ions from intracellular stores, leading to a rapid increase in intracellular calcium leve
• The elevated calcium levels are crucial for downstream signaling events.

Activation of Protein Kinases:

• Calcium signaling activates various protein kinases, including protein kinase C (PKC). PKC and other kinases contribute to the activation of transcription factors and other signaling pathways.

• Nuclear Factor of Activated T Cells (NFAT) Activation:

• Calcium signaling activates NFAT, a transcription factor that translocates to the nucleus. NFAT, along with other transcription factors, induces the expression of genes involves in B cell activation and differentiation.

• B Cell Activation and Proliferation:

• The combined signaling events lead to the activation and proliferation of the B cell. Activated B cells can differentiate into plasma cells or memory B cells, depending on the context and the presence of co-stimulatory signals.

General Results of Signal Transduction in B Cells:

• Proliferation and Clonal Expansion: B cell activation triggers the clonal expansion of antigen-specific B cells.
• Differentiation into Plasma Cells: Some activated B cells differentiate into plasma cells to produce antibodies
• Memory formation: A subset of activated B cells differentiates into memory B cells
• Class Switching: Depending on the cytokine environment and co-stimulatory signals, B cells may undergo class switching.
• Antibody Secretion: B cells, particularly plasma cells, secrete antigen specific antibodies.

ITAMs and B Cell Activation

• When two B cell receptors get cross linked their intracellular chains, the side chains, Igalpha and Igbeta and C19, all clustered together.
• Each of these chains has something called an immune receptor tyrosine based activation motif, or ITAM.
• The ITAM is a conserved sequence of amino acids that includes two tyrosine amino acids, the tyrosines.
• Both tyrosines need to be phosphorylated for the cell to get activated.
• These two tyrosines within the ITAM region of the protein get phosphorylated by three protein tyrosine kinases called BLK, Fyn and Lyn.
• Once the two tyrosines Get phosphorylated, there's a chain of events within the cell that ends with the activation of the major transcription factors NF, kappa B and NF.
• These transcription factors turn on the expression of genes resulting in cytokines, like interleukin one, interleukin two, interleukin six and tuber necrosis factor alpha.
• They also cause up regulation of anti apoptotic cell surface markers like BCL two, as well as the proliferation and differentiation of B cells.
• CD 21 which can also stimulate a B cell.

CD 21 and Complement Fragments

• A B cell can be stimulated using CD 21 which is also called CR two.
• CD 21 the second receptor identified for one of the complement fragments, C, 3d in the complement pathway.
• A variety of complement fragments are produced. Many would combine, non specifically to antigens. C, 3d can bind to an antigen and can then be bound by CD 21 on a B cell.

Activation of B Cells Goal

• An activated B cell has one goal in mind; to differentiate into a plasma cell and secrete antibody, initially the B cell will differentiate into an IgM secreting plasma cell, because that's ready to go.
• Five classes of antibodies that the plasma cell secretes exist, IgM is the basis for the B cell receptor.

TD, TI-1, and TI-2 Antigens and Humoral Responses Summary

• Activation and differentiation of B cells in response to thymus-dependent (TD) antigens requires TH cell, whereas the B-cell response to thymus-independent (TI) antigens does not.

Thymus-Dependent (TD) Antigens

• TD antigens require the help of T cells for the induction of effective immune responses.
• Structure: Complex proteins with multiple epitopes and diverse structures.
• Processing: Processed by antigen-presenting cells (APCs) to present peptides on MHC class II molecules to CD4+ helper T cells.

Humoral Response:

• B Cell Activation: CD4+ T cells help B Cels through direct interaction and cytokine release.
• Ig Isotypes: Can induce class-switching to various immunoglobulin isotypes (IgM, IgG, IgA, or IgE).
• Memory Response: Effective memory B Cell formation for long-lasting immunity.

Thymus-Independent Type 1 (TI-1) Antigens:

• TI-1 antigens directly activate B cells without the need for T cell help.
• Structure: Microbial products with repeating epitopes (e.g., bacterial lipopolysaccharides - LPS).
• Processing: B cells can recognize the antigens directly without antigen processing by APCs.

Humoral Response:

• B Cell Activation: Direct activation of B cells without T cell help.
• Ig Isotypes: Primarily induces IgM production.
• Memory response: Limited memory B cell formation.

Thymus-Independent Type 2 (TI-2) antigens:

• TI-2 antigens activate B cells directly but require repetitive epitopes for optimal activation.
• Structure: Large and repetitive structures on the surface of pathogens (e.g., bacterial capsules).
• Processing: B cells recognize repetitive epitopes without antigen processing.

Humoral Response:

• B Cell Activation: Direct activation of B cells, often more effective with repeated epitopes.
• Ig Isotopes: Primarily induces IgM production; some class-switching to IgG or IgA may occur.
• Memory Response: Limited memory B cell formation.
• Protein-based TD antigens and repetitive TI-2 antigens provide effective competence signal for B-cell activation.

T Cell-Dependent B Cell Activation

• B cell uses its BCR to bind and internalize the antigen (usually in the form of a pathogen or pathogen-derived molecules).
• Internalized antigens is processed into peptides within the B cell’s endosomal compartment, and aided by enzymes like cathepsins.
• These peptides are loaded onto MHC class II molecules in the endoplasmic reticulum (ER).
• MHC class II-peptide complex is transported to the cell surface through the Golgi apparatus for presentation to CD4+ T cells.

First Signal: TCR-MHC II Binding

• The TCR on the CD4+ T cell recognizes and binds presented antigenic peptide on MHC class II molecules of the B cell providing the first signal for T cell activation.
• The CD4 co-receptor on the T cell binds to the MHC class II molecule on the B cell, enhancing the TCR-MHC interaction, and stabilizes the interaction between the T cell and B cell.
• TCR-MHC binding activates protein tyrosine kinases (e.g., Lck, Fyn) in the T cell membrane. and phosphorylate CD3 and zeta chain initiating the downstream signaling cascade.
• The phosphorylation of CD3 and zeta chain activates phospholipase C-γ (PLC-γ) which catalyzes the conversion of PIP2 (phosphatidylinositol 4,5-bisphosphate) into IP3 (inositol trisphosphate) and DAG (diacylglycerol).
• IP3 binds to the IP3 receptor on the endoplasmic reticulum (ER), causing the release of calcium ions from the ER. DAG activates protein kinase C (PKC), which is involved in further signaling within the T cell.
• The increase in intracellular calcium activates calcineurin, which dephosphorylates and activates the NFAT transcription factor which translocates to the nucleus, and initiates the expression of genes involved in T cell activation.
• The Ras/Raf/MEK/ERK MAPK pathway is activated by DAG and leads to phosphorylation and activation of the MAP kinases, and promotes expression of genes necessary for T cell proliferation and differentiation.
• PKC and DAG activate the NF-κB pathway, which involves the degradation of IκB proteins and the release of NF-κB dimers and translocates to the nucleus to promote gene expression necessary for T cell activation, survival, and cytokine production.
• Activation of these pathways culminates in the full activation of the T cell, allowing it to produce cytokines and to express CD40L (CD154).

Second Signal: Co-Stimulatory Molecule Interactions (CD28-B7, CD40-CD154)

• Co-stimulation through CD28-B7:
• CD28 (on T cell) binds to B7 (CD80/CD86) on the B cell to provide the second signal required for full T cell activation.
• B7 molecules on the B cell are upregulated after antigen recognition, the binding of CD28 to B7 activates PI3K leading to T cell survival, proliferation, and cytokine production.
• Co-stimulation through CD40-CD154 (CD40L) Interaction:
• CD40 (on B cell) binds to CD154 (CD40L) on the T cell to provide a critical second signal for B cell activation, differentiation, and class switching.
• The binding of CD40 to CD40L activates the NF-κB pathway and other signaling cascades in the B cell.

Signal Cascade in the B Cell (via CD40):

• CD40 signaling activates various kinases, including IKK (IκB kinase), leading to the activation of NF-κB.
• NF-κB translocates to the nucleus and promotes the transcription of genes involved in B cell proliferation, survival, class switching, and affinity maturation.
• Activated T helper cells secrete cytokines like IL-4, IL-5, IL-21, which act on the B cell to promote class switching (e.g., from IgM to IgG, IgA, or IgE) and the formation of high-affinity antibodies.
• The CD40-CD154 interaction, along with the cytokine signals, drives the activation of the B cell and B cell differentiation in to plasma cells or antibody production.
• LFA-1 (Lymphocyte Function-associated Antigen-1) on the B cell binds to ICAM-1 on the T cell.
• This strengthens the adhesion between the T cell and B cell, promoting a stable T-B cell interaction, and ensures efficient TCR-MHC II signaling. and supports the antigen presentation process.
• The T-B Cell Interaction LFA-3 on the B cell binds to CD2 on the T cell providing additional co-stimulatory signals to the T cell activation process.
• The production and circulation of antibodies B cells differentiate into plasma cells.
• Plasma cells are differentiated B cells that secrete a globulin.
• Plasma cells lack detectable membrane bound immunoglobulin or IG molecules.
• Plasma cells synthesize high levels of antibodies that are secreted as high as 1000 molecules of IG per cell per second.

B Cell Differentiation

• B cells can be activated by T independent or dependent mechanisms.
• As B cells receive a help signal from T cells, cytokines are released and facilitate B cell class switching.
• Class switching describes the ability of B cells to express or secrete different IG isotypes, by which some activated B cells differentiate into plasma cells and others into memory cells.
• Germinal centers are structures within secondary lymphoid tissues, and are sites for B cell activation, proliferation, and differentiation during the adaptive immune response and are essential for generating high-affinity antibodies and memory B cells.
• Germinal centers form in response to antigen stimulation.
• In the germinal centers, B cells encounter their specific antigen in the presence of T helper cells, then undergo activation and proliferate rapidly, forming the germinal center.
• Somatic Hypermutation: B cells undergo somatic hypermutation by introducing point mutations into the variable regions of their immunoglobulin genes which leads to B cells with slightly different antibodies.
• The B cells with the highest affinity for the antigen are selected for survival through affinity maturation.
• B cells in the germinal center also undergo class switching to change the type of antibodies they produce, depending on the cytokine signals from T cells.
• Some B cells differentiate into plasma or memory cells for secreting antibodies.
• Plasma cells lack detectable membrane bound immunoglobulin and specialize in producing and secreting large amounts of antibodies.
• Memory B cells provide long-term immunity.

B Cell Cytokine Summary

• Focus is on the different cytokines involved in activating B cell differentiation into plasma cells.
• Cytokine signals are needed for differentiation and are important for understanding dysfunction in diseases
• This process of proliferation and differentiation is occurring in the Light Zone of thymus germinal centers.
• Memory B cells retain a membrane bound immunoglobulin.
• Memory B cells are important cells that differentiate in a similar way to plasma cells.
• Memory B cells do not change RNA processing to maintain as a consequence of class switching, and express different isotypes.
• Memory B cells provide a role in recognizing, processing and presenting specific antigens.

Structure and Function of Plasma B Cells vs. Memory B Cells:

Plasma B Cells:

• Cell Morphology: Differentiated with abundant cytoplasm and well-developed endoplasmic reticulum.
• Specialized for high rate antibody production and secretion produce large quantities of antibodies with high specificity for the encountered antigen.
• Expression: Express surface immunoglobulins (antibodies) identical to those on the naive B cell
• Life Span: Short lifespan, for immediate immune response.
• Antibody Isotypes: Secrete antibodies of various isotypes, including IgM, IgG, IgA, or IgE, after class-switching events.
• Do not undergo clonal expansion after activation. derived from the clonal expansion of activated B cells.

Memory B Cells:

• Cell Morphology: Like naive B cells.
• Cell Surface Markers: Express surface immunoglobulins specific to the encountered antigen and memory B cell markers.
• Long-lived to provide immune memory and persist until reactivated.
• Quiescent state but can rapidly differentiate.
• May be restimulated small point mutations that accumulate in the DNA encoding variable regions of both light and heavy chains resulting in generation of an antibody with increased binding affinity for its epitope.
• Can secrete antibodies of various isotypes, and retain the ability to undergo class-switching upon reactivation.
• Location: Circulate in the bloodstream and proliferate for robust results.
• B cells lock with antigens ultimately leading to antigens death.

First Time vs Subsequent Antibody Response

Difference between antibodies secreted by plasma cells during first exposure or subsequent exposure lies in their class, affinity, quantity, and speed of production:

• Plasma Cells (First Exposure/Primary Response): Arise from naive B cells activated by the pathogen initially.
• Produce antibodies for immediate, short-term defense.
• Memory B Cells (Subsequent Exposure/Secondary Response): Arise from naive B cells after they have undergone affinity maturation during the primary response.
• Rapidly re-activate and produce antibodies faster upon subsequent exposure to the same pathogen.

Characteristics of Antibodies:

• Feature Plasma Cells (From Naive B Cell Origin) Memory B Cells
• Affinity: Lower to moderate and less optimized. Mainly IgM, at first exposure. Subsequent exposures have specific specificity.
• Diversity: Broad reactivity but less optimized. Specific to previously encountered antigens with main IgG features.
• Production for long-term immunity is faster more abundant and more specific.

Antibody and Speed:

• First Plasma Cells: Produce antibodies gradually during the first immune response, peaking after days to weeks.
• Is a slower activation resulting peak production speed.
• Later Memory Cells: Produce large quantities of antibodies quickly after re-exposure.
• Memory B Cells are long-lived and are ready faster for stronger and more specific responses later if activated. Plasma Responsible for producing antibodies during the primary immune response, these antibodies providing the first and weaker line of defense. Memory cells produce stronger antibodies during secondary immune. This is the basis for long-term immunity.