Lecture 6: Generation of Antibodies

B-Cells

• B-cells must differentiate into plasma cells to produce antibodies

  • B-cells themselves do not secrete antibodies

  • Plasma cells, derived from antigen-specific B-cells, are the true antibody-producing cells

• Antibodies are made by the B lymphocyte lineage

• "B" stands for Bursa (of Fabricius) — a lymphoid organ in chickens where antibody-producing cells develop

  • Mammals don’t have a bursa, but the name stuck

• Cells originate in the bone marrow then migrate to secondary lymphoid tissues (e.g. lymph nodes, spleen)

• Naïve B- and T-cells circulate via the blood and enter lymph nodes through high endothelial venules (HEVs)
  → This ensures they’re in the right place to encounter antigens

Plasma Cell

• Very specific cellular structure

• Heterochromatic nuclei as large areas of the DNA are open, as the plasma cells are cells that pump out protein to generate lots of Ab

• Have lots of Golgi apparatus and ER to allow them to keep processing proteins

• Described as having a cartwheel-shaped nucleus and an unusual cellular make-up due to their specialised function in producing antibody

Development of Antigen-Specific B-cells

• B cells develop in the bone marrow, where they:

  • Rearrange immunoglobulin genes (antigen-independent)

  • Are supported by specialised stromal cells

• They express their rearranged immunoglobulin as membrane-bound IgM molecule, as a B-cell receptor (BCR)

  • This IgM can also be secreted later as an antibody

• If a BCR/ antibody binds strongly to self-antigens, the B cell is eliminated

  • This prevents autoimmunity and self-reactivity

• Upon maturation, B cells also begin to express IgD alongside IgM

  • Both have the same antigen specificity

Key stages in B-cell development and associated markers

• B-cell progenitor develops into a pro-B cell, which expresses CD19

  • CD19 is a clinical marker used to identify B cells (e.g. in flow cytometry)

• Pre-B cells begin to express IgM (via rearranged Igu heavy chain)

• This gives rise to an immature B cell with membrane-bound IgM

• Matures further into a mature B cell, expressing both:

  • Membrane IgM

  • Membrane IgD

  • Both have the same antigen specificity

B-Cell Fate After Development

• Cells leave the bone marrow and move around the body to populate secondary lymphoid organs and re-circulate

• When they encounter their specific antigen in the lymph nodes (in the cortex), they proliferate and eventually differentiate into plasma cells and long-lived memory B-cells (respond more quickly upon secondary challenge/infection)

B-Cell Development: Fate of Cells

• B-cells, following specific antigen recognition, undergo clonal proliferation

• It differentiates into:

  • Plasma cells that produce antibodies

  • Memory B-cells for long-term immunity

• All resulting cells retain the same antigen specificity

B-Cell Activation

• Require 2 signals for activation:

  1. Signal 1: Antigen recognition via membrane-bound Ig (IgM/IgD)

  2. Signal 2: Usually provided by CD4+ T cells (T cell-dependent activation)

T-Cell Independent Stimulation

• Certain antigens (e.g. bacterial polysaccharides) can directly activate B cells without T cell help → deliver strong enough antigens to stimulate B-Cels without T-cells

• Triggered by repetitive antigen structures on the pathogen surface → BCR activated as it sees ‘lots of the antigen close together’

• Results in low-affinity antibodies, as there is no affinity maturation

T-Cell Dependent Activation

• T-cell expresses CD40 ligand on its surface, which can ligate CD40. Of the B-cell to help it become activated

• T-helper cells can also produce cytokines e.g. IL-2 which can assist in B-cell proliferation and differentiation

• Conformational antigen recognition by B-Cell

Where Do B-Cells Meet Antigen

• B-cells meet antigens in the lymph node

• B-cells localised in the cortex (T-cells in the paracortex), in the absence of an antigen-specific response

• B-cells are in close proximity to subcapsular macrophages  in the subcapsular sinus

• B-cells near antigen-presenting cells

Antigen Recognition in the Lymph Node → Role of Macrophages

• Antigen entering lymph nodes or spleen are collected by specialised macrophages in the marginal zone or subcapsular sinus

• These macrophages preserve the conformational epitopes of antigens so that:

  • B cells in the cortex can directly recognise them via BCRs (no breakdown needed)

  • In contrast, T cells recognise peptide antigens presented by MHC II on dendritic cells

• Physical conformational antigens are often repetitive in structure, leading to simultaneous engagement (cross-linking) of many BCRs → recognition by many B-Cells at the same time

  • This cross-linking is crucial for B-cell activation

  • Macrophages help present antigens in a way that enables this cross-linking - need more that one BCR (Ig) on the surface that must be stimulated or cross linked

Marginal Zone Macrophages in the Lymph Node

• Located in the cortex close to where the B-cells are present

Actions Following B-Cell Activation

• Activated B cells move toward the border of the cortex and paracortex

• At the same time:

  • Dendritic cells (DCs) present peptide antigens via MHC II to CD4+ T cells in the paracortex, activating them

  • T cells and B cells that recognise the same antigen are activated simultaneously but via different forms (conformational vs peptide)

• Eventually, activated B cells and CD4+ T cells meet at the cortex-paracortex border for further interaction and differentiation

• 📍 Reminder: The paracortex is where CD4+ T cells are activated

Germinal Centre Formation

• Activated B and T cells first form primary foci in the medullary cords of the lymph node

  • These are areas of initial proliferation

• From the primary foci, some activated B cells migrate back into the cortex, entering primary follicles

• This leads to the formation of germinal centres within the follicles, where B cells undergo further maturation

How Do B-Cells Interact With T-Cells

• B cell binds antigen (e.g. from a macrophage), internalises the antigen, and processes it to become activated

• B cell then expresses MHC Class II with the processed peptide on its surface

  • B cells are one of the few cells that can produce MHC Class II!

• Meanwhile, T cells activated by dendritic cells recognise the same MHCII–peptide complex on the B cell

• This allows B cell–T cell interaction, enabling further B cell activation and support

T-Cell Co-Stimulation

• T-cells help the B-cells to drive the process forward and enhance the proliferation process

• B-cell bound to a viral coat protein and will then internalise and degrade the viral coat protein

• Peptide from this viral particle will be expressed by MHCII on the B-cell surface that interacts with a T-follicular helper cell, which can help to activate the B-cell by giving co-stimulation to the B-cell via CD40 ligand

T-Follicular Helper Cell (CD4+)

• If these cells have the right specificity, they deliver the second signal to B cells by recognising the peptide–MHCII complex presented by the B cell

• This Tfh–B cell interaction assists with antibody production

• Ensures only T cells with matching antigen specificity provide help to B cells, maintaining specificity in the immune response

T-Helper Cells Role in B-Cell Differentiation

• Helper T cells adhere to B cells and engage in CD40–CD40L interactions, initiating synthesis of IL-4, a cytokine important for B cell differentiation

• After antigen-specific recognition, the T cell cytoskeleton rearranges, directing the secretory apparatus toward the site of B and T cell interaction

  • This can be visualised using Talin staining

• IL-4 is selectively and directionally released toward the B cell, creating a high local concentration to ensure effective signalling

Clonal Expansion and Differentiation of B-Cell

• Occurs in response to a series of interactions of the antigen-specific B-cell.

• It involves the following signals:

  • co-stimulatory molecules eg CD154 (CD40L) on the T cell and CD40 on the B cell

  • Cytokines from the T follicular helper cell

• The results of these interactions in B-cell proliferation

Consequences of B-Cell Proliferative Events

• Some activated B cells become plasma cells/plasmablasts, rapidly secreting IgM

  • IgM is the first antibody produced against an antigen

  • It has low affinity and hasn’t undergone affinity maturation

• B cell proliferation, affinity maturation, and class switching occur after activation

  • Class switching allows plasma cells to produce other antibody types, e.g. IgG

How do activated B cells and Tfh cells contribute to affinity maturation in the lymph node?

• After 4–7 days, some activated B cells and T follicular helper (Tfh) cells move to the cortex of the lymph node

• They enter primary follicles, which are specialised regions

• Primary follicles contain Follicular Dendritic Cells (FDCs) – a unique type of antigen-presenting cell important for affinity maturation

Folicular Dendritic Cells

• These cells, not derived from haematopoietic origin, form a network throughout the primary follicle

• They are specially designated to hold antigen/ antibody complexes on their surfaces in small nodules - iccosomes

• The antibody intially comes fro the plasma cells in the extra follicular region of the cortex

Role of FDCs and Germinal Centres in Antibody Response

• FDCs in primary follicles hold antigens for extended periods to provide a sustained source of antigens for B cells.

• This supports the next stage of the antibody response: the formation of Germinal Centres.

• FDCs display antigens to B cells over time, helping maintain stimulation and supporting affinity maturation.

Germinal Centres

• Are the site of affinity maturation, where antibodies go from low to high affinity.

• They have a defined structure:

  • Centroblasts: proliferating B cells that express IgM

  • Centrocytes: non-dividing B cells that undergo selection

Affinity Maturation

• Activated B cells entering primary follicles down-regulate their Ig membrane receptors and proliferate (extensively) into centroblasts.

• During proliferation, B cells undergo affinity maturation, leading to high-affinity antibody production.

• As the centroblasts divide, affinity maturation involves hypermutation of heavy (H) and light (L) chains of the Ig molecule, randomly altering the structure of hypervariable regions.

• This process results in antibodies with either higher or lower affinity for the antigen.

What happens during the selection of high-affinity B cells in the germinal centre after affinity maturation?

• Centroblasts stop dividing and re-express surface Ig, becoming centrocytes.

• They compete for binding to antigens displayed by FDCs in the follicle.

• If centrocytes bind antigen with high enough affinity, they receive a survival signal; otherwise, they undergo apoptosis.

• Only B cells secreting high-affinity antibodies survive; low-affinity antibodies are still produced in small amounts.

• As antigen levels decrease with the progressive immune response, only B cells with higher affinity antibodies are selected.

• Affinity maturation can increase antibody affinity by 10,000 to 100,000 fold!

  • a small amount of low affinity Abs are still produced

Fate of Centrocytes After Selection in the Germinal Centres

• Surviving centrocytes interact with activated T helper (Th) cells again.

• They differentiate into:

  • Plasma cells – secrete large amounts of high-affinity antibody.

  • Memory B cells – provide long-term protection during secondary infection.

Structure of The Germinal Centre

• Dark zone – contains proliferating centroblasts.

• Basal light zone – contains selected centrocytes and Follicular Dendritic Cells (FDCs).

• Apical light/ Mantale zone – site of plasma cell and memory B cell differentiation.

Class Switching

• Occurs during the centroblast.centrocytes stage of B-cell differentiation process

• The B cell is able to change its heavy chain constant region from µ (IgM) to γ (IgG) or α (IgA) or ε (IgE) whilst keeping the same heavy chain variable and light chain (the antigen binding parts)

  • Changes the ‘section’ that binds to a cell

• Allows the generation of Abs with the same affinity but is a different class

Control of Class Switching

• It is mediated by CD4 T helper cells and cytokines

• Without CD40/CD40L only make IgM

• Different cytokines induce the production of different antibody classes e.g. IL-4, produced in response to allergens or worm infections induces IgE – effective in these defences

• N.B. cytokines also influence how much antibody is made

Combinations of Cytokines That Can Promote Class Switching

• Sub-combinations of cytokines that are effective in inducing Ab classes

  • IL21 + IL4 induce IgG1

  • IL21 Induces IgG3

  • IL13 gamma induces IgG3

  • IL10, IL21 +TGFb induce IgA

  • IL4 and IL13 induce IgE

• INF is effective at inducing

Plasma Cells and Memory B-Cells

• Cells formed in the final stage of differentiation

• Plasma cells secrete large amounts of antibodies (>2000 Ab molecules per second!)

• Memory B cells can survive for long periods – they have undergone class switch and affinity maturation – so when they see antigen for a 2^nd time, they can respond with a very quick and efficient response

Formation of Memory B-cells

• Occurs in the apical light zone of the germinal centre.

• When a B cell interacts with a CD4+ T follicular helper (Tfh) cell,

  • CD154 (on Tfh) binds CD40 (on B cell).

  • This interaction drives the B cell to become a memory B cell.

• These memory B cells can then exit the lymph node and enter circulation.

Why Do Some B-Cells Become Plasma Cells and Some Become Memory Cells

• We do not fully understand

• It’s suggested that in the absence of CD154–CD40 interaction, the default pathway is plasma cell differentiation.

• Plasma Cells can be:

  • short-lived (remain in lymph nodes/spleen, secrete Ab for a few weeks).

  • long-lived (migrate to the bone marrow, secrete Ab for months).

• The bone marrow is the main source of long-term antibody production — lots of Ab found in the body is derived from the long lived plasma cells → provides protection against invading pathogens.

Process of Germinal Centre Formation

• Occurs 4-14 days after antigen encounter

• 1. Antibody Class Switch

  • Cell surface IgM or IgD changes to IgG, IgA or IgE

• 2. Affinity Maturation of Antibody

  • Select for antibody with high affinity

• 3. Differentiation of B cells in memory cells

  • have undergone class switch and affinity maturation but not differentiated into plasma cells

• 4. Differentiation of B cells into Plasma Cells