Cell Cooperation in the Antibody Response

Md Hammadul Hoque
Assistant Professor
Dept. of GEB, SUST-3114
Overview of the Chapter

• The principles of B-cell

– Generation and Maturation
– Activation
– Proliferation and
– Differentiation

• The generation of
• Plasma cells and
• Memory cells
Overview of B cell Development

• In adults
– B cell development occurs in the bone marrow
– does not require contact with antigen
– During this time the B cells
• rearrange the genes for their heavy and light chains, and
• synthesize cell surface IgM which acts as their antigen
receptor (BCR)
– The BCR complex includes:
• membrane-bound immunoglobulin (mIg)
• the signaling chains Igα and Igβ (CD79a and CD79b)
Overview of B cell Development

– Immature transitional B-cells

• exit the bone marrow and
• enter the periphery where they further mature in
secondary lymphoid organs
– Antigen encounter
• If does not occur
– die within a few weeks by apoptosis
• If encounter specific antigen
– undergo
» activation
» proliferation and
» differentiation
– leading to the generation of
» plasma cells and
» memory B cells
B cell generation and maturation

• The generation of B cells first occurs in the embryo and continues throughout life.
– Before birth
• yolk sac
• fetal liver, and
• fetal bone marrow
– After birth
• bone marrow
• B-cell development begins
– lymphoid stem cells differentiate into the progenitor B cell (pro-B cell)
• expresses a transmembrane tyrosine phosphatase called CD45R
– Pro-B cells
• proliferate within the bone marrow
• filling the extravascular spaces between large sinusoids in the shaft of a bone
B cell generation and maturation

i. Progenitor B Cells Proliferate in Bone Marrow

– Proliferation and differentiation of pro-B cells into precursor B cells (pre-B cells)
• requires the microenvironment
• provided by the bone-marrow stromal cells
– interact directly with pro-B and pre-B cells, and
– secrete various cytokines (IL-7)
• pro-B cells require direct contact with stromal cells
– This interaction is mediated by several cell-adhesion molecules
» VLA-4 on the pro-B cell and
» its ligand, VCAM-1, on the stromal cell (Figure 11-2)
 B cell generation and maturation

i. Progenitor B Cells Proliferate in Bone Marrow (Contd.)

– VLA4 (Very Late Antigen-4/Integrin α4β1)
– VCAM-1 (Vascular cell adhesion molecule-1 (CD106)
– C-Kit/SCFR (Stem cell growth factor

receptor/tyrosine-protein kinase KIT)

– SCF (Stem cell factor)
B cell generation and maturation

i. Progenitor B Cells Proliferate in Bone Marrow (Contd.)

– After initial contact is made
• c-Kit interacts with stem-cell factor (SCF)
• activates c-Kit (tyrosine kinase), and
• pro-B cell
– begins to divide and differentiate into a pre-B cell and
– begins expressing a receptor for IL-7
» drives the maturation process
» inducing down-regulation (decrease of sensitivity) of the adhesion molecules on the pre-B cells
» proliferating cells can detach from the stromal cells.

– pre-B cells
• no longer require direct contact with stromal cells
• continue to require IL-7 for growth and maturation
B cell generation and maturation

ii. Ig-Gene Rearrangment Produces Immature B Cells

– B-cell maturation depends on rearrangement of the immunoglobulin DNA
– First to occur in the pro-B cell stage heavy-chain gene rearrangement
• DH-to-JH gene
• VH-to-DHJH gene
• VH-DH-JH rearrangement continues on the other chromosome
– Upon completion of heavy-chain rearrangement, the cell is classified as a pre-B cell
– Continued development of a pre-B cell into an immature B cell
• requires a productive light-chain gene rearrangement
– Because of allelic exclusion, only one light-chain isotype is expressed on the membrane of a B cell.
B cell generation and maturation

ii. Ig-Gene Rearrangment Produces Immature B Cells (Contd.)

B cell generation and maturation

ii. Ig-Gene Rearrangment Produces Immature B Cells (Contd.)

– Completion of a productive light-chain rearrangement

• commits the now immature B cell to a particular antigenic specificity

– Immature B cells express mIgM (membrane IgM) on the cell surface

– Two recombinase enzymes RAG-1 and RAG-2

• required for both heavy-chain and light-chain gene rearrangements

• are expressed during the pro-B and pre-B cell stages

– Enzyme terminal deoxyribonucleotidyl transferase (TdT)

• catalyzes insertion of N-nucleotides at the DH-JH and VH-DHJH coding joints

• active during the pro-B cell stage

• ceases to be active early in the pre–B-cell stage

B cell generation and maturation

iii. The Pre–B Cell Receptor Is Essential for B-Cell Development

– In the pre-B cell

• the membrane chain is associated with the surrogate light chain

• a complex consisting of two proteins

– a V-like sequence called Vpre-B and

– a C-like sequence called λ5

– Only pre-B cells

• are able to express membrane bound heavy chains in association with surrogate light chains

• able to proceed along the maturation pathway

B cell generation and maturation
B cell generation and maturation

iv. Cytokines required for B cell development

– Common lymphoid progenitors (CLP) are responsive to IL-7
• promotes B-cell lineage development
– Blys (B-lymphocyte stimulator) signaling through its receptor BR3
• survival of pre-immune B-cell stages from transition stage onwards
• required for the later survival of mature B-cells
– Cytokines that are important in initiating the process of B-cell differentiation
• IL-4
• IL-3 and
• Low-molecular-weight B cell growth factor (L-BCGF)
– Some other factors are active in the later stages
 B cell generation and maturation

v. Transcriptional controls in B-cell development

– B-lymphopoiesis is regulated by multiple transcription factors (Nearly a dozen) including:
• E2A and Early B-cell Factor (EBF)
– do not express RAG-1
– are unable to make DHJH rearrangements, and
– fail to express λ5
• Pax5 (B-cell– specific activator protein (BSAP))
– restricting transcription of lineage inappropriate genes
– activating expression of B-lineage signaling molecules;
• Sox4 and LEF1
– promote the survival and proliferation of pro-B cells
• IRF4 and IRF8
– terminate pre-BCR signaling by IRF4
– promote differentiation to small pre-B cells;
• Bcl-6 expression
– required for germinal B-cell differentiation and generation of memory B-cells.
B cell generation and maturation

vi. Cell-Surface Markers Identify Development Stages

– Pro-B stage
• CD45R (tyrosine phosphatase)
• Ig-α/Ig-β (signal-transducing molecules)
• CD19 (part of the B-cell coreceptor)
• CD43 (leukosialin), and
• CD24 (heat stable antigen (HSA))
• c-Kit (a receptor for a growth-promoting ligand present on stromal cells )
– Pre-B stage
• many of the same markers that were present during the pro-B stage
• cease to express c-Kit and CD43
• express CD25 (α chain of the IL-2 receptor)
B cell generation and maturation

Abnormalities in B-cell development

– XLA (X-linked agammaglobulinemia) in humans and XID (X-chromosome-linked
immunodeficient) in mice
• Several mutations in man have identified key molecules in the ontogeny of B-cells

• lead to a block at the pro-B cell to large pre-B cell transition in the bone marrow

• mostly caused by a mutation in the gene encoding the enzyme Bruton’s tyrosine
kinase (Btk)
• Btk is a key enzyme involved in signal transduction downstream of
– pre-BCR and
– BCR
 B cell generation and maturation

Abnormalities in B-cell development (Contd.)

– XLA patients have
• very few circulating B-cells and
• negligible serum immunoglobulin (Ig)
– XLA accounts for 85% of the cases of
agammaglobulinemia
– The rest (15%) are characterized by mutations (affecting
pre-BCR functions) in
• µHC
• λ5
• Igα (CD79a)
• Igβ (CD79b), and
• BLNK (SLP- 65)
B cell generation and maturation

Abnormalities in B-cell development (Contd.)

– Common variable immunodeficiency (CVID)
• impacts later stages of B-cell development
• manifests in reduced
– serum Ig
– memory B-cells
– class switch recombination and
– B-cell activation
• Mutations identified in CVID patients
– CD40 ligand on T-cells
– B-cell surface receptor CD19
– activated T-cell co-stimulatory molecule ICOS, and
– TACI (another receptor for Blys)
B-Cell Activation and Proliferation

• Overview
• B cells
– export from the bone marrow

– Reach in the periphery

– Require antigen for subsequent

• Activation (antigen driven)
• Proliferation (clonal selection), and
• Differentiation to plasma cell and memory cells

– In the absence of antigen-induced activation

• naive B cells in the periphery have a short life span
• dying within a few weeks by apoptosis
B-Cell Activation and Proliferation

• Types of B cells: Three main types

– Follicular B‐cells (B2-B cells)
• express highly specific monoreactive B‐cell receptors (BCRs)
• are present in the lymphoid follicles of the spleen and lymph nodes
• typically require T‐cells in order to generate high affinity antibodies
• undergo class switching
– B1- B‐cells and Marginal zone (MZ) B‐cells
• Both are “innate like” B‐cells
• express polyreactive BCRs
– are of broad specificity
– recognize multiple different kinds of evolutionarily conserved microbial antigens
• express TLRs
• can be directly activated by PAMPs
• act as APCs, and
• secrete cytokines
B-Cell Activation and Proliferation

• T-independent antigens do not require T cell help to stimulate B cells

– Small in number
– Can activate B cells without MHC class II-restricted T cell help
– Many TI antigens are particularly resistant to degradation
– Based on the manner of B cells activation, TI antigens can be divided into two
groups
• TI-1 antigens
• TI-2 antigens
B-Cell Activation and Proliferation

• T-independent antigens do not require T cell help to stimulate B cells (Contd.)

• TI-1 antigens
– predominantly bacterial cell wall components
– lipopolysaccharide (LPS)
– At high concentrations
» mitogenic (induce proliferation) for most B cells
» Have ability to bind to pattern recognition receptors (PRRs) on the surface
of the B cell
– At lower concentrations
» activate only those B cells that bind antigen with their Ig receptors
B-Cell Activation and Proliferation

• T-independent antigens do not require T cell help to stimulate B cells (Contd.)

• TI-2 antigens
– predominantly large polysaccharide molecules
– have repeating antigenic determinants
» Ficoll
» Dextran
» Polymeric bacterial flagellin
» Poliomyelitis virus etc.
– are not inherently mitogenic
– Have ability to cross-link a large fraction of the Ig receptors on the surface of a B cell
– allows them to deliver an activation signal in the absence of T-cell help
– Many TI-2 antigens
» specifically and covalently bound by the complement component C3d
» mice depleted of C3d mount poor responses to TI-2 antigens
B-Cell Activation and Proliferation

• T-dependent antigens require T cell help to stimulate B cells

– The immune response to most antigens depends on both T cells and B cells
– Recognized antigens in a linked fashion
• Polysaccharides
• Glycolipids
• nucleic acids
• serum proteins
• Erythrocytes
• haptens
– mediated by B-2 B cells binding to TD antigens
• B-2 B cells
– represent the majority of B cells
– refer to the B-2 B-cell subset simply as “B cells,”
– distinguish the other B-cell subclasses by their particular names as
» B-1 (marginal zone) cells or
» B-10 B cells
B-Cell Activation and Proliferation
 B-Cell Activation and Proliferation

• T-independent antigens induce poor memory

– Primary antibody responses to TI antigens
• slightly weaker than those to TD antigens
• peak fractionally earlier and
• generate mainly IgM

– The secondary response to TI antigens

• resembles the primary response
• do not usually induce the maturation of a response leading
– to class switching or
– to an increase in antibody affinity
• most likely due to the lack of CD40 activation
• Memory induction to TI antigens is also relatively poor
B-Cell Activation and Proliferation
B-Cell Activation and Proliferation

• T-independent antigens induce poor memory (Contd.)

– There are potential survival to escape the complex cell interactions

• Could be more rapid
• Very effective inducers of cytokine production by macrophages
• Macrophages secrete
– IL-1
– IL-6 and
– tumor necrosis factor-α (TNFα)
• short-lived response
• lack of
– IgG
– CD40L (co-stimulation)
– IL-2
– IL-4 and
– IL-5
B-Cell Activation and Proliferation

• T-independent antigens tend to activate the CD5+ subset of B cells

– TI antigens predominantly activate the B-1 subset of B cells
– found mainly in the peritoneum
– B-1 cells
• can be identified by their expression of CD5

– signal transducing molecule

– involved in tyrosine phosphorylation of intracellular proteins

• have the ability to replenish themselves

B-Cell Activation and Proliferation

Activation of B cells by T-dependent antigens

i. T cells and B cells recognize different parts of antigens

– To induce an optimal secondary antibody response to hapten

– Experimental animal immunized with hapten–carrier conjugate

– Referred to as the carrier effect

– By manipulating the cell populations, it was shown that:

• TH cells are responsible for recognizing the carrier

• B cells recognize hapten

– These experiments were later reinforced by details of how:

• B cells use antibody to recognize epitopes

• T cells recognize processed antigen fragments

 B-Cell Activation and Proliferation

Activation of B cells by T-dependent antigens

ii. B-cell activation and T-cell activation follow similar patterns

• If membrane Ig
– becomes cross-linked (e.g. by a T-independent antigen)
– tyrosine kinases become activated
• Lck (lymphocyte-specific protein tyrosine kinase)
• Lyn (Lck/Yes novel tyrosine kinase)
• Fyn and
• Blk
– They phosphorylate the ITAM domains in the Igα and Igβ chains of the
receptor complex
– These can then bind another kinase, Syk, which activates
phospholipase C
– This acts on membrane PIP2 to generate IP3 and diacyl glycerol
(DAG) which activates protein kinase C
– Signals from the other kinases are transduced to activate nuclear
transcription factors
 B-Cell Activation and Proliferation

Activation of B cells by T-dependent antigens

ii. B-cell activation and T-cell activation follow similar patterns

(contd.)
– B-cell activation is augmented by
• the ‘co-receptor complex’
• comprising three proteins:
– CD21 (complement receptor-2, CR2)
– CD19 (IgSF surface glycoprotein) and
– CD81 (target of anti-proliferative antibody, TAPA-1)
– Antigen with covalently bound C3b or C3d can cross-link the
membrane Ig to CD21 of the coreceptor complex
– This greatly reduces the cell’s requirement for antigen to activate
it.
– CD19 can associate with tyrosine kinases including Lyn, Fyn, Vav
B-Cell Activation and Proliferation

Activation of B cells by T-dependent antigens

iii. Direct interaction of B cells and T cells involves costimulatory molecules

– Membrane immunoglobulin (mIg)
• takes up antigen (Ag) into an intracellular compartment
• degraded and
• peptides can combine with MHC class II molecules
– Arrows A and B are the antigen
• receptor signal transduction events involving tyrosine phosphorylation and phosphoinositide
breakdown
• The antigen receptors also regulate LFA-1 affinity for ICAM-1 and ICAM-3, possibly through the
signal transduction events.
B-Cell Activation and
Proliferation

iii.
Direct
interacti
on of B
cells
and T
cells
involves
costimu
latory
molecul
es
B-Cell Activation and Proliferation

Activation of B cells by T-dependent antigens

iii. Direct interaction of B cells and T cells involves costimulatory molecules (Contd.)
– Arrows C
• In the T cell, CD28 also sends a unique signal to the T cell
• In the later phases of the response CTLA-4 can supplant CD28 to cause downregulation.

– Arrows D
• In the B cell, stimulation via CD40 is the most potent activating signal
– Arrows E
• Class II MHC molecules appear to induce distinct signaling events (E)
– Not shown is the exchange of soluble interleukins and binding to the corresponding
receptors on the other cell
B cell differentiation and the antibody response

• Following activation, antigen-specific B cells can follow either of two separate developmental
pathways:
– First pathway
• Proliferation and differentiation into antibody forming cells (AFCs)
– Occurred in lymph nodes or in the periarteriolar lymphoid sheath of the spleen
– AFCs function to rapidly clear antigen
– Most of the AFCs die via apoptosis within 2 weeks
– AFCs are unlikely responsible for long-term antibody production
– Second pathway
• some members of the expanded B-cell population migrate into adjacent follicles to
form germinal centers before differentiating into memory B cells
 Germinal Center

• When an antigen is introduced into the

body
– Becomes concentrated in various
peripheral lymphoid organs
– Bloodborne antigen is filtered by the
spleen
– Antigen from tissue spaces drained
by the lymphatic system is filtered by
regional lymph nodes or lymph
nodules
i. B cell affinity maturation
occurs in germinal centers

• Germinal center
– provides a microenvironment
– B cells can undergo developmental events
– arise within 7–10 days after initial exposure to a
TD antigen
– initially contains only dividing centroblasts
– Subsequently polarize into two regions
• Dark zone
– containing centroblasts
• Light Zone
– containing nondividing (resting)
centrocytes
 i. B cell affinity maturation occurs in germinal centers

• Germinal centers
– Centroblasts
• appear dark zone
• large size
• expanded cytoplasm
• diffuse chromatin, and
• absence or near absence of surface Ig
• Centroblasts eventually give rise to centrocytes
– centrocytes
• small
• nondividing B cells
• express membrane Ig
• move from the dark zone into a region containing follicular dendritic cells
called the light zone
• Some centrocytes make contact with antigen displayed as antigen-
antibody complexes on the surface of follicular dendritic cells
– Three important B-cell differentiation events take place in germinal centers
• affinity maturation
• class switching, and
• formation of plasma cells and memory B cells
i. B cell affinity maturation occurs in germinal centers

• Centroblasts
– proliferate rapidly in the dark zone
– Downregulate the expression of their surface immunoglobulin (sIg)
• Somatic hypermutation
– occurs to diversify the rearranged variable region genes
– allows a single B cell to give rise to variants with different affinities for the antigen
• Isotype switch recombination
– occurs following somatic hypermutation
– requires cell cycling
– Receptor editing of immunoglobulin light chain genes also occurs in centroblasts
i. B cell affinity maturation occurs in germinal centers

• Following these developmental changes

– the centroblasts migrate to the follicular dendritic cell- (FDC-) light zone of the
germinal center and
– give rise to centrocytes which then re-express surface immunoglobulin BCR
– In the light zone, centrocytes encounter antigen bound to the FDCs and antigen specific
TH2 cells
– FDCs and T cells interact with centrocytes
• After the centrocytes have stopped dividing, they are selected according to their ability to
bind antigen
– Those with high-affinity receptors for foreign antigen are positively selected
– those without adequate affinity are induced to undergo apoptosis by ligation of the
surface molecule Fas.
 ii. B cell isotype switching

• Class switching
– B cells produce
• antibodies of five major classes
• IgM, IgD, IgG, IgA, and IgE

– In humans, there are also

• four subclasses of IgG and
• two of IgA

– occurs during maturation and Proliferation (Majority)

– can also take place before encounter with exogenous antigen during
• early clonal expansion and
• maturation of the B cells
 ii. B cell isotype switching

• Class switching (Contd.)

– Each terminally differentiated plasma cell
• derived from a specific B cell and
• produces antibodies of just one class or subclass (isotype)
– The first B cells to appear during development
• carry surface IgM as their antigen receptor
• Upon activation, other classes of immunoglobulin are seen
– When a mature AFC switches antibody class
• all that changes is the constant region of the heavy chain
• expressed V(D)J region and light chain do not change
• Antigen specificity is therefore retained
ii. B cell isotype switching
ii. B cell isotype switching

• X-linked hyper-IgM syndrome

– an immunodeficiency disorder

– TH cells fail to express CD40L

– Patients with this disorder

• produce IgM but not other isotypes

• fail to generate memory cell populations

• fail to form germinal centers, and

• their antibodies fail to undergo somatic hypermutation

iii. Memory B Cells and Plasma Cells are Generated in
Germinal Centers

• After B cells are selected in the germinal center for those bearing high-affinity mIg for antigen displayed on follicular

dendritic cells, some B cells differentiate into plasma cells and others become memory B cells (see Figure 11-17).

• While germinal centers are important sites of plasma cell generation, these Ig-secreting cells are fomed in other

sites as well.

• Plasma cells generally lack detectable membrane-bound immunoglobulin and instead synthesize high levels of

secreted antibody (at rates as high as 1000 molecules of Ig per cell per second).

• Differentiation of mature B cells into plasma cells requires a change in RNA processing so that the secreted form of

the heavy chain rather than the membrane form is synthesized.

• In addition, the rate of transcription of heavyand light chain genes is significantly greater in plasma cells than in

less-differentiated B cells.
iii. Memory B Cells and Plasma Cells are Generated in
Germinal Centers

• Several authors have suggested that the increased transcription by plasma cells might be explained by the
synthesis of higher levels of transcription factors that bind to immunoglobulin enhancers.
• Some mechanism also must coordinate the increase in transcription of heavy-chain and light-chain genes, even
though these genes are on different chromosomes.
• As indicated above, B cells that survive selection in the light zone of germinal centers also differentiate into
memory.
• Some properties of naive and memory B cells are summarized in Table 11-6.
• Except for membrane-bound immunoglobulins, few membrane molecules have been identified that distinguish
naive B cells from memory B cells.
• Naive B cells express only IgM and IgD; as a consequence of class switching, however, memory B cells express
additional isotypes, including IgG, IgA, and IgE.
 Neutralization by antibodies

• Generated antibodies
– circulate in the blood stream and lymphatic system
– binding with the antigen
• The neutralized antibody-coated pathogens
– can then be filtered by the spleen
– to be eliminated in urine or feces
• The antigen-antibody complex
– stimulates the complement system
– destroying the cell bearing the antigen
• Antibodies opsonize pathogen cells
– wherein they mark them for destruction by phagocytic cells, such
as macrophages or neutrophils.
• Antibodies
– stimulate inflammation,
– their presence in mucus and on the skin prevents pathogen