Chapter 9: The Humoral Immune Response

I. B cell Activation and antibody production

1. The humoral immune response is mediated by

antibody molecules secreted by plasma cells

• Ags bound by a B cell  presented to T cells

• TH cells activate the B cell  help it to
differentiate into plasma cells  secrete Abs
• Abs can cause:
i. Neutralization
ii. Opsonization
iii. Complement activation

2. B cells are activated by two types of Antigen

1. Thymus-dependent (TD) Ags

• most common: examples= Dipth.
Toxin, foreign proteins
• Requires TH cells
• Primarily foreign peptides
• Signal 1: from Ag
• Signal 2: CD40L from TH cell triggers
CD40 on B cell
2. Thymus-independent (TI) Ags
2 types:
A. TI-1 antigens

• possess an intrinsic activity that can directly induce B-cell

activation.
• An example of a TI-I antigen is LPS.
• At high LPS concentrations polyclonal B cells are activated
via the ‘mitogen’ receptor. Mechanism of action:
a. LPS is bound by LPS-binding protein (LBP)
b. LBP delivers LPS to LPS receptor (CD14)
c. CD14 associates with and activates TLR4
d. Leading to B cell activation and differentiation into
IgM producing plasma cells.
 At low LPS concentrations only B cells with
specific receptors for LPS can concentrate
enough LPS on B cells to activate TLR4

B. TI-2 antigens

• egs bacterial polysaccharides that possess

highly repetitive structures
• In the absence of helper T cells, TI-2
antgiens activate B cells by BCR cross-
linking, leading to B cell activation and
differentiation into IgM producing plasma
cells
• TI-2 responses can also include switching to
other Ab classes such as IgG3 in the mouse.
This is possibly the result of help from DCs,
which provide secreted cytokines such as
BAFF
• The IgM and IgG Abs induced by TI-2 Ags
are likely to be an important part of the
humoral immune response in many bacterial
infections.
• Wiskott-Aldrich syndrome patients fail to
make antibody against polysaccharide Ags
and are highly susceptible to infection with
encapsulated bacteria.
The properties of different classes of Ag (TD, TI- 1 and TI-2) that elicit Ab responses are summarized:

3. B cell responses to Ag are enhanced by co-ligation of the B cell coreceptor.

• The B cell coreceptor complex is composed of three proteins: CD19, CD21 and CD81. CD21 is a
receptor for the complement fragments C3d and C3dg.
• The linking of BCR to the B cell coreceptor complex generates signals through CD19 that
activate a PI3- kinase signaling pathway and costimulate the B cell response
• ~10,000 less Ag are required for B cell activation by engaging the B cell coreceptor.

4. Hepler T cells activate B cells that recognize the same Ag.

• For B cell to make antibodies to a pathogen, it must get help from a CD4 TH cell specific for
peptides from this pathogen.
• The epitopes recognized by the T
cell and the B cells are distinct. However,
the T and B cell epitopes must be found in
the same Ag in order to be recognized by
antigen-activated B and T cells, a process
referred to as “linked recognition”

5. Use of linked recognition to raise

antibodies to polysaccharide Ags in H. influenzae type B vaccine

• Haemophilus influenzae B (HIB) causes meningitis in young children

• Vaccine is a complex of bacterial polysaccharide + tetanus toxoid
• Although adults have TI response to bacterial polysaccharide, infants do not
 • Polysaccharide is linked
covalently to a protein
• infants are immunized to and
respond well to (i.e.) tetanus toxoid.
• Immunization with this complex
allows recruitment of memory TH cells to
the toxoid protein that maximize Ab
response to the polysaccharide.

• NOTE: accidental coupling of a

small molecule to a protein is responsible for the allergic responses shown by many people to
the antibiotic penicillin, which forms complexes with host proteins, leading to an Ab response.

6. Focused release of cytokines by TH2 cells

Step 1- binding of activated TH cells to Ag-

stimulated B cells requires:
 LFA-1/ICAM-1
 TCR/MHC class II-
peptide
 increased synthesis of CD40
ligand and IL-4.

Step 2- reorientation of cytoskeleton and

secretory apparatus toward B cell

Step 3-polarized release of cytokines such as

IL-4.

7. Ag-binding B cells meet T cells at the border between the T and B cell zones in secondary
lymphoid tissues

• Where and how do rare Ag-specific T- and B-cells meet?

• In a lymph node (LN), T and B cells usually home to different regions
• Circulating B cells may encounter Ag in the blood stream or Ag that are brought into lymphoid
tissues by the lymph.
• Ag activated B cells express activated adhesion molecules such as LFA-1 and the CCR7
chemokine receptor for migration to T cell zone.
• After their initial encounter, the B cells and their cognate T cells migrate from the T zone – B
zone border to continue their proliferation and differentiation. In the spleen they move to the
border of the T cell zone and the red pulp; in lymph nodes they migrate to the medullary cords
and form a primary focus.
• Primary foci appear about 5 days after an infection. Ag stimulated B cells that fail to interact with
T helper cells that recognize the same Ag die within 24 hours.

8. Ab-secreting plasma cells differentiate from activated B cells.

• Proliferating B cells differentiate into Ab- synthesizing plasmablasts in the primary focus. Others
migrate into the lymphoid follicle and differentiate further there before becoming plasma cells.
Plasmablasts stop dividing after a few days and either die or differentiate into plasma cells.
• Plasma cells may remain in the lymphoid organs, where they are short-lived, while the majority
migrate to the bm and continue Ab production there.
• The properties of resting B cells, plasmablasts and plasma cells are compared in Fig. 9.8, 7ed
(9.10, 6ed).
9. Some activated B cells form germinal centres in lymphoid follicles

• Some early activated B and T cells migrate into a primary follicle, where they continue to
proliferate and form a germinal center.
• The germinal center is a
specialized
microenvironment in
which intense B cell
proliferation (dividing
every 6 to 8 hours),
somatic hypermutation,
and selection for B cells
that express high affinity
BCRs for Ag occurs.

• Closely packed centroblasts (proliferating cells) formed the so-called ‘dark zone’ of the germinal
center. The dense network of follicular dendritic cells mainly occupy the light zone. FDCs
attract both naive and activated B cells into the follicles by secreting the chemokine CXCL13,
which is recognized by the receptor CXCR5 on B cells.
• The slower proliferating B cells found in the light zone are called centrocytes.
• The germinal center is surrounded by CD4 T helper cells.
• Engagement of CD40 (expressed by B cells) with CD40L (on T helper cells) is essential for the
survival of germinal center B cells.
10. Selection of high-affinity mutant B cells in the germinal centre

• Rapidly proliferating B cells in the germinal

centre initiate V-region somatic
hypermutation at a very high rate (one
mutation per 103 base pairs per cell
division (vs a rate of 1010 for normal cells)
• Requires:
o BCR engagement
o T cell help involving cytokines
o CD40 ligand
o  survival signals, Bcl-xL
• involves individual point mutations that
change a single amino acid  generates
related B cells with subtle changes in
specificity and affinity for the Ag they first
encountered.
• Mutations affect the individual B cell’s fate:
• Most are deleterious  B cell eliminated by
apoptosis (negative selection)
• Rarely, a mutation improves affinity for Ag
 selectively expanded and selected
(positive selection)
• Selection is repeated with each division and
mutation in both centroblasts and
centrocytes  Ab affinity is continually refined for higher affinity  “affinity
maturation”

Fate of activated B cells in GCs:

• Some germinal centre cells differentiate into plasmablasts  plasma cells  migrate to bone
marrow, long-lived  source of high affinity Abs
• Others differentiate into memory B cells  long-lived, low rate of division, stop producing
antibodies

11. Cytokines from TH cells direct isotype switching in B cells Isotype Switching
• unlike IgM vs IgD which occur
by differential RNA processing,
this involves irreversible change
in DNA  specialized
homologous recombination
between “switch” regions
upstream of C-region gene
segments (Fig 4.21, 6ed; 4.27,
6ed)
• Isotype switching occurs during
an Ab response. Message: T cells
direct the show
• TH cells provide cytokines that
enhanced Ab production and
induces Ig class switching
 • Isotype switching is preceded by transcription of H- chain C regions

• B cells + LPS increased IgM and IgD

• B cells + LPS + IL-4  IgG1 + IgE
• B cells + LPS + TGFβ  IgG2b + IgA
• IL-5 promotes IgA secretion by cells that have already undergo switching
• TGF-β induces switching to IgG2b and IgA
• IFN-γ (produced by TH1 cells) preferentially induces switching to IgG2a and IgG3 (opsonizing
Abs. Why?)
• The roles of cytokines in Ig class switching is summarized:

12. Isotype Switching and HyperIgM Immunodeficiency

Hyper IgM immunodeficiency:

Type 1: defect in CD40L; no Ig isotype switching, Abs produced primarily of the IgM class.

Type 2: defect in either

a. Activation Induced Cytidine Deaminase (AID) or
b. Ku protein  required for Ig isotype switching; animals deficient in Ku protein produces only IgM.

13. Surviving germinal center B cells differentiate into either plasma cells or memory cells.
• Germinal center B cells differentiate first into plasmablasts, at which stage they undergo somatic
hypermutation and some also may undergo class switching.
• Some then differentiate into plasma cells under the control of a regulatory protein, BLIMP-1 (B
lymphocyte-induced maturation protein 1).
• BLIMP-1 is a transcriptional repressor in B cells that switches off genes required for B cell
proliferation, and for class switching and affinity maturation. B cells in which BLIMP-1 is
induced become plasma cells and change their cell-surface properties, enabling their migration
to peripheral tissues.
• Other germinal center B cells differentiate into memory B cells (non-proliferating, long-lived,
express cell surface Ig).

II. The distribution and function of Ig isotypes

1. Ig isotypes are selectively distributed in the body.

Abs must be widely distributed to combat pathogens wherever they enter.
• The distribution of Abs is determined by their isotype.
o IgM- plasma (first Abs produced, and of low affinity since have not undergone somatic
hypermutation- compensated by pentamers with 10 Ag binding sites)
o IgG- plasma (principal blood isotype) and extracellular fluid
 o Dimeric IgA- epithelia and breast milk
o IgE- sensitization of mast cells

2. Ig isotypes have specialized functions

Don’t need all the little details just note:

Neutralization: IgG and IgA
Opsonization: IgG
Sensitization for killing
by NK cells: IgG
Sensitization of mast cells: IgE
Activation of complement: IgM and IgG
Transport across epithelium: IgA
Transport across placenta: IgG

3. Transport of IgA across epithelia

• IgA is synthesized in plasma cells lying just beneath epithelial basement membranes of the gut,
respiratory epithelia, tear and
salivary glands
• Dimeric IgA diffuses across the
basement membrane
• Dimeric IgA is bound by poly-Ig
receptor
• Dimeric IgA is transported in a
vesicle across the cell to the
apical surface
• The poly-Ig receptor is cleaved,
IgA is secreted as a dimer +
secretory component (part of the
poly-Ig receptor)

4. Mechanism for transporting IgG across placenta (Fig. 9.21, 6 and 7ed).
• Facilitated by the IgG transport protein, FcRn (related to MHC class I molecules)
• FcRn binds Fc of IgG
• Two molecules of FcRn bind to one molecule of IgG andbear it across the placenta.

5. Ab-Ag complex activation of classical pathway of complement by binding C1q

• started this chapter iterating that Abs confer humoral immunity by neutralization, opsonization
and complement activation.
• Focus on how Abs induce the complement pathway
• You saw the details of the cascade of proteolytic cleavage events that define the three different
complement pathways:
o Classical
o Alternative
 o Mannose-binding lectin pathway (Fig 2.25, 7ed; 2.19, 6ed).
• Complement can be activated without Abs as part of the innate immune response
• Abs however, can also initiate complement via the classical pathway as part of adaptive immune
response.

Initiation of pathway by Abs:

• 1st component of classical pathway is C1
= C1q, C1r (x2) and C1s (x2). C1q has 6
globular heads on a long stem; each can
bind one Fc.
• Abs (IgM or IgG) bound to a pathogen
surface will bind C1q. Structural
requirements prevent Abs in solution
from binding C1q  activated only by
bound Ab.
• IgM is much more efficient at activating
C1 than IgG (higher number of molecules
required).
• IgM exists as planar conformation (non-
activating) in plasma and as staple
conformation that binds C1q  activates
C1r then C1s and the rest of the
complement cascade. The binding of C1q
to a single bound IgM molecule leads to activation of C1r, triggering the complement cascade.

III. The destruction of antibody-coated pathogens via Fc Receptors

• Many pathogens are not neutralized by Ab; must be destroyed by other means.
• Fc receptors (specific for Fc portion of Abs) play an important role in pathogen destruction.
• Accessory effector cells such as macrophages, neutrophils and eosinophils express Fc receptors.

a) Types of Fc receptors

• Distinct Fc receptor expression on different accessory cells

• Accessory cells may be phagocytic (macrophages and neutrophils) or secrete stored cytotoxic
molecules and inflammatory mediators (NK cells, eosinophils, basophils and mast cells)
• Different accessory cells, have Fc receptors for different isotypes
 • Distinct FcR’s bind IgG, IgE, or IgA
molecules with differing affinities
o Egs FcγRI binds IgG1 with a
KA=10 8 M-1
o FcγRII-B1 binds IgG1 with a
KA=2 x 10 6 M-1
o FcεR1 binds IgE with a KA=10
10 M-1

b) Function of FcR’s:
(i) Activation of accessory cells

• FcγRI activates phagocytes

• FcεRI activates mast cells

• FcαRI activates phagocytes

• Some activating FcR’s (specific for IgG, IgE and IgA) use a γ chain for signaling

(ii) Negative regulation of B cell activation

• Some FcRs have inhibitory function.
• FcγRII-B1 contains ITIM (immunoreceptor tyrosine-based inhibitory motif)  recruits SHIP
phosphatase  neutralize action of PLCγ1  inhibit B cell activation

(iii) Fc receptor and CR1 act in concert to facilitate phagocytosis

• Most important phagocytes are macrophages and neutrophils.

• Some (capsular) pathogens resist direct engulfment.
• Are susceptible if become coated with Ab and complement which synergize to engage FcγR or
FcαR and CR1 complement receptor.
 • Internalization and destruction are enhanced by engagement of FcR’s and CR1.
• Once engulfed, the compartment is acidified  phagosome  fuses with lysosomes 
phagolysosome  destruction of pathogen.

(iv) Destruction of Ab-coated cells by NK cells (ADCC) (Antibody-dependent cell-mediated

cytotoxicity (ADCC)

• NK cells express FcγRIII which recognizes IgG

• Abs that bind to the surface of a cell can engage the FcR on NK cells
• Triggers cytotoxic attack by NK cell on Ab-coated cell  same mechanism as cytotoxic T cells
(granzyme and perforin release)  death of target cell

c) IgE has an important role in resistance to parasite infection.

• Monomeric IgE binds with very high affinity to Fc receptors on mast cells
• Mast cells have at least three important functions in host defense:
(i) located near body surfaces
(ii) increase the flow of lymph when activated
(iii) when activated produce mediators that trigger muscular contraction that lead to the
expulsion of pathogens from the lungs or the gut.
• W/W mutant mice have a found mast cell deficiency caused by mutation of the gene c-kit
v

 These mutant mice show impaired clearance of intestinal nematodes.