This document outlines the mechanisms of humoral immune response, focusing on the structure and function of immunoglobulins (antibodies), including their classes and roles in immune defense. Key topics include VDJ recombination, class switching, antibody specificity, and effector functions such as opsonization and complement activation. The document emphasizes the importance of B cell activation in generating diverse antibody responses and transferring immunity from mother to fetus.

1. Mechanisms of humoral immune response:
Mechanisms of humoral immune response:
indution $ effector mechanisms
indution $ effector mechanisms
 By Dr Oyaro
By Dr Oyaro

2. Objectives
Objectives
 Structures of immunoglobulins and their
Structures of immunoglobulins and their
proteolytic digestion
proteolytic digestion
 Difference in biologic properties among antibody
Difference in biologic properties among antibody
classes
classes
 VDJ recombination and class switching of
VDJ recombination and class switching of
immunoglobulin
immunoglobulin
 Transfer of IgG from mother to fetus (via FcRn)
Transfer of IgG from mother to fetus (via FcRn)
 Mucosal secretion of IgA (via poly-Ig receptor)
Mucosal secretion of IgA (via poly-Ig receptor)
 Primary and secondary immunoglobulin responses
Primary and secondary immunoglobulin responses
 Effector functions of antibody
Effector functions of antibody
To understand;

3. Humoral immune responses
Humoral immune responses
 “
“Antibody” = immunoglobulin is the primary
Antibody” = immunoglobulin is the primary
component of the humoral immunity
component of the humoral immunity
 “
“Antigen” = a molecule recognized by
Antigen” = a molecule recognized by
antibody or T cell
antibody or T cell
 “
“Epitope” is the molecular structure in an
Epitope” is the molecular structure in an
antigen that interacts with antibody or T cell
antigen that interacts with antibody or T cell
 Proteins (best antigen) >> carbohydrates
Proteins (best antigen) >> carbohydrates
(weak) >> lipids, nucleic acids (poor antigen)
(weak) >> lipids, nucleic acids (poor antigen)

4. Antibodies are composed of polypeptides with both
Antibodies are composed of polypeptides with both
variable and constant regions
variable and constant regions
The immunoglobulin G (IgG) molecule
Hinge
Note: 2 identical heavy chains (green)
2 identical light chains (yellow)
Note: Location of the variable (red)
and constant (blue) regions in
the antibody molecule.

5. The immunoglobulin constant (C) regions
The immunoglobulin constant (C) regions
 
 
Any of two identical light chains can pair with any two identical
heavy chains to make a complete immunoglobulin molecule.
The constant (C) regions have little variability in
amino acid sequence.
Two different kinds of C regions for light chains :
kappa (κ)
lambda (λ)

6. Five antibody classes (
Five antibody classes (isotypes
isotypes), determined by the
), determined by the
constant region of the heavy chain
constant region of the heavy chain
The Structural Organization of the Human Immunoglobulin Classes
Five different kinds of C regions for H chains :
gamma (γ) chains, IgG
mu (µ) chains, IgM
delta (δ) chains, IgD
alpha (α) chains, IgA
epsilon (ε) chains, IgE

7. Each chain is folded into multiple ‘Ig’ domains
Each chain is folded into multiple ‘Ig’ domains
The heavy chain constant domains (CH) :
The “biological activity” region
Responsible for interactions with other
proteins (eg, complement), cells (eg, mast
cells), and tissues that result in the effector
functions of immunoglobulins
Both H and L chains contain multiple loops of around 110 amino acids each.

8. Each chain is folded into multiple ‘Ig’ domains
Each chain is folded into multiple ‘Ig’ domains
The variable domains (VH, VL) :
The “antigen-binding” region
Together, these domains compose the
sole antigen-binding region of the
immunoglobulin molecule.
Both H and L chains contain multiple loops of around 110 amino acids each.
The V regions are the first 110 amino acids at the N-terminus of both H and L
chains. This is the region that gives antibody specificity for a given antigen.

9. Proteolytic digestion of IgG
•Papain treatment produces Fab fragments and Fc fragment
•Fab, antigen-binding; Fc, crystalizable or constant region
•Fc portion interacts with host systems
•Complement activation
•Binding to Fc receptor (FcR) on macrophages and other
cells

10. Immunoglobulins
Immunoglobulins
 Basic structure
Basic structure
 2 identical heavy chains
 2 identical light chains
 Heavy and Light chains
Heavy and Light chains
 variable (V) and constant (C) regions
 disulfide bonds link the chains together
 light chains have 2 domains, 1V and 1C
 heavy chains have 4-5 domains, 1V and 3-4 C
 heavy chains: 5 classes, μ, γ, α, δ, ε
 light chains: 2 classes, κ and λ
 Membrane bound (mIg) or secreted (sIg)
Membrane bound (mIg) or secreted (sIg)

11. •Antibody recognizes only a small region (epitope) on
Antibody recognizes only a small region (epitope) on
the surface of a large molecule
the surface of a large molecule
•Antibody binds an epitope formed by a specific
Antibody binds an epitope formed by a specific
sequence (linear epitope) or a three-dimensional
sequence (linear epitope) or a three-dimensional
structure (conformational epitope)
structure (conformational epitope)

12. Antigens can bind in pockets, grooves, extended surface, or
Antigens can bind in pockets, grooves, extended surface, or
protruding surfaces in the binding sites of antibodies
protruding surfaces in the binding sites of antibodies

13. The process of
rearrangement of
the Ig locus – called
VDJ recombination.
During B cell development, V, D, and J gene segments
During B cell development, V, D, and J gene segments
rearrange, creating functional genes that encode
rearrange, creating functional genes that encode
immunoglobulin transcripts.
immunoglobulin transcripts.
Genetic recombination at the DNA level and processing at the RNA level to
produce functional mRNA

14. 51 27 6
variable genes constant gene
For example, a rearranged heavy chain locus is composed of :
• one V gene segment (from 51)
• one D gene segment (from 27)
• one J gene segment (from 6)
• one C gene segment
During B cell maturation, V, D, and J gene segments
During B cell maturation, V, D, and J gene segments
rearrange, creating functional genes that encode
rearrange, creating functional genes that encode
immunoglobulin transcripts.
immunoglobulin transcripts.
*After maturation, the variable gene segment rearrangment
(and therefore the epitope specificity) does not change.
V23 D14 J3

15. Thus, 2.64 x 10
Thus, 2.64 x 106
6
DIFFERENT specificities are possible from the process of
DIFFERENT specificities are possible from the process of
VDJ recombination alone
VDJ recombination alone
8262 x (200 + 120) = 2643840 = 2.64 x 10
8262 x (200 + 120) = 2643840 = 2.64 x 106
6
8262 x (200 + 120) = 2.64 x 10
8262 x (200 + 120) = 2.64 x 106
6
A given immunoglobulin has either
A given immunoglobulin has either κ
κ chains or
chains or λ
λ chains, never one of
chains, never one of
each
each

16. No two B cells are likely to secrete the same Ig (unless
No two B cells are likely to secrete the same Ig (unless
they are clonal progeny), due to the number of different
they are clonal progeny), due to the number of different
rearrangements that are possible within the variable
rearrangements that are possible within the variable
region of the immunoglobulin locus
region of the immunoglobulin locus
Note
Note – There are several other forms of antibody
– There are several other forms of antibody
diversification (eg, somatic hypermutation) that
diversification (eg, somatic hypermutation) that
greatly increase the number of distinct antibodies
greatly increase the number of distinct antibodies
that the immune system can generate.
that the immune system can generate.
Thus, 2.64 x 10
Thus, 2.64 x 106
6
DIFFERENT specificities are possible from the process of
DIFFERENT specificities are possible from the process of
VDJ recombination alone (more than 10
VDJ recombination alone (more than 1011
11
different specificities in total)
different specificities in total)

17. Murray
textbook, p75
Number of gene
segment, wrong
information:
κ chain: 300 V,
5J
H chain: 300-
1000 V, 12D, 9J

18. Naïve B cells die if they do not
Naïve B cells die if they do not
see antigen (Ag)
see antigen (Ag)
Naïve B cells that encounter
Naïve B cells that encounter
antigen (plus T cell help) :
antigen (plus T cell help) :
1. become activated
1. become activated
2. undergo class switching
2. undergo class switching
3. differentiate into either :
3. differentiate into either :
plasma cells [antibody (Ab)
plasma cells [antibody (Ab)
secreting]
secreting]
or
or
memory cells (non-secreting)
memory cells (non-secreting)
The Life of a B cell
The Life of a B cell

19. When membrane-
bound Ig (mIg) on the
B cell surface binds
to it’s specific
antigen, the B cell
becomes activated.

20. Proper activation of the B cell
typically requires contact
with CD4 helper T (TH) cells.

21. Activated B cells differentiate into
Activated B cells differentiate into :
:
plasma cells (Ab secreting)
plasma cells (Ab secreting)
or
or
memory cells (non-secreting)
memory cells (non-secreting)

22. Class switching (IgM to IgG, IgE, or
Class switching (IgM to IgG, IgE, or
IgA) occurs in response to different
IgA) occurs in response to different
cytokines produced by CD4 helper
cytokines produced by CD4 helper
T cells
T cells
Class switching does not change
Class switching does not change
the variable region
the variable region

23. The usage of heavy chain
The usage of heavy chain constant
constant gene segments changes with B
gene segments changes with B
cell activation in a process termed
cell activation in a process termed class switching
class switching
2
3 4 5
1
but ...... epitope specificity does
but ...... epitope specificity does not
not change !
change !

24. Structures of the five major classes of secreted antibody
Structures of the five major classes of secreted antibody
Important because antibody classes differ with regard to biological properties
Important because antibody classes differ with regard to biological properties

25. IgG and IgA are divided into
IgG and IgA are divided into subclasses
subclasses
Subclasses differ in the number
and arrangement of the interchain
disulfide bonds.

26. 13.5 mg/ml 0.0003 mg/ml
vs.
The principal immunoglobulins in plasma are IgG >> IgA > IgM

27. Function IgG IgM IgA IgD IgE
Complement
Activation
+ +++ - - -
Opsonization + - - - -
ADCC + - - - -
Placental transport + - - - -
B cell antigen
receptor
Naïve - + - + -
Memory + - + - +
Mucosal secretions - - + - -
Mast cell degranulation - - - - +
Antigen binding + + + + +
Biological properties of antibody classes differ
Biological properties of antibody classes differ

28.  IgG is the only isotype to cross the placenta
 The transfer of IgG from mother to fetus (placental transfer) is
mediated by an IgG transport protein in the placenta, FcRn,
which binds to the Fc portion of IgG
 Infant produces its own IgG at 6 months
IgG levels are low from the age of 3 months to 1 year
IgG levels are low from the age of 3 months to 1 year

29. to the fetal
circulation
Maternal
blood
Syncytio-
trophoblast
Endothelial
cells of fetal
capillary
Villi

30. FcRn: neonatal Fc receptor for IgG

31. Syncytiotrophoblasts are bathed in maternal blood and internalize serum
containing maternal IgG. FcRn is expressed in the internal vesicles of the
syncytiotrophoblast. On acidification in the endosome, FcRn binds to
maternal IgG and transcytoses it to the fetal circulation where it is released
at physiological pH.

32. Dimeric IgA is the predominant in mucosal secretions
The delivery of antibody
to mucosal surfaces
requires their
movement across
epithelial cell layers
Transport is mediated
by the polymeric
immunoglobulin
receptor (pIgR)
IgA in colostrum,
intestinal and
respiratory tracts,
saliva, tears
Some pathogens
produce proteolytic
enzymes that can
cleave IgA
Haemophilus influenzae
(respiratory), Neisseria
gonorrhoeae (genital mucosa)
Secretory
component:
part of the
cleaved pIgR
associated
with the IgA

33.  IgE is found associated with mast cells
beneath epithelial surface, including
respiratroy tract, gastrointesitnal tract,
and skin
 IgD exists as membrane IgD, which
serves with IgM as an antigen receptor
on B cells, and activate B cell growth
 In healthy individuals the Ig
concentration in the central nervous
system (CNS) is low.
Immunoglobulin isotypes
Immunoglobulin isotypes
are selectively distributed
are selectively distributed

34. The humoral immune response
The humoral immune response
to infectious pathogens
to infectious pathogens

35. The
The primary response
primary response to an initial antigen exposure
to an initial antigen exposure
• long lag phase
long lag phase
• IgM is the first reponder
IgM is the first reponder
• gradual switch to IgG
gradual switch to IgG
• low affinity antibodies
low affinity antibodies

36. The
The secondary response
secondary response to antigen exposure reflects
to antigen exposure reflects
immunological
immunological memory
memory.
.
• short lag phase
short lag phase
• very rapid rise in titer
very rapid rise in titer
• IgG predominates
IgG predominates
• high affinity antibodies
high affinity antibodies

37. EFFECTOR FUNCTIONS OF ANTIBODIES
EFFECTOR FUNCTIONS OF ANTIBODIES
 Opsonization – the promotion of phagocytosis of antigens by macrophages
and neutrophils. Protein molecules called Fc receptors (FcR), which bind the
constant region of antibody, are present on the surfaces of phagocytes.
 Activation of complement – IgM (most effective) and most subclasses of IgG
can activate the classical complement pathway
 Antibody-dependent cell-mediated cytotoxicity (ADCC) – The linking of
antibody bound to target cells (virus infected cells, or some tumor cells) with
FcR of natural killer cells (NK cells), neutrophils, macrophages, or eosinophils
can result in killing of the target cell.
 Neutralization of viruses and bacteria – Prevent attachment to cell
receptors.
 Neutralization of toxins

38. Antibody-dependent
Antibody-dependent opsonization
opsonization of bacteria
of bacteria
facilitates phagocytosis of the pathogen
facilitates phagocytosis of the pathogen
opsonization (op´sŏn-ī-zā´shŭn) The process by which bacteria and other cells are altered in such a
manner that they are more readily and more efficiently engulfed by phagocytes.
opsonin (op´sŏ-nin) Any blood serum protein that binds to antigens, enhancing phagocytosis (e.g., C3b
of the complement system, specific antibodies). [G. opson, boiled meat, opsonin: to prepare for a meal]
IgG
FcγR

39. Antibody-dependent activation of the complement pathway
Antibody-dependent activation of the complement pathway
Potential outcomes : - lysis of bacteria via MAC
- induction of inflammatory response
- phagocytosis of bacteria
IgM is the most efficient immunoglobulin for fixing
(binding) complement (Murray p 102)
“Complement fixation” = “Complement activation”

40. Antibody-dependent cellular (cell-mediated)
Antibody-dependent cellular (cell-mediated)
cytotoxicity (ADCC) kills the infected target cell
cytotoxicity (ADCC) kills the infected target cell
=FcγR

41. Infection of cells can be blocked by
Infection of cells can be blocked by
neutralizing antibodies
neutralizing antibodies

42. Infection of cells can be blocked by neutralizing
Infection of cells can be blocked by neutralizing
antibodies
antibodies

43. Antibody can prevent
Antibody can prevent
the attachment of
the attachment of
bacteria to cell surface
bacteria to cell surface

44. Many common
Many common
diseases are
diseases are
caused by
caused by
bacterial toxins
bacterial toxins

45. Neutralization by IgG antibodies can protect cells from toxins
Neutralization by IgG antibodies can protect cells from toxins

46. Neutralization by IgG antibodies can protect cells from toxins
Neutralization by IgG antibodies can protect cells from toxins

47. Function IgG IgM IgA IgD IgE
Complement
Activation
+ +++ - - -
Opsonization + - - - -
ADCC + - - - -+
Placental transport + - - - -
B cell antigen
receptor
Naïve - + - + -
Memory + - + - +
Mucosal secretions - - + - -
Mast cell degranulation - - - - +
Antigen binding + + + + +
Biological properties of antibody classes differ
Biological properties of antibody classes differ
Murray p73, Table 9-4 is inaccurate
helminth
killing

48. Naïve B cells die if they do not
Naïve B cells die if they do not
see antigen (Ag)
see antigen (Ag)
Naïve B cells that encounter
Naïve B cells that encounter
antigen (plus T cell help) :
antigen (plus T cell help) :
1. become activated
1. become activated
2. undergo class switching
2. undergo class switching
3. differentiate into either :
3. differentiate into either :
plasma cells [antibody (Ab)
plasma cells [antibody (Ab)
secreting]
secreting]
or
or
memory cells (non-secreting)
memory cells (non-secreting)
The Life of a B cell
The Life of a B cell
IgM, IgD

49. The usage of heavy chain
The usage of heavy chain constant
constant gene segments changes with B
gene segments changes with B
cell activation in a process termed
cell activation in a process termed class switching
class switching
2
3 4 5
1
but ...... epitope specificity does
but ...... epitope specificity does not
not change !
change !

50. Every B cell expresses a single type of immunoglobulin.
Every B cell expresses a single type of immunoglobulin.
The specificity of that immunoglobulin is determined by
The specificity of that immunoglobulin is determined by
the process of VDJ recombination – rearrangement of
the process of VDJ recombination – rearrangement of
the DNA that encodes the variable region.
the DNA that encodes the variable region.

51. THANK YOU
THANK YOU