Innate Immunity

Specific Immunity
 2 competing theories for
antibody-antigen specificity

Ab-Ag specificity

Selective Theory Instructional Theory

interaction as "lock and key fi t" antigen serves as tem plate, around which, antibody folds
induces cell to produce/ release m ore antibodies disproved in 1960's by genetic discoveries in B cells

1950's - selective theory changed to become

C lonal Selection Theory

Paradigm of Modern Im m unology

binding of Ag to specifi c receptor (Ab) on specifi c B cell stim ulates that cell line
Paul Erlich’s side chain
hypothesis for antibody
formation (1900)
 Pluripotent blood cells
with variety of
receptor “side chains”
 Contact with foreign
molecules (antigen)
stimulated increased
receptor production
 Specific receptors
produced on cells
prior to contact with
antigen
Foundation of selective theory
Mediated by 2 very specialized cells

B lymphocyte

It gives Humoral immunity

(Antibody production)

T lymphocyte

It gives Cellular immunity

(Immune regulation and Cell mediated immunity)
Types of immunity
Classification …

 On the basis Mode of action

 Humoral or antibody mediated
 Cell mediated
 On the basis of How it is acquired
 Natural
 Active
 passive
 Artificial
 Active
 Passive
ON BASIS MODE OF
ACTION

Antibody mediated
•

Cell mediated
 I. Antibody mediated acquired immunity
( Humoral)

B lymphocyte is activated to transform

into Plasma cells

Plasma cells then produce antibodies

Antibodies act on antigens

Antibodies are mainly effective in

Extracellular pathogens (Bacteria)

Exotoxins (Diphtheria, tetanus)
Neutralization of virus (Hepatitis B, Rabies)
 2. Cell mediated Acquired immunity

Here, T lymphocytes are activated

2 types of T lymphocytes CD 4
molecule

1. Helper (CD4) T cells

It has CD4 molecule on its surface CD 8

molecule

2. Cytotoxic (CD8) T cells

It has CD8 molecule on its surface

 Action of Helper (CD4) T cells

Stimulated Helper T cells release

Lymphokines

Lymphokines stimulate other cells such as:

B cells
Macrophages
Cytotoxic T cells

Stimulated B cells produce antibody

Stimulated Macrophage engulf pathogens more efficiently

Stimulated Cytotoxic T cells kill target cells

 Action of Cytotoxic(CD8) T cells

Stimulated Cytotoxic (CD 8) T cells

kill following cells

Virally infected cells

Tumour cells
Macrophages containing intracellular pathogens
Mismatched transplant cells
Humeral immunity Cell mediated immunity

= MHC class I
= MHC class II

Proliferation IL 4 IL 12
& maturation
of B cells

IL- 4, 5
Lymphokines IL-2
Activated
Gamma IFN
T 8 cell

perforin

Antibody formation Increased phagocytic & Killing of Virus infected cell &
Killing power Tumor cell
ANTIBODIES AND B
LYPHOCYTES
From Jenner experiment-
protection of smallpox was due to some small proteins
that circulated in the blood of immunized animal

This proteins are antibodies and

the agent that elicited the antibodies was called the
antigen
 ANTIBODIES
Antibodies are a group of glycoprotein
present in the serum and tissue fluids of
all vertebrates which are produced from
activated B cell in response to antigens
with which it react specifically
 Immunoglobulin
Immunoglobulin (Ig) are globulin of animal origin produced
from activated B cells in response to antigenic stimulation
which are endowed with or without antibody activity

Structurally related proteins without antibody activity are also

immunoglobulin (myeloma protein)
 Immunoglobulin
By definition all antibodies are immunoglobulin but
all immunoglobulins are not antibodies

Immunoglobulins are produced by activated B cells or

plasma cells known as antibody factories
 B cell

The term B lymphocytes derived from its

site of maturation

 in bursa of fabricius in birds and

in bone marrow in mammalian species
 B cell

 Mature B cells are

distinguished from T-cell
by the membrane bound
antibody (BCR) which
serves as receptor for
antigen

1.5 x105 antibody with

identical binding site are
present on B cell known
as B-cell receptor (BCR)
 Function
B cells perform two important
functions

 Differentiate into Plasma cell - Plasma cell

then produce antibodies
Produce memory cell – provide future

protection
 Origin of B lymphocytes

During embryogenesis B cell

precursors recognized in fetal liver
Than they migrate to bone marrow
 Stages of B cell development

Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell

Peripheral

Small pre-B cell Immature B cell Mature B cell

Each stage of development is defined by

rearrangements of IgH chain genes, IgL chain genes,
expression of surface Ig, expression of adhesion
tages of differentiation in the bone marrow ar
defined by Ig gene rearrangement

B CELL STAGE Stem cell Early pro-B Late pro-B Large pre-B

IgH GENE
Germline DH to JH VH to DHJH VHDHJH
CONFIGURATION

Pre-B cell
receptor
expressed

Ig light chain gene has not yet rearranged

 Origin of B lymphocytes

• B cell development begins as

haematopoietic stem cells become Pro-
B cell
• Than they express transmembrane
tyrosine phosphatase called CD45R

CD45R

PRO-B CELL
HAEMATOPOITIC STEM CELLS
 Origin of B lymphocytes
Pro-B cell proliferate &differentiate into precursor B cell (pre-B cell)

Pre-B cells lack surface Ig and light chains but have the µ heavy chains in
the cytoplasm

Then become immature B cells expressing monomer IgM on surface

 Origin of B lymphocytes

Full maturation is signaled by co-

expression of IgD & IgM on the
membrane

µ HEAVY CHAINS IgM IgM

IgD

PRE-B CELL
IMMATURE B CELLS MATURE B CELL
B Menu F
 Development of B
lymphocytes
In the bone marrow immature B cell with
only surface IgM is not functional and

Ag induces death rather than

differentiation
 Development of B
lymphocytes
Most of the immature B cell (Possessing IgM) leave the bone
marrow & migrate to the spleen

 In the spleen the immature B cell acquire IgD and become

mature B cell (possessing both IgM and IgD) and leave the
spleen

 Approximately 1 billion B cells are produced each day

and migrate to peripheral lymphoid organ

 Naïve B cell in the periphery have a short life span

 Dying within a few weeks by apoptosis, if not activated

Maturation of B cell
 Maturation of lymphocytes from bone
marrow stem cells consists of three
processers
 Proliferation of immature cells
 Expression of Ag receptor gene
 Selection of B cells that express useful Ag
receptor

 IL-7 stimulate survival and proliferation

earliest lymphocytes – produced by stromal
cells in bone marrow and thymus
Maturation and Selection of
B cell
 Selection is based on the
 Expression of intact antigen receptor
components, and
 What they recognize
 Pre-lymphocytes that fail to express
useful receptors die by apoptosis
 Mature lymphocytes are selected by the
process of
 Positive selection
 Negative selection
Positive selection and Negative selection

 Positive selection
 Immature B cells with weak recognition and
interaction with self Ag are selected to survive
 Immature T cells with weak recognition and
interaction with self MHC + peptide Ag are
selected to survive
 Negative selection
 Immature B cells with strong recognition and
interaction with self Ag are negatively selected
and eliminated
 Immature T cells with strong recognition and
interaction with self MHC + peptide Ag are
negatively selected and eliminated
Receptor editing
 Negatively selected B cells
 Either die by apoptosis, or
 may generate a second light chain by
editing the light chain gene and
 change the specificity of the Ag receptor
 a process called receptor editing
 Development of B
lymphocytes
 IN LYMPHOID ORGAN B CELL DEFEND US
IN 3 PHASES-

 recognition of danger
 production of weapons specific for the

invader
 transport of this weapons to the battle

field
 B cells
Types
 B1 cells
 B2 cells

 Marginal zone B cells

 Naive B cells
 Memory B cells
 B1 B cells

 Subset of B cell – 5% of total B cell

population
 Arises before the B2 B cell (follicular

B cell, conventional B cell, the

major group of B cell)
 Called B2 B cell because develop
after B1 cell
B1 B cells
 B1 B cell arise from stem cell during
fetal life
 Bear surface IgM but little or no IgD
 Self renewing and can generate naive B1
cell
 Display CD5 marker on the surface
 Major B cell population in peritoneal and
pleural cavities
 Activated in response to carbohydrate Ag
 Do not require T cell help for activation
B2 B cell
 The major group of B cell, called B2 B
cell because of their later appearance
 Also called –
 follicular B cell – because they reside in the
follicles of lymphoid organ
 Make the major bulk of T-dependant, class-
switched, high-affinity Ab response
 Respond against both protein and
carbohydrate antigen
 Give rise to long-lived memory cell
Characteristics of B1 and B2 cells

Characteristics B2 cells B1 cells

Source of new B cells Bone marrow Fetal liver, yolk sac - Self
renewing
Major sites and % 2nd lymphoid organ, 95% Peritoneal & Pleural cavity, 05%

Somatic hyper mutation Yes No

T cell help Required No

Isotype of Ig produced High level of IgG High level of igM

Response to CHO Ag Poor Definite & strong

Response to protein Ag Definite and strong Poor

Memory Yes Very little or none

sIgD on mature cell Present None or little

CD5 marker on the surface absent Present

Marginal zone B cells
 Located in the marginal zones of the
splenic white pulp
 Respond to blood-borne polysaccharide
antigens
 Like B1 B cells, express Ag receptors of
limited diversity
 Make predominantly IgM response
Characteristics of Naïve and Memory B cells

Characteristics Naïve B cells Memory B cells

Membrane markers
Ig IgM and IgD IgM, IgG, IgA IgE, IgD (?)
Complement R Low expression High expression
Anatomic location BM, LN and BM, LN and Spleen
Spleen
Life span Short lived Long lived

Receptor affinity Lower Higher

Adhesion molecule Low ICAM-1 High ICAM-1

ACTIVATION OF B CELL

Activation involves two “ways”

one way depends on T cell help, i.e., T-dependant activation

other is called T independent activation

 T-dependant activation
 Most proteins are T-dependent antigens.

 For activation, B-lymphocytes must

interact with effector T4-lymphocytes.

 All classes of antibody molecules can be

made against T-dependent antigens and

 There is usually a memory response

 Steps involved in T-dependant activation

5 general steps required

 Contact of antigen with B cell
 Clonal selection.
 Clonal expansion
 Differentiation of selected clones of B cells.
 Production of Memory cells .
 Contact of antigen with B
cell
 Antigens
encounter the B-
lymphocytes, and
bind with B cell
receptor in the
secondary
lymphoid organs
of the lymphoid
system
Clonal Selection
Clonal selection
Animation of Clonal Selection and Clonal Expansion
 Activation of naive B-lymphocytes by T-dependent
antigens…

 Once bound, antigen is

engulfed and degraded
with lysosomes

 During this process,

antigens are broken down
into peptide epitopes

 These peptides bind to

grooves in MHC-II
molecules which are then
transported to the surface
of the B-lymphocyte
 Activation of naive B-lymphocytes by T-
dependent antigens…

 Than the activated B-

lymphocyte will
interact with
subclasses of T4-
lymphocytes, primarily
Tfh cells, that
promote antibody
production
 Animation of An Effector T4-Lymphocyte
Recognizing Epitope/MHC-II on an Activated B-
Lymphocyte

 T4-lymphocyte uses
its TCR and CD4
molecule to bind to
MHC-II molecule with
epitope on activated
B-lymphocyte.

 This interaction,
triggers Th2-cell to
produce cytokines
 Fig. 3: Clonal Expansion

Cytokines from an activated T4-lymphocyte now enable the

activated B-lymphocyte to proliferate into a large clone of
identical B-lymphocytes. During this time, "fine-tuning" of the
B-cell receptor occurs through affinity maturation.
 Activation of naive B-lymphocytes by T-dependent
antigens…

Tfh cells produce cytokines

such as Il-4, Il-5, Il-6

Collectively these cytokines:

a. enable activated B-
lymphocytes to proliferate.
b. stimulate activated B-
lymphocytes to synthesize
and secrete antibodies.
c. promote the differentiation
of B-lymphocytes into
antibody-secreting plasma
cells.
d. enable antibody producing
cells to switch the class of
antibodies being produced
 Memory cells
 During proliferation
and differentiation
some of the B-cells
stop replicating and
become circulating,
long-lived memory
cells

 Memory cells are

capable of
anamnestic or
"memory“ response
 Memory cells
 These memory cells will initiate a rapid,
heightened secondary response against if
the same antigen again enters the body
 This is why the body sometimes develops
a permanent immunity after an infectious
disease and is also the principle behind
immunization.
 Many plasma cells migrate to the bone
marrow where they continue to secrete
antibodies for months or years after the
antigen has been eliminated
 Anamnestic Response
 After primary exposure,
there is an inductive
period of generally 1-2
weeks when no
measurable antibodies
are detected in the
serum
 In this period the antigen
is exposed to ICC,
processed by APCs,
clonal selection occurs,
and B-cells differentiate
into plasma & memory
cells
 a second exposure to the
same antigen results in
more antibodies being
made faster for a longer
 T-INDEPENDENT ANTIGENS

 T-independent (TI) antigens are usually large

carbohydrate, lipid and other protein molecules
with multiple, repeating subunits
 B-lymphocytes mount an antibody response to T-
independent antigens without the requirement of
interaction with T4-lymphocytes
 non-protein antigens cannot bind to MHC
molecules
 So, cannot be seen by T cells
ACTIVATION OF B-LYMPHOCYTES BY T-
INDEPENDENT ANTIGENS

 Bacterial LPS and capsular polysaccharides are

examples of T-independent antigens
 The resulting antibody molecules are generally
of the IgM isotype and do not give rise to a
memory response
 There are two basic types of T-independent
antigens: TI-1 and TI-2
 TI-1 activation

 2 ways
 Monoclonal activation
 Polyclonal activation
 TI-1 antigens include
lipopolysaccharide and bacterial
nucleic acid
 LPS can interact with two
different receptors on B cells
 Polyclonal activation - Toll-
like receptors (TLR 4,
TLR5,TLR7,TLR9)
 Monoclonal activation - B cell
receptor (BCR) – present only
in few B cell surface
 Flagellin – viaTLR 5
Toll-like receptors
 These antigens
activate B-cells by
binding to their
specific toll-like
receptors rather
than to B-cell
receptors
 TI-1 ANTIGENS
 TI-1 antigen are polyclonal B cell activators
(mitogens)
 They are able to activate B cells without
antigenic specificity
 Antibody generated against TI-1 antigens are
often called "natural antibodies" because they
are always being made against bacteria present
in the body
 T Independent Antigens (TI-1)

LPS binding
protein Bacterial Lipopolysaccharides, (TI-1
antigens), bind to host LPS binding
LPS protein (LPSBP) in plasma
TLR 4
LPS/LPSBP is captured by CD14
on the B cell surface

Toll - like receptor 4 (TLR4) interacts

with the CD14/LPS/LPSBP complex

CD14

Activation of B cell
B Cell
 T Independent Antigens (TI-1 e.g. LPS)
LPS complexes with CD14, LPSBP & TLR4

B B B B B B
Y Y Y Y Y Y
Six different B cells will require 6 different antigens to activate
them
At high dose TI-1 antigens (like LPS) will POLYCLONALLY ACTIVATE
all of the B cells irrespective of their specificity.
TI-1 antigens are called MITOGENS
YY YY YY YY YY YY
YY YY YY YY YY YY
Y YY YY YY YY YY Y
 TI-2 antigens
 TI-2 antigens, such as
capsular polysaccharides,
bacterial flagellin are
molecules with multiple,
repeating subunits

 These repeating subunits

activate B-lymphocytes
by simultaneously cross-
linking a number of B-cell
receptors
 TI-2 Antigen

Y
Mature

Y
B-1

Y
Y
Y Y

Y
Y Y
Y
Y Y
Y

Y
Y Y
Y
Y Y
Y Y
Y
Y
Y IgM
B cells are directly stimulated
by antigens containing
multivalent epitopes.
No T cells are necessary

Induces the expression of

natural antibodies specific for
 TI-2 DIFFER FROM TI-1 IN DIFFERENT RESPECTS:

TI-1 antigens TI-2 antigens

 TI-1 are B cell
 Are not B cell
mitogens mitogens
 do not act as
 Act as both polyclonal activator
monoclonal and
polyclonal activator
 TI-2 activate only
 TI-1 antigen mature B cell and
activate both inactivate immature
mature and B cell
immature B cell
 DIFFERENCE BETWEEN T- INDEPENDENT (TI)
AND T -DEPENDENT ANTIGEN

 TI antigen is generally
weaker
 No memory cell are
formed
IgM is the predominant antibody
secreted

These differences highlight the important role played by

TH cells in generating memory B cell, affinity
maturation and class switching
Why are babies unresponsive to TI-2 antigens?
In Adults: TI-2 Antigen

Y
Immature
B-2 Cell

Y
YY Mature

Y
B-1

Y
Y
Y Y

Y
Y Y
Y
Y Y
Y

Y
Y Y
Y
Y Y
immature B cells that bind to
Y
Y

Y
Y Y
multivalent self Ag undergo
apoptosis IgM
mature B cells are directly
stimulated by antigens containing
multivalent epitopes produce IgM
WITHOUT T cell help.
Why are babies unresponsive to TI-2 antigens?

In babies:
All B cells, B-1 & B-2, are immature

TI-2 Antigen

Immature
B-1 Cell
YY
Immature B cells that bind to
multivalent self Ag undergo
apoptosis
As with adult B cells, immature B cells that bind multivalent self Ag
undergo apoptosis
Hence babies do not respond to TI-2 antigens.
Babies are, therefore susceptible to pathogens with multivalent
antigens such as those on pneumococcus
 B Lymphocytes
Memory
cell
Binding of antigen to the Parent B
cell surface receptor
triggers proliferation Cell
and differentiation
Plasma cells
release antibodies
(AB)
PC PC

PC PC PC PC

Fate of B-cells after activation

 Immunoglobulin/ Antibody

 Antibodies are specific glycoprotein produced

by plasma cells in response to a specific
antigen and capable of reacting with that
antigen

 Immunoglobulin structurally similar animal

proteins that may or may not be endowed with
antibody activity.

Thus all antibodies are immunoglobulin but all

immunoglobulin are not antibodies however,
the terms are used interchangeably
Non functional immunoglobulins are produced in
multiple myeloma (plasma cell cancer)

These immunoglobulins can not bind with specific

antigen and are not antibodies.

Functional antibody means those who can

bind with antigen
Chemically-
Immunoglobulin is a glycoprotein composed
mainly of polypeptides and carbohydrate (3-18%)

Sometimes called γ- globulin, because

they are predominantly present in γ fraction
of globulin (after electrophoresis)

(A small proportion of immunoglobulin

is also found in α and β globulin fraction)

20% of proteins of plasma are γ- globulin

Serum Proteins

Figure 17.17
Immunoglobulin (antibody) is present

1. On surface of B lymphocyte

2. When activated, B lymphocyte is

transformed into plasma cell and
secretes immunoglobulin
Immunoglobulin/ Antibody
Surface immunoglobulin
on B cell

There are 10 lac immunoglobulins

on each B cell surface!!!

Stimulated B cell is transformed

into antibody forming plasma cell

plasma cell secretes

Immunoglobulin (Antibody)

Function of antibody:
It binds with antigen
 Antibody Structure

 Porter and Edelman –

won noble prize in 1972
 Composed of "Y"-
shaped macromolecules
called monomers
 A monomer is composed
of four peptide or
glycoprotein chains
 two identical heavy
chains and two
identical light chains

IgG
Antibody Structure
 The light chains are
of two varieties:
 kappa or lambda
 The Heavy chains are
of 5 types
γ α μ δ ε
 The four glycoprotein
chains are connected
to one another by
disulfide (S-S) bonds
and noncovalent
bonds
 Heavy chain of Ig - denoted by lower case Greek letter
µ for IgM, δ for IgD, γ for IgG, α for IgA and ε for IgE heavy
chain

Light chains of Ig belong to κ (kappa) or λ (lambda).

Any one of Ig contain only one type of light chain, either
κ or λ

Chromosomal location for human Ig

genes:

λ (lambda) light chain chromosome no. 22

 κ (kappa) light chain chromosome no. 2
 heavy chain chromosome no. 14
Basic Structure of Antibody
 Immunoglobulin G (IgG) can
be considered as the
prototype antibody
 It has a common structure
of 4 peptide chain:

Li

He
gh

av
tc
 Two identical light (L)

y
ha
in
chains about 25,000

(2
5k
d)
molecular weight
 Two identical heavy

chain (50kd)
chain (H) of molecular Bound together
Hinge region Disulphide bon
weight 50,000 or more
 Each L chain bound to a H
chain by a disulfide bond
 Similar disulfide bridges
link the two H chains
Antibody molecule comprises three
equal sized globular portions

Formed by two ARM and one TRUNK/ STEM

Joined by a flexible stretch known as

hinge region to form a crude “y”
shape

Each arm of “y” is form by association of a

light chain

with amino terminal half of heavy chain

 Carbohydrate

Model of Immunoglobulin:
Based on x-ray
crystallographic analysis Protein+ carbohydrate
= Glycoprotein
 Basic Structure of
Antibody
The trunk of the
“y” is formed
by pairing of
carboxy
terminal half of
the two heavy
chain
Structure of IgG Antibody
 Amino terminal

Fab

If treated with
Papain Fc
(an enzyme)
Above the Disulphide bonds

Carboxy terminal
 2 Fabs (separate, monovalent)

Papain treated
immunoglobulin

1 Fc
 Fab is the antigen binding site
(“Fragment, antigen binding”)

It is composed of part of heavy chain and

whole of light chain at amino terminal

Amino terminal
antigen

Fab

Function: antigen binding

 Fc is the crystalizable fraction
(“Fraction, crystalizable”)
( because it forms crystal on refrigeration)
It is the part of heavy chain at carboxy terminal

Fc region

Carboxy terminal

Function: 1.complement activation,

2. binding with phagocytes, NK cells etc
3. Other: passing to placenta, secretion
 Antibody molecule-
Bifunctional and Divalent
• Papain treated
antibody produce:
Bifunctional
• Two identical Fab-
fragments for antigen
binding
• One Fc fragment for
different biologic
activities
Divalent
• Bind two antigens
with two Fab
 If treated with
Pepsin
Below the Disulphide bonds
 Bivalent (Fab’)2

Pepsin treated
immunoglobulin

Fc
is disintegrated
 Amino terminal
VH

Variable region exists in amino

VL

terminal of both H and L chains

CH
1

Constant region exists in

CL

Carboxy terminal in both H and L

CH2

chain

Variable region: In Amino terminal

Their amino acid sequence varies for
different paratopes (106 idiotypes)
CH3

Carboxy terminal Constant region: amino acid sequence

is more or less constant
( 5 isotyoes: γ, µ, α, δ, ε and a few
allotypes)
 HYPERVARIABLE REGIONS

Hypervariable regions
Each of Variable domains of
H and L chain
Amino terminal contains 3 Hypervariable regions
Where they show exceptional variability
VH
VL

CH
1
CL

CH2

Hypervariable region is also called

Complementarity Determining Region (CDR)

Because it specifically binds with epitope

CH3

These are antigen binding sites (paratope)

Carboxy terminal
These segments are located near amino acid positions 30, 50 and
 HYPERVARIABLE REGIONS
•Only 5-10 amino acids in each hyper variable region form
the antigen binding site

Variable regions of the L and H chain are folded in such a

way that the regions of hyper variability are brought
together to create the surface structure that binds antigen
 Heavy chain variable
region (VDJ)
Light chain variable region(VJ)
Light chain variable
Constant regions of the light region(VJ)
and heavy chains

Class switching, while keeping the same variable region

(antigen binding site), is achieved by gene rearrangements

Makes  for
IgM

Makes  for
IgG
 Domains

Each heavy and light chain is folded into globular

segments. Each segment is called domain

Each domain consists of 100-120 amino acid

Foldings are held together by disulfide bonds

Amino terminal
VH VH

s
-s
s

s
-s
VL

-s
VL CH CH

s
s

s
-s
s -s

-s
-s
CL
CL
CH2 s-s CH2
s-s
s-s

s-s
CH3 CH2
s-s

s-s

Carboxy terminal
 Carbohydrate

Model of Immunoglobulin:
Based on x-ray
crystallographic analysis Protein+ carbohydrate
= Glycoprotein
 Domains
 Variable region of light
chain is called VL domains

 Variable region of heavy

chain is called VH domains

 Light chain has got only

One constant domain: CL
One variable domain: VL

 Heavy chain has got

3 to 4 constant domains:
CH1, CH2, CH3 or CH4
One Variable domain: VH
There are 4 Domains in
IgG, IgA and IgD:

1 VH
3 CH

& a Hinge region

Between CH1 and CH2

There are 5 Domains in

IgM and IgE:

1 VH
4 CH

No Hinge region
 VARIABLE REGION
DOMAIN
• First 110 amino acid of the
amino terminal domain in a
H and L chain is called
variable region, abbreviated
VH and VL respectively
• Each VH domains positioned
directly beside a VL domain,
and this pair of domains
together forms a single
antigen binding site
• Ag specificity of Ab is
determined by combined
sequences of its VH and VL
domains
 CONSTANT REGION
DOMAINS
 Take part in various
biological functions
determined by amino acid
sequence of each domain
• CH1 & CL Domains -serves to
extend the Fab arms of the
antibody molecule
• Thus facilitating interaction
with antigen and increasing
maximum rotation of Fab arms
• Other Domains ( CH2, CH3,
CH4) act as Fc receptor and
complement binding site for
ICC.

•
 CONSTANT REGION
DOMAINS
Depending on the class and
subclass of antibody,
biological activities of the Fc
portion of antibodies include
the ability to:
 activate the
complement
pathway (IgG & IgM);
 bind to phagocytes
(IgG); or
 bind to mast cells,
basophils, and
eosinophils (IgE);
 bind to NK cells (IgG).
 HINGE REGION DOMAIN
• IgG, IgD, and IgA contains
an extended peptide
sequence, called the
hinge region between
CH1 AND CH2 domains
• This region, is rich in
prolin residues and is
flexible giving IgG, IgD,
and IgA segmental
flexibility
• As a result, two Fab arms
can assume various
angles to each other
when antigen is bound
• It has no homology with
the other domains
THE CORRESPONDING DOMAIN OF THE TWO GROUPS
ARE AS FOLLOWS

IgA, IgD, IgG IgM, IgE

CH1/CH1 CH1/CH1

HINGE REGION CH2/CH2

CH2/CH2 CH3/CH3

CH3/CH3 CH4/CH4

Although CH2/CH2 domain in IgE and IgM occupy the

same position in the polypeptide chains as the hinge region
in the other classes of the Ig -
function of this extra domain has not determined
 Trans-membrane and Secreted
Immunoglobulin
Immunoglobulins can exist, either as-
Membrane Bound Immunoglobulin (MIg),

also known as B cell receptor (BCR); or as

Secreted Immunoglobulin (SIg)
1. Membrane –bound immunoglobulin (mIg)
(on B cell surface)

2 types of
immunoglobulins

2. Secreted immunoglobulin (SIg)

(secreted by plasma cell)
 Trans-membrane Immunoglobulin

• Displayed on surface of B cell as surface antibody (BCR/MIG)

• Through these BCR/MIg, the B cell is able know that its

cognate antigen is “out there”

• When BCR recognize and binds to its cognate antigen, that B

cell is selected to proliferate and make a clone of B
cells

• Most members of this clones differentiate into plasma B cell,

and

• Produce secreted form of immunoglobulin (SIg) with the

same specificity
 Figure 43.8 Antigen receptors on lymphocytes

Antigen-
Antigen- Antigen- binding
binding binding site site
Disulfide
site V bridge

V
Variable

V
Light
V

regions V V
chain C
C

C C Constant C C
regions
Transmembrane
region

Plasma
Heavy chains membrane  chain  chain
Disulfide bridge T cell
B cell Cytoplasm of B cell Cytoplasm of T cell
(a) A B cell receptor consists of two identical heavy (b) A T cell receptor consists of one
chains and two identical light chains linked by  chain and one  chain linked by
several disulfide bridges. a disulfide bridge.

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

 Fc domain in SIg differs in structures &
function from the corresponding domain
in MIg
Fc domain of MIg contain three
regions:
 An extra cellular hydrophilic
sequence
 A hydrophobic
Transmembrane sequence
 A short cytoplasmic tail
The hydrophobic
Transmembrane sequence is
25 amino acid residues
This anchors it to the surface of
the B lymphocytes
 Difference between SIg and MIg

 Secretory Ig (SIg)  Membrane Ig (MIg)

 Transmembrane domain  Transmembrane
is absent in SIg domain is present in
MIg
 SIg, e.g., IgM and IgA
are pentamer and  MIg are monomers
dimmer respectively

 5 types of SIg
 IgM and IgD act as
MIg in naïve B cell
 Bi-functional  Function of MIg is
Ag recognition
A MEMORY B CELL can express MIgM, MIgG, MIgA,
or MIgE

All MIg in a single cell is identical, so Ab

molecule binds to same Epitope
Sequencing of H chain DNA revealed - all
the CH segments have two additional M1
and M2 exones
These M1 and M2 exones are responsible
for encoding the Transmembrane and
cytoplasmic segments respectively in
MIg
 HOW THE BCR SIGNALS

 Function of BCR
 Recognition of the epitope
and
 to signal this recognition to
the nucleus of the B cell to
activate the genes
involved
Transmission of signal from
BCR to the genes of
nucleus involved two
accessory proteins, Igα
& Ig β
 HOW THE BCR SIGNALS
 These Igα & Igβ associate with the H
chain and protruded in Cytoplasm of B
cell

Thus, the complete BCR has two part

 The H/L chain (BCR) - outside the cell

recognize the Ag but can’t signal,

and the
 Ig α & Ig β proteins that can signal,
but cannot recognize antigen
 HOW THE BCR SIGNALS
• In order for a B cell to signal, many BCRs must be
brought close together on the surface of B cell
• This clustering of BCRs can result when BCRs
bind to an epitope
• The reason for necessity of clustering is that, the
tails of Ig α and Ig β signaling molecules interact
with enzymes inside the cell
• When enough of these interactions are
concentrated in one region, it sets of an
enzymatic chain reaction
• This sends a message to the nucleus of the cells
saying BCR engaged
 Typing of Antibody
Isotype
Idiotype
Allotype
 Isotype
Depending on the difference in
amino acid sequence in H
chain constant region –
• Abs are Classified into 5
types known as isotype
 each class plays a different
role in the immune
response
 IgG
 IgM
 IgA
 IgD
 IgE

Isotype -typing of Ig depending

Classes (isotypes) of Immunoglobulins

Immunoglobulin G (IgG):
have 2 identical γ heavy chains
(& 2 κ or 2 λ light chains)
Immunoglobulin M (IgM):
have 2 identical µ heavy chains
(& 2 κ or 2 λ light chains)
Immunoglobulin A (IgA):
have 2 identical α heavy chains
(& 2 κ or 2 λ light chains)
Immunoglobulin E (IgE):
have 2 identical ε heavy chains
(& 2 κ or 2 λ light
Immunoglobulin D (IgD):
chains)
have 2 identical δ heavy chains
(& 2 κ or 2 λ light chains)
Immunoglobulin (Ig)

IgG

IgA IgM
Isotypes are antigenically different.

Hence, antibody against each isotype can be

produced: such as anti- IgG, anti-IgM etc

γ isotype is subdivided into

4 sub-isotypes: γ 1, γ 2, γ 3, γ 4

α isotype is subdivided into

2 sub-isotypes: α 1, α 2
Allotype

Due to 1 or 2 amino acid substitutions, alternative

antigenic forms of IgG or IgA or κ- light chain occurs

Like blood group, each person have one of

the several allotypes of heavy or light chains
Each person inherits different allelic genes
(codominant) for such allotype

(Allele: Each of many alternative forms of a

gene)

Allotype exists in
IgG ( expressed as Gm1, Gm2…Gm20)
IgA ( expressed as Am1…Am3)
κ chain ( expressed as κm1…km3)
 Idiotype

It is the amino acid sequence responsible for

paratope formation in hypervariable region

Each clone of immunoglobulin has got different

antigen binding site (paratopes) at its Fab portion

Each is called idiotype

There are approximately 108
idiotypes in an individual
This means there are
approximately 108 B cell clones in
an individual
Antibodies against idiotype can be
Idiotypes
produced: (Anti idiotype antibody)
 IMMNOGLOBULIN G (IgG)
Most abundant (80%) class in
serum (13.5mg/ml)

Consists of two γ H chain and

two κ or λ L chain

Molecular formula γ2κ2 or

γ2λ2)

γ H chain contain one variable

and three constant domain
(Cγ1, Cγ2, Cγ3 ) - involved
in the different effector
functions

Produced during secondary

 Physical Property
molecular wt. - 150,000


Sedimentation Coefficient - 7s


serum half life, 7- 21 days (IgG3- 7 days)


 Subclasses
• Four subclasses,
distinguished by
differences in γ chain
sequence
• Numbered according
to decreasing serum
concentrations
 IgG1-60-70%
 IgG2-14-20%
 IgG3-4-8%
 IgG4-2-6%
 Biologic activity

1. activate the classical complement pathway

2. bind to macrophage and neutrophils for enhanced

phagocytosis

3. bind to NK cells for antibody-dependent cytotoxicity

(ADCC).

4. enables it to cross the placenta (IgG is the only class of

antibody that can cross the placenta and enter the
fetal circulation)

5. Co-Agglutination – IgG has reactivity with

Staphylococcal Protein A and mediate co-agglutination
 IgM (Immunoglobulin M)
•Is the first antibody produced during primary immune
response

Also first antibody produced by neonates

•

First to appear in phylogeny (Phylogeny means evolutionary

•
development; Lamprey eel has IgM
 Physical Property

• Molecular formula µ2κ2 or µ2λ2

• molecular weight of monomer

MIgM :1,80,000

• pentamer SIgM has mol. wt. of 9,00000

• Sedimentation Coefficient (S) -19

• Contain 15% carbohydrate

 IgM (Immunoglobulin M)

• Approximately 13% of the serum antibodies

(1.5 mg/ml).
• Present as receptor (monomer) on B cell
surface
• Secreted from plasma cell as pentamer
(µ2L2)5 Linked by disulfide bond at CH3
and CH4 with an additional J chain
• IgM has 10 antigen binding sites
On B cell surface

In serum
 IgM (Immunoglobulin M)

 Each pentamer
contains a J (Joining)
chain - a
polypeptide linked
with Carboxyl-
Terminal end of  H
chain
 J chain is required for
polymerization of
monomer to form
pentamer IgM before
secretion
 Biological Properties

Complement activation-
 Most efficient in activating complement

Complement activation requires close

proximity of 2 Fc regions. Pentavalent
IgM fulfills that need

Neutralization of virus-
 less IgM than IgG is required to

neutralize viral infectivity

 Biological Properties

Agglutination of RBC-
 more efficient in binding with RBC than with

any other isotypes

 100-1000 times more molecules of IgG than

IgM is required to achieve the same level of

agglutination

Like IgA, it can also be transported to external

secretions
 But not as abundant as IgA (because of larger

size )
 The Secretory component allows IgM to be

transported across epithelial linings

 IMMUNOGLOBULIN A (IgA)
 Most abundant class of Ab in the human body
(both external and internal body fluids)
 Foecal matter contain 30% of IgA
 IgG is the most abundant antibody class in the
blood

 Everyday human secretes - 5 to10gm SIgA into

mucus secretions to defend 400 sq. m of mucosa

 4 mg/ml, i.e. 10% - 15% of total IgA are secreted

in the blood.
 Physical Property

 Molecular formula α2κ2 or α2λ2)

 Mol wt. 160, 000 to 600, 000 Dalton

 Serum half life of 6 days

 Contain 10% carbohydrate

 Contain two subclasses- IgA1 and IgA2

present in a ratio of up to nine to one
 Secretory IgA

• Present in blood as monomer

• Present in secretion (Milk,

colostrum, tear, saliva, mucous
of GIT, RT and genital tract) as
dimer or polymer

• Secretory IgA is a dimer and has

4 Fab sites

• polymerized by a polypeptide
chain called J-Chain (MW 15000)

• Function of J-Chain is to
Polymerization of IgA
IgA
 Structure
 Serum - monomer
 Secretions (sIgA)
 Dimer (11S)
J chain
 Secretory
component

Secretory J
Piece Chain
 Secretory Component

• Secretory dimer of IgA contains a polypeptide

chain (70,000 mw) called secretory component
• produced by epithelial cells of mucous membrane

• responsible for transporting polymeric IgA across

the cell membranes into the mucous secretions

• It masks Hinge region from protease cleavage in

Protease- rich mucosal environment

• Pentameric IgM also transported into the mucous

secretions by same mechanism
Origin of Secretory Component of sIgA
 Transcellular Transport

Plasma cell migrate to sub epithelial

tissue

Secreted IgA binds with poly- Ig

receptor on outer surface of
mucosal epithelium

Endocytosis of the complex occurs,

a vesicle is formed

enzymatic cleavage of receptor

occurs

Portion of the receptor remains

attached to IgA as secretory
component
 Biological function of IgA

IgA plays important role in mucosal

immunity provide first line of defense
specially against
 Salmonella

 Vibrio

 N. gonorrhoeae

 Polio

 Influenza

 Rota virus
 Biological function of IgA
Breast milk contains secretory IgA and protect the newborn against
infection during the first month of life

Binds to bacterial and viral antigen and prevent attachment to

mucosal cells
IgA cannot bind to complements
 IMMUNOGLOBULIN E (IgE)
IgE makes up about 0.002% of the serum antibodies


 Present in extremely low concentration due to

 short half life of two days

slow rate of synthesis and

rapid binding to mast cell and basophil

 Physical Properties

 IgE is a monomer and has 2 epitope-binding sites

 Consists of two light chain and two heavy chain

with an extra domain (CH4) similar to IgM

 Molecular formula ε2 κ2 or ε2λ2

 Molecular weight - 1,90,000 Dalton with a

sedimentation coefficient of 8

 Contain 18% carbohydrate

 Biological Functions

 The Fc portion of IgE made against parasitic worms

(helminths) and arthropods can bind to eosinophils
enabling opsonization

 This is a major defense against helminths

 Strongyloides
 Ascaris
 Trichinella
 Hookworm

 IgE may protect external mucosal surfaces by

promoting inflammation, enabling IgG, complement
proteins, and leucocytes to enter the tissues
Parasitic infection cause production of IgE

IgE is attached to parasites

Eosinophil has Fc receptor for IgE

Through Fc attachment
parasite comes close to eosinophil

Eosinophil release
lethal substances that kill parasites

This is an example of ADCC

(antibody dependant cellular
cytotoxicity)
How IgE helps host against parasites

helminth Epitope for attachment of IgE

FcR for
IgE
 Biological Functions
 Mediates type 1 hypersensitivity
(anaphylactic) reaction

 The Fc portion of IgE can bind to mast

cells and basophils where it mediates
many allergic reactions.

 Cross linking of cell-bound IgE by

antigen triggers the release of
vasodilators for an inflammatory
response
 How IgE mediates
Type 1 hypersensitivity (anaphylaxis)

Allergen

IgE
Further exposure of allergen
causes attachment of and
IgE
cross bridging with adjacent IgE
IgE
receptor

Mast cell

Granules
containing
Histamine
& other

Deregulation and release of histamine

and other substances

Histamine and other products

are responsible for anaphylaxis
 IMMUNOGLOBULIN D (IgD )
Physical Properties

 IgD is a monomer and has 2 epitope-

binding sites

 Consists of two heavy chains and two

light chains
 Molecular formula δ2κ2 or δ2λ2

 Present in minute concentrations in blood

and other body fluids (30 µg/ml)
 Biological Functions
 Commonly found on the surface of B-
lymphocytes (along with monomeric IgM) as
a B-cell receptor or sIg where it may control
of B-lymphocyte activation and suppression
• binds with antigen and transmit signals to
the cell interior and activate B cells
• when B cells become activated, surface IgD
expression ceases
 IgD may play a role in eliminating B-
lymphocytes generating self-reactive auto-
antibodies
 Biological Functions
 Role in the physiology of B cells is under
investigation
• Isolated reports of IgD with Ab activity
towards insulin, penicillin, milk protein,
diphtheria toxoid, nuclear components, and
thyroid antigens
• Its presence on many mature naïve
lymphocytes has suggested as an unproven
role in B cell differentiation or tolerance
IgG IgA IgM IgD IgE
 Generation of Antibody Diversity

• 100 million different types of invaders are

present in the environment
• 100 million different types of recognition
molecules are required to recognize them
• Each B cell makes only one kind of antibody
• So, the question is how to make 100 million
different B cell that can recognize the
invaders
• This riddle solved by Susumu Tonegawa,
received Nobel prize in 1987
 Diversity

 Tonegawa, hypothesized that genetic arrangement for

receptors of B and T cells in mature and immature B cell
and T cells are different

 This change is enough to generate 100 million different

antibodies

• He compared DNA sequence of light chain from a mature B

cell with the DNA sequence of the light chain from an
immature B cell

• And found that, mature Immunoglobulin genes are made by

modular design
 DNA for the heavy chain in the immature B-cell
includes four types of modules (gene segment)

Variable (v)
Diversity
(d),
Joining (j)
and
Constant
(c)
There are multiple copies of these modules, each of which
is slightly different from the other

100 different v segments, 4 different d segments

6 different j segment and about 10 c segments
 To make a mature heavy chain, the B cell chooses one of each kind of
segment and paste them
IMMATURE B CELL DNA
V D J CM CD CG3 CA1 CG2 CG4 CG3 CE CA2

100 >4 6

choice of gene segments by recombination

V1 D2 J1 CM CD

MATURE B CELL DNA

The magic of this “mix and match” strategy is that by using modular
design only a small number of gene segments is required to create 100
million different B cell
Animation of Gene Translocation

Gene translocation determines the amino acid sequence of the

variable portion of the heavy and light chains that form the Fab of
an antibody molecule. The variable portion of the heavy chain is
determined after one of the multiple genes of the VH region join
with one of the genes of the DH region and one of the genes of
the JH region. Likewise, the variable portion of the light chain is
determined after one of the multiple genes of the VL region join
with one of the genes of the JL region. (The numbers of the V, D,
and J genes are approximations.)
One progeny (clone) of B cell
will multiply and produce
similar cell with similar paratope

Clone: means an exact copy of an

animal or a plant
 One cell will always
produce similar paratope

But the class may change

from IgM to IgG or IgA or IgE
from time to time

This is called class switching

IgM

Paratope is same but

IgG Class is different

IgA
 Class switch

IgM
IgG
 heavy chains
 heavy chains

In order to change the function of the

molecule but keep the specificity, one
needs to conserve the light chain and the
heavy chain variable region and replace
the heavy chain constant region
Animation of Gene Translocation

Gene translocation determines the amino acid sequence of the

variable portion of the heavy and light chains that form the Fab of
an antibody molecule. The variable portion of the heavy chain is
determined after one of the multiple genes of the VH region join
with one of the genes of the DH region and one of the genes of
the JH region. Likewise, the variable portion of the light chain is
determined after one of the multiple genes of the VL region join
with one of the genes of the JL region. (The numbers of the V, D,
and J genes are approximations.)
 Class switching
• Class switching occur among constant region
genes
• i.e., the same VH exon can associate with
different CH genes in the course of immune
response
Class switching, while keeping the same variable region
(antigen binding site), is achieved by gene rearrangements

Makes  for
IgM

Makes  for
 How does class switching
occur?
Arrangement of VDJ (for
heavy chain) and VJ (for
light chain) occurs first

 VDJ or VJ for one

particular B cell always
remains same
 Then a constant C gene

is added to make it VDJC

or VJC
This constant C gene
comes from any one of
µ,γ,α,ε,δ genes
 VDJ for one particular B cell always remains same

But any of different C (µ,γ,α,ε,δ) is attached with VDJ.

Later, another isotype C gene is attached

Cµ with VDJ by splicing

Cγ1
splicing
Cε
 Why IgM at primary
immune response?
 IgM is formed first because the µ constant region is closest to
the VDJ-DN

 Later the µ constant region can be switched with a γ ε or α

constant region to from the heavy chain of IgG, IgE or IgA
respectively

 That’s how different classes of immunoglobulins are produced

from the same clone
Class switching, while keeping the same variable region
(antigen binding site), is achieved by gene rearrangements

Makes  for
IgM
 Class switching
 Further exposure
of B cell with same
Ag will induce
constant H-chain
DNA to undergo
rearrangement
• So same variable
region VH DH JH
unit can combine
with any CH gene
 Class switching
Thus plasma cells producing IgM in
primary immune response will switch to
 IgE in parasitic infestation or
 IgA in respiratory tract infection

This change is known isotype switch and is

stimulated by cytokines release by TH
cells
 Control of Isotype switch
Isotype switch is controlled by the

cytokines of TH cells that

B cells encounter during class switching
Cytokines controlling Isotype
switch
Certain cytokines Of T helper cells influence B cells to
switch to one isotype or another

If B cells exposed to an environment rich in IL-4 and IL-5,

they switch isotype from IgM to IgE appropriate for
protection against worms
 Cytokines controlling
Isotype switch
 If there is INF γ around, B cells switch to
produce IgG 3, that work against bacteria or
viruses

 Or if TGF β is present during class switching,

change from IgM to IgA appropriate for the
common cold

 Once a B cell has class switched a certain H

chain gene, it can no longer make that
class of H chain, as the intervening DNA
is excised
 Isotype switch
Class switching occurs with H chain,
L chain don't undergo class
switching
 Isotype switch
In hyper IgM syndrome, the failure to interact properly results in

•inability of B cell to switch to production of either IgG,

IgA or IgE
therefore only IgM is made
•
 SOMATIC HYPERMUTATION
The overall mutation rate of DNA in
mammalian cell is extremely low,
about one mutated base per trillion base per
DNA replication cycle
SOMATIC HYPERMUTATION
 However, in the chromosomes of B cells-the
regions that contain the v, d and j segments,
 an extremely high rate of mutation can take
place

In these regions, mutation rates are as high as

 one mutated base per thousand bases per

generation

This high rate of mutation is called somatic

hyper- mutation
 SOMATIC HYPERMUTATION
Somatic hyper mutation occur after class switching has taken
place
So, hypermutation is a relatively late event in the maturation B
cells
B cells that still make IgM Abs have not undergone hypermutation
 SOMATIC HYPERMUTATION
Somatic hypermutation changes the part
of the rearranged antibody gene
•that encodes the antigen binding region
of the antibody
 SOMATIC HYPERMUTATION
Depending on mutation there are 3 possible outcome can occur

The affinity of the Ab

 may remain unchanged or
it may be increased or

it may be decreased

 SOMATIC HYPERMUTATION
If the affinity of the Ab is increased

B cell with higher affinity Ab are stimulated more often

(because their BCRs bind better)
They proliferate more than B cell with lower affinity receptors

AFFINITY MATURATION
The result is a collection of B cell
whose receptors have a higher
average affinity for their Ag

This process is called “affinity

•

maturation”
 Affinity Maturation
By going through the processes of class switching and
affinity maturation cells can change both their

 Fc region (by class switching) and

Fab region (by somatic hypermutation)
 Antibodies are divalent
and bi-functional

Divalent - Ab bind two Ag at the same time by

Fab fragments

Bi-functional - in addition to binding to Ag,

Fc fragment posses special receptors for

complement, Placenta & cells like neutrophils,
mast cells, macrophage etc.
 Surface immunoglobulins

A. Surface immunoglobulins present on B cell

act as receptor for binding with antigen.

After binding, B cells are stimulated to be

transformed into plasma cell

Plasma cells produce and secrete

Immunoglobulins called antibodies
B) These secreted Antibodies binds with antigen
and ultimately neutralize, remove or destroy them

But this mere binding can not destroy the antigen

Antigen Antibody

Antibody act as a target setter

After attachment with antigen, it makes itself

a target of macrophage, NK cell or complement
Destruction or removal of antigen is done by

Collaborative interaction between antibody and

some cells, proteins and tissues

Fab is responsible for antigen binding only

Fc region is responsible for

collaborative interactions
 1. Opsonization

It is the enhancement of phagocytosis

Fc of antibody causes opsonization

(Other example of opsonin: C3b, a product of complement)

Macrophage and neutrophil have

Fc receptors (FcR) on their surface

When Fab of antibody is attached with an antigen,

its Fc can be attached to Fc receptor of the phagocyte

Phagocytosis becomes easy due to close approximation

 Antigen

Macrophage

Macrophage
 Fc receptor
Antigen

antibody

Macrophage

Phagocytosis
 Activation of complement

A Antigen antibody reaction causes activation

of classical pathway of complement
End result is:
formation of tube like structure called
MAC (membrane attack complex) bacteria

MAC perforates the organism and

causes lysis

B C3b is a by product of
complement activation

It also act as an opsonin

 Antibody dependent
cell mediated cytotoxicity (ADCC)

Virus infected cells can be killed by this way

Antiviral antibody becomes attached to virus (by Fab)

Fc region of antibody becomes attached to

NK cell (NK cell has got Fc receptor )
cell

NK cells then release perforin which

perforates virus infected cell
Neutralization
antibodies bind to and block specific sites on viruses
or exotoxins

thus preventing these antigens from binding to

receptors on tissue cells
 Fate of B cell after
activation
 B cell has two fates
to choose after
activation

 To become
 a plasma cell or
 a memory cell
 Plasma cells
Plasma cells are antibody factories
If a B cell chooses to become a plasma cell, it
begins to produce the secreted form of the
BCR, antibody molecules

Plasma cells produce about

 two thousand antibodies each second, and as

a result,
 live only for a few days
 MEMORY B CELLS

Memory B cells remember the

• first exposure to a pathogen, and
•defends against subsequent
exposures
 MEMORY
 Once interaction with antigen occurs: Memory
cells will be produced

 Develops from previous exposure to foreign

material

 Memory cells are long lived B and T cells

 During further exposure memory cells will be

activated and will give heightened and more
effective immune response

 This attributes can provide life long immunity

 B Lymphocytes
Memory
cell
Binding of antigen to the Parent B
cell surface receptor
triggers proliferation Cell
and differentiation
Plasma cells
release antibodies
(AB)
PC PC

PC PC PC PC
 Example

A. Many infectious diseases do not occur twice.

Infection gives rise to memory cells which will
prevent further infection.
e.g., measles, mumps, polio, diphtheria, hepatitis
B. Vaccines (artificial immunity) can provide Life long
immunity

In Innate immunity
no such memory exists.

Immunity does not improve

on repeated exposure
 Memory B cells
Memory B cells have undergone class switching

Their constant region of class switched Ab is

appropriate to defend
against the invader they remember
 Memory B cells
Memory B cells have reduced requirements
for activation relative to naïve B cells
They are “ready to go” to defend against a
second attack
 Life long immunity
Memory B cell can confer life long immunity
to infection
• In 1782 Swedish traders brought measles virus
to the Faeroe islands
• In 1846, another ship carrying sailors with
measles visited the island
• People who were older then 64 years did not
contract the disease
• This indicate that they still had antibodies
against the measles virus
 Maintenance of Life long
immunity
How this long lasting B cell immunity
is maintained is under debate
•Some evidence suggests that memory B cells
can live a long time
 Maintenance of Life long
immunity
Other experiments indicates that
• memory B cells are short lived,
• but can be periodically proliferate when
re-stimulated by Ag
• These could be antigen that is left
around after the attack,
• These Ags are similar to the invader to
re-stimulate memory B cells
 NATURAL ANTIBODIES

A high proportion of circulating antibodies

normally able to bind to
• pathogen specific molecules such as
bacterial LPS or Mannan

 Because these antibodies are continually

expressed even without specific
immunization
 they are called natural antibodies
 NATURAL ANTIBODIES
• Other natural Abs react with endogenous
Ags that are
• expressed by injured or distressed host
cells when a tissue is deprive of O2
• preexisting Abs may bind the injured
cells activate complement, and
immediately trigger an immune
response
 Preparing Antibodies

 Typically BALBc mice

 Sometimes Rat (ascites fluid)
 Monoclonal:
 Individual B lymphocyte hybridoma is
cloned and cultured.
 Secreted antibodies are collected from
culture media.
 Antibodies recognize one antigenic
binding site of the antigen.
 Antibodies are prepared in
animals by
• injecting the animal with
a specific antigen and
• collecting the serum after
antibodies are produced
 Monoclonal Antibodies

 Major breakthroughs in immunology

occurred when monoclonal antibody
technique was developed

 Monoclonal antibodies are antibodies of

a single specific type coming from a clone
of identical B-lymphocytes.
 Techniques

 an animal is injected
with the specific antigen
for the antibody desired
 After an appropriate
time for antibody
production, the animal's
spleen is removed
 The spleen is rich in B-
lymphocytes and plasma
cells
 each plasma cell is
genetically programmed
to produce only one
specificity of antibody
 Techniques

 Plasma cells , however, will not

grow artificially in cell culture
 Therefore, a plasma cell
producing the desired
antibody is fused with a
myeloma cell –
 a cancer cell, which will grow
rapidly in cell culture - to
produce a hybridoma cell
 The hybridoma cell has the
characteristics of both parent cells
 it will produce the specific antibody
of the plasma cell and will
 grow readily in cell culture like the
myeloma cell.
Fig. 1: Production of Monoclonal Antibodies, Step-1
Fig. 2: Production of Monoclonal Antibodies, Step-2
Fig. 3: Production of Monoclonal Antibodies, Step-3
 Monoclonal
antibody
+

B cell Myeloma cell

(cancerous plasma cell)

Hybridoma

Hybridoma will produce unlimited

number of immunoglobulin of from single clone
Monoclonal Antibodies

Monoclonal antibodies are now used routinely

 in medical research and diagnostic serology
 also being used for modulating immune responses to produce immunosuppression for treatment of
 autoimmune diseases and graft versus host disease,
 prevention of allograft rejection
 Monoclonal antibodies against normal antigens or receptors on human B-lymphocytes and/or T-
lymphocytes are used to
 destroy the lymphocytes
 Monoclonal antibodies have also been tried clinically against a number of viruses and bacteria.
Human Monoclonal Antibodies

 Most monoclonal antibodies used to date have been produced using rodent
cells
 but these antibodies will not activate the human complement pathways or
participate in opsonization
 They also have a short survival time in humans
 Genetically engineered antibodies are now being produced whereby
 the Fab portion from rodent antibodies is attached to the
 Fc portion of human antibodies
 The bacteriophages are subsequently mixed with an antigen to select for
those producing complementary Fabs
 Those bacteriophage genomes are then converted into plasmids that
can subsequently produce soluble specific Fabs in bacteria.
Human Monoclonal Antibodies

 Human monoclonal antibodies have also been produced by

 "immortalizing" human B-lymphocytes with the Epstein-Barr virus and
 by using special immunodeficient mice immunologically reconstituted
with human immunocompetent cells or tissues
 Monoclonal antibodies are also being produced by
 recombinant DNA techniques by inserting random human genes coding for
the variable Fab portion of human antibodies
 into the genome of filamentous bacteriophages
 As the bacteriophages replicate, they display the Fab portion of the
antibody on their surface
 Blocking Type-1 Hypersensitivity Using Monoclonal Antibodies
Against IgE

A new experimental approach to treating and preventing Type-I

hypersensitivity involves giving the person with allergies injections of
monoclonal antibodies that have been made against the Fc portion of
human IgE. This, in turn, blocks the attachment of the IgE to the Fc
receptors on mast cells and basophils and the subsequent release of
histamine by those cells upon exposure to allergen.
Types of acquired immunity

On basis mode of action

On basis of acquirement
 Humoral
Acquired Immunity
Cell mediated

Natural Artificial

Active Passive
Active Passive

Clinical or Vaccination
sub clinical
infection ADS, TIG
Mother to fetus
ON THE BASIS OF
ACQUIREMENT
Natural

Artificial
Active Naturally Acquired Immunity

 refers to the natural

exposure to an infectious
agent

 body responds by making

its own antibodies
 Passive Naturally Acquired
Immunity
The placental transfer of IgG from mother to fetus during
pregnancy

These antibodies generally lasts 4 to 6 months after birth

 The IgA and IgG found in human colostrum and milk

of babies who are nursed
 Human milk…
• In addition to the IgA and IgG, human milk
also contains:
• gamma-interferon, a cytokine that enhances the
activity of certain immune cells;
• hormones and growth factors that stimulate the
baby's gastrointestinal tract to mature faster and
be less susceptible to infection;
• lysozyme to break down peptidoglycan in bacterial
cell walls.
• Breast-fed infants have a lower incidence of
gastrointestinal infections, respiratory infections,
urinary tract infections, and meningitis
Artificial

Active
Passive
 Active immunity

Body actively takes part in production of

immunity

•Develops after giving a safe form of the

antigen artificially
•Produce immune response (e.g.,
antibodies ) and, develop circulating, long-
lived memory cells
 Active immunity
If we again exposed to the same antigen, the memory cells will cause
immediate and rapid production of the appropriate antibodies for
protection

Since body produces its own immunity: it takes some time (few weeks) to
develop

But it remains in body for a prolonged period

 Active immunity- Types
With artificially acquired active immunity, one is immunized
with one or more of the following:
attenuated microbes

Killed vaccines

 Toxoid

Vaccines produced by recombinant DNA technology

Attenuated vaccines
Attenuated microbes are living, nonvirulent strains of a microbe

Attenuated vaccines tend to be immunologically quite effective

Microbes can multiply in the body, thus increasing the amount

and persistence of the antigen for a greater antibody response
Attenuated vaccines
Living attenuated microbes can
sometimes be potentially dangerous to
highly immunosuppressed individuals

Examples
 the MMR vaccine
 oral polio vaccine
 the yellow fever vaccine
 varicella zoster virus vaccine
Killed vaccines

 killed organisms, fragmented microorganisms,

or antigens produced by recombinant DNA
technology

a. Whole cell
 IPV or inactivated poliomyelitis
 Rabies vaccines
 Influenza vaccines
b. Fragments (polysaccharides)
 Meningococcal
 Pneumococcal
 H.influenzae type b
 Vaccines produced by
recombinant DNA technology
– Hepatitis B vaccine (HepB),
 the first human vaccine produced by
recombinant DNA technology;

– Experimental HIV vaccines for AIDS;

– Acellular pertussis (aP)
– Hepatitis A vaccine (HepA);
– vaccine against Lyme disease
 Toxoid
Toxoid is an exotoxin treated so as to be nonpoisonous but still
immunogenic

The body responds by making antibodies capable of neutralizing

the exotoxin

the diphtheria and tetanus components of the DT vaccines

 Adjuvant
The antigen may be adsorbed to an adjuvant, a
substance such as aluminum hydroxide or aluminum
phosphate

Adjuvant is not immunogenic but increases the

persistence of the antigen, to heighten the response
 Passive artificially acquired
immunity
Sometimes we need antibodies on immediate basis

In such cases preformed antibodies are introduced

Body does not takes part in production of immunity

 Passive immunity
Passive artificially acquired immunity acquired from
the injection of

 antibody-containing serum or
 immune globulin, from another person or animal or
 the injection of monoclonal antibodies

The injection of serum carries a greater risk of

allergic reactions - than the injection of antigens
during active immunization.
 These allergic reactions are referred to as serum
sickness
 Passive immunity
Since the body is not making its own antibodies and memory cells are not
produced

So, passive artificially acquired immunity is

short lived and

offers only immediate, short term protection.
Examples

 the use of pooled adult human immune globulin (IG)

to prevent hepatitis A and measles and to prevent
infections in people with certain immunodeficiency
diseases;

 human HBIG to prevent hepatitis B in those not

actively immunized with the HepB vaccine;

 human TIG to prevent tetanus in those not actively

immunized with the DTP, DTap, or Td vaccines;

 RhoGAM to prevent Rh hemolytic disease of

newborns;
 Examples

 VZIG to prevent varicella;

 CMV-IGIV to prevent cytomegalovirus infections in highly

immunosuppressed individuals;

 RIG to prevent rabies, given concurrently with active

immunization with the rabies vaccine;

 the antisera used for botulism;

 IVIG (intravenous immune globulin), used to reduce

infections in people with certain immunosuppressive
diseases
 primary immunodeficiency syndrome
 chronic lymphocytic leukemia
 certain autoimmune diseases
 immune thrombocytopenia purpura (ITP)
 and Kawasaki disease.
 Active- Passive artificial
immunity
Tetanus provides a nice example of how active
immunization (tetanus toxoid) and passive immunization
(TIG) may be used in preventing a disease
 Active immunity Passive immunity

Antigen is introduced Preformed antibody is introduced

Takes time to develop Readymade, so, Immediate effect

Immunity: long lasting Short lasting

(B and T cells are active) (B and T cells do not take part)

Memory: present Absent

Hypersensitivity: May occur

Does not occur (foreign serum can do it)

Example: Example:
Infection Antibody of mother (placenta/milk)
Vaccination Antisera (horse sera, human globulin)
 Probable questions

What do you mean by specificity and diversity

of adaptive immunity ?

Write in brief difference between active and passive immunity

Give examples of active and passive immunity

Why passive immunity does not last long?

How does cell mediated immunity work?

What do you mean by self and non self differentiation ?