Microbiology & Immunology [BBT302]

Introduction to Unit 3

[What, Why, Where of Immunology]

What: The contents of the Unit 3 include Introduction to immune system: Innate and Adaptive
immunity, Humoral and Cell mediated immune response, Cells and Molecules of the immune
system, Primary and Secondary lymphoid organs, T cell maturation and its activation & B cell
maturation and its activation, Characteristics and types of Antigens, Haptens, adjuvants and
Epitopes, Antibodies: Structure, functions and characteristics of different classes of antibodies.
Monoclonal antibodies.
Why: The study of immunology explains the features, components and processes of immune
system. It explains about the process of infection and defense of our body against the infectious
agents. It also explains the importance of defense proteins of our body i.e. Antibodies.
Where: Immunology explains the host-pathogen interaction. It also helps in understanding the
mechanism of immunity which prevents the occurrence of diseases. The study of classes of
antibodies helps in disease diagnosis and cure. The immune-pathology labs routinely use this
knowledge for diagnostics purpose. The immune-techniques relied on structure and features of
antibodies.
 INNATE IMMUNITY (First line of defense)
The four components of innate immunity are:
1. Anatomical (or Physical) Barriers
2. Physiological and Chemical Barriers
3. Phagocytic Barriers
4. Inflammatory response
A. Physical Barriers :
Physical barriers that prevent pathogens from entering the body, destroy them after they enter, or flush
them out before they can establish themselves in the hospitable environment of the body’s soft
tissues. The different modes of barrier defenses are associated with the external surfaces of the body
from where pathogens may try to enter.
Skin: The skin is physical barrier of body. Its outer tough layer prevents the entry of bacteria and viruses.
Additionally, alkaline sweat and other skin secretions may lower pH, contain toxic lipids, and physically
wash microbes away, Keratin a water-proof protein etc.
Another barrier is the saliva in the mouth, which is rich in lysozyme—an enzyme that destroys bacteria
by digesting their cell walls. The acidic environment of the stomach, which is fatal to many pathogens, is
also a barrier.
Mucus layer of the gastrointestinal tract, respiratory tract, reproductive tract, eyes, ears, and nose traps
both microbes and debris, and facilitates their removal.
Respiratory tract: waxy nasal hair, desquamation, mucus lining that traps and prevents infection;
mucociliary escalator system upward motion away from the lungs. Microorganisms and dust particles
can enter the respiratory tract with air during breathing which are trapped in the mucus. The cilia sweep
the mucus loaded with microorganisms and dust particles into the pharynx (throat).
GU tract: urine flushing action, urethra length.
GI tract: peristalsis
B. Physiological and Chemical Barriers :
The physiologic barriers that contribute to innate immunity include temperature, pH, and various soluble
and cell associated molecules.
High Temperature (Fever) is characterized with increase in body temperature which inhibits creates
unfoavorable conditions (temp.) for the growth of pathogens.
Low pH also creates unfoavorable conditions (temp.) for the survival of pathogens. Highly acidic pH of
gastric juice in stomach kills most of the ingested pathogens.

Chemical barriers (Secretions) for Innate defense

Site Source Secretions Components of secretions
Eyes Lachrymal glands Tears lysozyme, IgA, IgG
Ears Sebaceous gland Cerumen (ear wax) Waxy substance
Oral cavity Salivary glands Saliva lysozyme, digestive enzymes, IgA
Skin Sweat glands Sweat High NaCl (alkaline pH)
sebaceous glands Sebum Low chain-fatty acids (acidic pH)
Stomach Gastrointestinal tract Gastric juice acidic pH
Urine Urea
Vagina Lactic acid acidic pH
Anti-bacterial peptides Cercopins, Defensins, beta-lysin, lactoferrin
Interferons (chemokines) Maintains anti-viral state (limits the spread of viral
infection to nearby healthy cells)
 C. Phagocytic Barriers:
The function of ingesting and destroying microbes is mediated by phagocytes, like neutrophils,
macrophages and natural killer cells (NK cells).
Neutrophils: Neutrophils or polymorphonuclear leukocytes are the most abundant population of circu-
lating white blood cells. Neutrophils are spherical cells of about 12-15 μm diameter and with numerous
ciliary projections. The cytoplasm is granular. The granules are of two types. Specific granules are filled
with degradative enzymes, such as lysozyme, collagenase and elastase.
Macrophages: Macrophages and their precursors, called the monocytes play a central role in both
innate and acquired immunity. Macrophages may assume different morphologic forms, some develop
abundant cytoplasm and are called epitheloid cells. Macrophages are found in sub-epithelial connective
tissue, in the interstice of parenchymal organs, in the lining of the vascular sinusoids in the liver and
spleen and in the lymphatic sinuses of lymph nodes.
Process of Phagocytosis and killing of microbes:
Innate immunity is that defence mechanism which ingests pathogens via phagocytosis by the
invagination of the plasma membrane (Fig. A). During phagocytosis the plasma membrane expands
around the particulate material to form large vesicle called phagosome. The phagocytic vesicles or
phagosome contain the ingested foreign particles Then these vesicles fuse with primary lysosomes to
form phagolysosome which later form secondary lysosomes. Primary lysosomes includes degrading
enzyme with proteases, nucleases, lipases and hydrolytic enzyme, which digest macromolecules within
secondary lysosomes and break into certain small products.
Before forming phagosome, macrophages and neutrophils are attracted towards a variety of substances
generated in an immune response.

Fig. A - PHAGOCYTOSIS
 D. Inflammatory Response :
Tissue damage caused by a wound or by an invading pathogenic microorganism induces a complex
sequence of events collectively known as the inflammatory response. Inflammation is a defensive
response of the body to tissue damage. The signs and symptoms of inflammation are redness, pain, heat
and swelling. Inflammation can also cause the loss of function in the injured area, depending on the site
and extent of the injury. These signs were described by the Roman physician Celsus in the first century
AD, as the “four cardinal signs of inflammation” rubor (redness), tumor (swelling), calor (heat), and
dolor (pain), functio laesa (loss of function).
The cardinal signs of inflammation reflect the major events of an inflammatory response : (Fig B)
Tissue Injury- The injured cells stimulate the release of mast cell granules and their potent inflammatory
mediators such as histamine, leukotrienes, and prostaglandins. The chemical mediators released from
the cells initiate the series of events which are as follows-
1. Vasodilation – It is an increase in the diameter of blood vessels of nearby capillaries. Histamine released
from mast cells increase the diameter of local blood vessels (vasodilation), causing an increase in blood
flow. The engorged capillaries are responsible for tissue redness (erythema) and an increase in tissue
temperature.
2. Increased Vascular Permeability- At the same time, inflammatory mediators increase the permeability
of the local vasculature, causing leakage of fluid into the interstitial space, resulting in the swelling, or
edema, associated with inflammation.
3. Influx of phagocytes - Neutrophils and monocytes from the blood are attracted to the site of infection
by chemotaxis. The emigration of phagocytes is a multistep process that includes adherence of the cells
to the endothelial wall of the blood vessels (margination), followed by their emigration between the
capillary endothelial cells into the tissue (diapedesis or extravasation), and, finally, their migration
through the tissue to the site of the invasion (chemotaxis). As phagocytic cells accumulate at the site
and begin to phagocytose bacteria, they release lytic enzymes, which can damage nearby healthy cells.
The accumulation of dead cells, digested material, and fluid forms a substance called pus.

Fig B. Major events of inflammatory response

One of the principal chemical mediators of the inflammatory response is histamine. Another important
group of inflammatory mediators are (i) small peptides called kinins, are normally present in blood
plasma in an inactive form. Tissue injury activates these peptides like Bradykinin, which stimulates pain
receptors in the skin, (ii) enzymes of the blood-clotting system which gets activated and results in the
blood clot formation. It is the first step towards wound repair. (iii) acute-phase proteins (ex. C-reactive
protein) activates complement system resulting into pathogen destruction and clearance.

Types of inalammation-
Acute inflammation is a short-term inflammatory response against tissue damage which develops
immediately. Phagocytes are mainly involved in the process. If the cause of the inflammation is not
resolved, however, it can lead to chronic inflammation, which is associated with major tissue destruction
and fibrosis.
Chronic inflammation is ongoing or persistent inflammation which may extend upto months or years. It
can be caused by foreign bodies, persistent pathogens, and autoimmune diseases such as rheumatoid
arthritis. Phagocytes and lymphocytes are involved in the process.

x x
 CELLS OF IMMUNE SYSTEM
Primary cells-
Primary cells of IS are Lymphocytes, which are also the central cells of the immune system. These
are responsible for adaptive immunity and the immunologic attributes of diversity, specificity,
memory, and self/nonself recognition. Lymphocytes constitute 20%–40% of the body’s white blood
cells and 99% of the cells in the lymph.
Two types:
 B lymphocytes (B cells) —
Bone marrow is the major site of synthesis and maturation of B cells in a number of mammalian
species, including humans and mice.
They produce antibodies and help alert the T lymphocytes.
Express surface receptor- membrane-bound immunoglobulin (Antibody) molecules which has an
identical binding site for antigen. Also have many other surface molecules (MHC II, CD40, B220
etc.).
B cell types:
i) Naïve B cells- cells that have not interacted with
antigen are referred to as naïve. They have a
short life span. On interaction of with antigen, B
cells proliferate and differentiate into effector
cells or into memory cells.
ii) Effector cells or Plasma cells- the antibody-
secreting effector cells of the B cell lineage.
They function in various ways to eliminate
antigen. These cells have short life spans,
generally ranging from a few days to a few
weeks.
iii) Memory cells- express membrane bound
antibodies. They have long life (~20 years) and
thus is responsible for life-long immunity to
many pathogens.
Role of B lymphocytes- Once lymphocytes spot the antigen, they begin to secrete antibodies.
Antibodies are special proteins that lock on to specific antigens which generates humoral response for
eliminating antigen.
It protects against extracellular antigens.

 T lymphocytes — they destroy compromised cells in the body and help alert other leukocytes. T
lymphocytes derive their name from their site of maturation in the thymus.
These cells have membrane rec ptors for antigen known as TCR (T-cell re eptor). It does not
recognize free antigen but recognizes antigen that is bound to MHC molecules. Also express other
membrane molecules (CD3, CD28, either CD4 or CD8).

T cell types:
i) Naïve T cells- cells that have not interacted with antigen are referred to as naïve. They have a short
life span. On interaction of with antigen, T cells undergo proliferation and differentiation into Th and
Tc cells.
ii) Th (T helper) cells – have CD4 membrane-
bound receptor. It is of two types: Th1 and Th2
cells which provide help for both humoral and
cell-mediated immune response.
iii) Tc (T cytotoxic cells) - have CD8 membrane-
bound receptor. These cells mediate killing of
infected or tumor cells. Two types: effector cells
and Memory cells.
The role of T lymphocytes- They destroy the infected cells and protects against intracellular
antigens.

Secondary cells
These cells surround and absorb pathogens and break them down, and work together for destroying
them. There are several types, including:
 Mononuclear phagocytic cells -
Monocytes — these are mobile mononuclear phagocytic cells circulating in the blood. Monocytes are
smaller than macrophages and have lesser organelles especially lysosomes.
Macrophages — these are mononuclear phagocytic cells located in the tissues, hence also known as
fixed phagocytic cells. These cells are differentiated/ originated from blood monocytes. Free
macrophages travel by amoeboid movement throughout the tissues and patrol for pathogens to
remove dead and dying cells
Fixed macrophages serve different functions in different tissues and are named to reflect their tissue
location. examples are-
i. Pulmonary alveolar macrophages in the lung
ii. Histiocytes in connective tissues
iii. Kupffer cells in the liver
iv. Glomerular Mesangial cells in the kidney
v. Microglial cells in the brain
vi. Osteoclasts in bone
vii Synovial A cells in joints
viii Sinus or circulating cells in spleen and lymph nodes

Function: Macrophages are capable of ingesting and digesting exogenous antigens, such as whole
microorganisms and insoluble particles, and endogenous matter, such as injured or dead host cells,
cellular debris, and activated clotting factors, by the process of phagocytosis.
Mcarophages destroy the pathogen by two ways:
1) oxygen dependent: formation of byproducts of respiration: O2-, H2O2, OH-, O-
2) oxygen independent: lysosomal enzymes, acidic hydrolases etc.

The macrophage membrane has receptors for certain classes of antibody (Fc.R). If an antigen coated
antibody binds to FcR (antibody receptors) on the macrophage membrane then antigen is
phagocytosed more readily and phagocytosis is enhanced. Thus, antibody functions as an opsonin, a
molecule that binds to both antigen and macrophage and enhances phagocytosis. The process by
which particulate antigens are rendered more susceptible to phagocytosis is called opsonization.
 Dendritic cell- These cells are called so because the cell is covered with long membrane
extensions that resemble the dendrites of nerve cells. There are many types of dendritic cells, which
have the same major function, the presentation of antigen to TH cells.
Four types of dendritic cells are:
Langerhans cells in skin (epithelial and mucous membrane)
Interstitial dendritic cells in heart, lungs, liver, kidney, GIT etc.
Circulating dendritic cells (veiled cells) in blood and lymph nodes
Lymphoid dendritic cells in T cell areas of lymphoid tissue.
express high levels of membrane receptors for
They all constitutively express high levels of antibody, which allows the binding of antigen-
both class II MHC molecules and members of antibody complexes and help in generating B cell
the co-stimulatory ‘B7’. They are more potent responses.
antigen-presenting cells. Dendritic cells acquire
antigen by phagocytosis or endocytosis; the
antigen is processed, and mature dendritic cells
present it to TH cells which is required for the
initiation of immune responses against the
antigen.
Another type of dendritic cell, the follicular
dendritic cell, does not arise in bone marrow and
has a different function from the antigen-
presenting dendritic cells. These are located in B
cells rich lymph follicles in the lymph node. FDC
 Antigen Presenting Cells (APCs) – Three cells of immune system are characterized as APCs
namely B cells, Macrophages and Dendritic cells. APCs process the antigens and present it to MHC
molecules for the recognition by Th or Tc cells to generate immune response for the elimination of
antigen.

Granulocytes: These are a type of white blood cells that contain small granules in their cytoplasm.
There are mainly three types – Neutrophils, Eosinophils and Basophils, which derived their names
from their staining characteristics. Mast cells are also a type of granulocyte that is present in tissues.

 Neutrophils — The most common type of phagocyte and are the most abundant granulocyte (50-
70%) in blood circulation.
They are stained by neutral dyes and are characterized by having multi-lobed nucleus, hence also
known as Polymorphonuclear (PMN) cells.
They move through the capillary endothelium and are first to reach the site of inflammation. They are
chemically drawn to bacteria by chemotaxsis and migrate through tissue to the site of infection.
Neutrophils are phagocytic in that they engulf the target cell. Neutrophilic granulocytes play
important role in inflammation.
Neutrophils have two types of granules; primary (azurophilic) granules (found in young cells)
and secondary (specific) granules (which are found in more mature cells). Primary granules are
larger and denser and a type of lysosome containing cationic proteins and defensins that are used to
kill bacteria, proteolytic enzymes and cathepsin G to break down (bacterial) proteins, lysozyme to
break down bacterial cell walls, and peroxidases (used to generate toxic bacteria-killing
substances). The small secondary granules contain compounds that are involved in the formation of
toxic oxygen compounds, lysozyme and lactoferrin (used to take essential iron from bacteria).
 Eosinophils - make up approximately 1-3% of circulating leukocytes and are stained by eosin
dye. Their nucleus is bi-lobed.
Eosinophils become increasingly active during parasitic infections and allergic reactions. Eosinophils
are involved in allergic reactions and can attack multicellular parasites such as worms.
Eosinophils play a crucial part in the killing of parasites (e.g., enteric nematodes) because their
granules contain a unique, toxic basic protein and cationic protein (e.g., cathepsin) receptors that bind
to IgE to promote pathogen killing bound to Ig E.
 Basophils are the least common type of granulocyte, making only 0.5% of the circulating blood
leukocytes. The cytoplasm of basophils contains a varied amount of granules; these granules are
 usually numerous enough to partially conceal the nucleus. Granule contents of basophils are abundant
with histamine, heparin, chondroitin sulfate, peroxidase, platelet-activating factor, and other
substances. Basophils are also involved in allergic reactions and are able to release histamine, which
helps to trigger inflammation, and heparin, which prevents blood from clotting. Histamine dilates
blood vessels, increases the permeability of capillaries, and increases blood flow, which helps to
transport leukocytes to infected areas. Heparin thins the blood and inhibits blood clot formation.

 Mast cells are a type of granulocyte whose

granules are rich in heparin and histamine.
Mast cells are important in many immune
related activities from allergy to response to
pathogens and immune tolerance. These cells
found to to be associated with basophils
during inflammatory response.

 Natural killer cells (NK cells)- These large, non-phagocytic, granular lymphocytes. They account
for 5% to 10% of all lymphocytes in the circulation. These are lymphocytes by origin but are
involved in innate immunity.
Natural killer (NK) cells are so named because they easily kill cells infected with viruses. They are
said to be “natural killer” cells as they do not require the same thymic education that T-cells require.
They contain cytoplasmic granules which are rich in granzyme and perforins (similar to T cells). NK
cells kill virus-infected cells by injecting it with chemicals released by granules. They are important
in defending against viruses and possibly preventing cancer as well. They are particularly important
in the defense against herpes viruses.

Molecules of immune system

 Complement proteins- These are large number of secreted proteins which works in a cascade
manner to activate complement pathway for antigen killing. The activated component such as C3b,
C4b, C5b of the complement protein mediate antigen killing and the smaller fragments like C5a, C4a
and C3a released on activation, mediate inflammatory response against the antigen.
 MHC molecules- These are glycoproteins which are expressed on the cell surface. These are the
antigen presenting molecules which present the processed antigenic fragment to the respective T
cells. It is of two types namely: Class I and Class II MHC molecules.
 Cytokines- These are the chemical mediators involved in the immune response. These are of variety
of types; Interferons, Interleukines, TNF etc. which play important role in regulating different
processes of immune system such as hematopoeisis, B & T cell activation and inflammatory
response.
 Organs of Immune System
Primary and Secondary Lymphoid Organs
A number of diverse organs and tissues which are involved in the development of immune responses
are known as lymphoid organs. These can be distinguished by function as the primary and
secondary lymphoid organs.
1) Primary lymphoid organs- The thymus and bone marrow are the primary (or central) lymphoid
organs because these are the site of production and maturation of lymphocytes (B and T cells).
2) Secondary lymphoid organs The lymph nodes, spleen, and various mucosal-associated lymphoid
tissues (MALT) such as gut-associated lymphoid tissue (GALT) are the secondary (or peripheral)
lymphoid organs, which trap antigen and provide sites for mature lymphocytes to interact with that
antigen.
3) In addition, tertiary lymphoid tissues such as Cutaneous associated lymphoid tissues (CALT),
which normally contain fewer lymphoid cells than secondary lymphoid organs.

Fig.A- Lymphoid organs

Primary lymphoid organs

a) BONE MARROW
Bone marrow is the site of B-cell origin and development. Arising from lymphoid progenitors,
immature B cells proliferate and differentiate within the bone marrow. The stromal cells within the
bone marrow interact directly with the B cells and secrete various cytokines that are required for
their development. During B cell maturation, a selection process within the bone marrow eliminates
B cells with self-reactive antibody receptors.

b) THYMUS
The thymus is the site of T-cell development and maturation.
Structure: It is a flat, bilobed organ situated above the heart. Each lobe is surrounded by a capsule
and is divided into lobules, by connective tissue called trabeculae. Each lobule is organized into two
compartments: the outer compartment, or cortex, is densely packed with immature T cells, called
thymocytes, whereas the inner compartment, or medulla, is sparsely populated with mature
thymocytes.
Medulla is sparsely populated because only 5%
of the immature thymocytes reach maturity.
Both the cortex and medulla also consists of
epithelial cells, dendritic cells, and
macrophages, which make up the framework of
the organ and contribute to the growth and
maturation of thymocytes.

Function of the thymus is to generate and select a repertoire of T cells that will protect the body
from infection.
The activity of thymus increases with age and reaches to maximum at puberty and then decline/
atrophies.

Fig B – Cross section of lobule of thymus

Secondary lymphoid organs

These are the organized lymphoid tissues which consist of aggregates of lymphocytes called
lymphoid follicles, and nonlymphoid cells (macrophages). The lymphoid follicle called a primary
follicle, comprises a network of naïve lymphocytes. It is a tightly packed cluster of cells. After an
antigenic challenge, a primary follicle becomes a larger secondary follicle consisting of
proliferating lymphocytes. Secondary follicle have central cavity called germinal center where
lymphocyte activation and differentiation occur.
Naïve lymphocyte + Ag ---------> Activated lymphocyte
 Lymph nodes & Spleen – Both are the most highly organized secondary lymphoid organs.
Mucosal-associated lymphoid tissue (MALT) found in various body sites are less-organized sec.
lymphoid tissue.

a) LYMPH NODES
Lymph nodes are the sites where immune responses are mounted against antigens carried in
lymph.
They are encapsulated bean-shaped structures containing a reticular network packed with
lymphocytes, macrophages, and dendritic cells. Lymph nodes are the first organized lymphoid
structure to encounter antigens that enter the tissue spaces.
Structure: A lymph node can be divided into three regions: the cortex, the paracortex, and the
medulla. The outermost layer, the cortex, contains lymphocytes (mostly B cells), macro-phages, and
follicular dendritic cells arranged in primary follicles. Beneath the cortex is the paracortex, which is
populated largely by T lymphocytes and also contains interdigitating dendritic cells. These
interdigitating dendritic cells express high levels of class II MHC molecules,which are necessary for
presenting antigen to TH cells. The innermost layer of a lymph node, the medulla, is more sparsely
populated with lymphoid-lineage cells mainly antibody- secreting plasma cells.

Function: As antigen is carried into a regional node by the lymph, it is trapped, processed, and
presented together with class II MHC molecules by interdigitating dendritic cells in the paracortex,
resulting in the activation of TH cells. Once activated, TH and B cells form small foci consisting
largely of proliferating B cells. Some B cells within the foci differentiate into plasma cells secreting
IgM and IgG.
b) SPLEEN
The spleen plays a major role in mounting immune responses to antigens in the blood stream.
While lymph nodes are specialized for trapping antigen from local tissues, the spleen specializes in
filtering blood and trapping blood-borne antigens; thus, it can respond to systemic infections.
Bloodborne antigens and lymphocytes are carried into the spleen through the splenic artery.
Structure: It is a large, ovoid secondary lymphoid organ situated high in the left abdominal cavity.
The spleen is surrounded by a capsule that extends a number of projections (trabeculae) into the
interior to form a compartmentalized structure.
The compartments are of two types, the red pulp and white pulp, which are separated by a diffuse
marginal zone.
The splenic red pulp consists of a network of macrophages and numerous red blood cells
(erythrocytes) and few lymphocytes; it is the site where old and defective red blood cells are
destroyed and removed. Many of the macrophages within the red pulp contain engulfed red blood
cells or iron pigments from degraded hemoglobin.
The splenic white pulp surrounds the branches of the splenic artery through which blood-borne
antigens enter the spleen. The white pulp is organized into periarteriolar lymphoid sheath (PALS)
which is populated mainly by T lymphocytes and is attached to B cells rich area. The marginal
zone, located peripheral to the PALS, is populated by lymphocytes and macrophages.
Function: Blood-borne antigens and lymphocytes enter the spleen through the splenic artery, which
empties into the marginal zone. In the marginal zone, antigen is trapped by interdigitating dendritic
cells, which carry it to the PALS. Here lymphocytes interact with antigens and undergo proliferation
and differentiation generating immune response against it.

Fig C- Cross section of Spleen

c) MUCOSAL-ASSOCIATED LYMPHOID TISSUE

The mucous membrane lining of the digestive, respiratory, and urogenital systems are the major sites
of entry for most pathogens. Therefore these membrane surfaces are defended by a group of
organized lymphoid known collectively as mucosal-associated lymphoid tissue (MALT). This
includes- lamina propria of intestinal villi, tonsils and appendix, Peyer’s patches. These lymphoid
tissues are populated with lymphocytes, macrophages, granulocytes, and mast cells. The B cells are
organized into follicles and germinal centers.
The components of MALT are sometimes subdivided into the following:
 GALT (gut-associated lymphoid tissue. Peyer's patches which are found in the lining of the
small intestines.)
 BALT (bronchus-associated lymphoid tissue)- lungs
 NALT (nasal-associated lymphoid tissue- Tonsils)- nasopharynx
 CALT (conjunctival-associated lymphoid tissue)- eyes
 LALT (larynx-associated lymphoid tissue)
 SALT (skin-associated lymphoid tissue)
 VALT (vulvo-vaginal-associated lymphoid tissue)
 TALT (testis-associated lymphoid tissue)
The tonsils defend against antigens entering through the nasal and oral epithelial routes. The mucous
membrane of gastrointestinal tract, the respiratory and urogenital tracts, has the capacity to
endocytose antigen from the lumen.
The epithelial cells of mucous membranes play an important role in promoting the immune response
by delivering small samples of foreign antigen from the lumina of the respiratory, digestive, and
urogenital tracts to the underlying mucosal-associated lymphoid tissue. This antigen transport is
carried out by specialized M cells. The structure of the M cell is striking: these are flattened
epithelial cells lacking the microvilli and in addition have a deep invagination, or pocket. This
pocket is filled with a cluster of B cells, T cells, and macrophages. Luminal antigens are
endocytosed into vesicles that are transported from the luminal membrane to the underlying pocket
membrane. The vesicles then fuse with the pocket membrane, delivering the potentially response-
activating antigens to the clusters of lymphocytes contained within the pocket.
Antigens transported across the mucous membrane by M cells can activate B cells within these
lymphoid follicles. The activated B cells differentiate into plasma cells, which leave the follicles and
secrete the IgA class of antibodies. These antibodies then are transported across the epithelial cells
and released as secretory IgA into the lumen, where they can interact with antigens. Mucous
membranes are therefore an effective barrier to the entrance of most pathogens and thereby
contribute to nonspecific immunity.

Fig D- (a) Structure of M cell , (b) Function of M cell

 Tertiary lymphoid tissue
Cutaneous-Associated Lymphoid Tissue (CALT)
Also referred as Skin Associated Lymphoid Tissue (SALT)
The skin is an important anatomic barrier to the external environment. It is composed of epidermis
and dermis.
The epidermal (outer) layer of the skin is composed largely of specialized epithelial cells called
keratinocytes. These cells secrete a number of cytokines to induce a local inflammatory reaction,
express class II MHC molecules and may function as antigen-presenting cells. Among the epithelial-
cell of the epidermis Langerhans cells, a type of dendritic cell are also found, which internalize
antigen by phagocytosis or endocytosis. The Langerhans cells then migrate from the epidermis to
regional lymph nodes, where they differentiate into interdigitating dendritic cells and function as
potent activators of naive TH cells.
The underlying dermal layer of the skin contains scattered CD4+ and CD8+ T cells and macrophages.
Most of these dermal T cells were either previously activated cells or are memory cells.
 B cell Development- Maturation, Activation and Differentiation
B cells or B lymphocytes, fu ction in the humoral immunity by secreting antibodies. B-
cells present antigen (they are also classified as professional antigen-presenting cells (APCs) and
secrete cytokines. B cells express B cell receptors (BCRs) on their cell membrane. BCRs allow the
B cell to bind to a specific antigen, against which it will initiate an antibody response.

B cell Development- It occurs in 3 stages-

a) Maturation - Antigen Independent phase
b) Activation and Proliferation - Antigen dependent phase
c) Differentiation - Antigen dependent phase

B cell Maturation
In mammals, B cells are produced and matured in the bone marrow, which is at the core of
most bones. In birds, B cells mature in the bursa of Fabricius, a lymphoid organ.
B cells develop from hematopoietic stem cells (HSCs) that originate from bo e marrow. HSCs
first differentiate into Myeloid and Lymphoid stem cells. From lymphoid cell mass, B & T
lymphocytes are originated. The stages of development of lymphocytes are marked by
various gene expression patterns.

- B cells proceed through a number of distinct maturational stages:

1) Pro-B cell 2) Pre-B cell 3) Immature B cell 4) ature B cell

Pro-B cells –
Committed to the B lineage
- Express B220 (CD45R) - a B-lineage specific isoform of CD45,
- Express no Ig but the process of rearranging the heavy chain genes (D-J) occurs.
They are Igα/Igβ positive - Co pletion of heavy chain rearrangement marks the transition to
the pre-B cell stage.
Pre-B cells –
Express rearranged the heavy (H) chain locus but have not yet rearranged a light chain locus.
Express the µ heavy chain on their surface in association with the “surrogate light chain” to
form the “pre-BCR”. - Are also positive for CD25 (IL-2Rα).
Immature B cells –
Have successfully rearranged b th a IgM heavy chain allele and a light chain llele (κ or λ light
chains )- Express mIgM (not IgD) on their surface –
Still express RAG-2 and low levels of RAG-1. This allows for receptor editing. Eventually RAG-1
and RAG-2 expression terminat s and the cell differentiates into a mature B cell.
Negative Selection - Negative s lection occurs through the binding of self-anti en with the BCR;
If the BCR can bind strongly to self-antigen, then the B cell undergoes one of four fates: clonal
deletion, receptor editing, anergy or ignorance (B cell ignores signal and continues
development). Self-reactive immature B cells (mIgM) binding to self antigens are deleted in the
B.M. Only 10% exit the B.M. Receptor editing rescues cells that failed negati e selection edits
light chain.
This negative selection process leads to a state of central tolerance, in which the mature B cells
don't bind with self antigens will only survive.

NEGATIVE SE ECTION

Mature B cells –
Express both mIgM and mIgD o their surface

B cell development: from stem cell to immature B cell

To complete development B cells migrate from the bone marrow into the spleen as transitional
B cells, passing through two tr nsitional stages: T1 and T2. Throughout their migration to the
spleen and after spleen entry, they are considered T1 B cells. Within the spleen, T1 B cells
transition to T2 B cells. T2 B cells differentiate into either follicular (FO) B cells or marginal zone
(MZ) B cells depending on signals received through the BCR and other receptors.Once
differentiated, they are now considered mature B cells, or naive B cells.
 B cell activation
B cell activation occurs in the secondary lymphoid organs such as the spleen and lymph nodes
which receive a constant supply of antigen through circulating lymph. B cell ac ivation begins
when the B cell binds to an anti en via its BCR.
B cell activation process is dependent on antigens which are of two types
1) Thymus-independent (TI) antigens - Independent of TH cells
2) Thymus-dependent (TD) antigens- Dependent on TH cells

TI Antigens - Antigens that acti ate B cells without T cell help are known as T cell-independent
(TI) antigens.
 These are simple and repetitive antigens- usually polysaccharides or lipopol saccharides (e.g.
bacterial capsules).
 Don’t generate strong immu e response.
 No memory cells produced. IgM is the only antibody class produced, a d the immunity
doesn’t last long.
 B cell response to these antigens is rapid, though antibodies generated tend to have lower
affinity and are less functi nally versatile than those generated from T cell-dependent
activation.
 They are further of two types: (A) TI-type 1= LPS (B) TI-type 2= polymers (flagellin, bacterial
cell wall components, etc)
Type I T-independent antigens: are mitogens (polyclonal activators) such as
lipopolysaccharide (LPS) that activate B cells via nonspecific binding to B cell s rface molecules.
Any B cell, irrespective of its antigen specificity, can be activated by such molecules.
Type II T-independent antigens: are usually linear polymeric antig ns that have a
repeating unit structure such a polysaccharides. The repeating structure allows simultaneous
binding to, and cross-linking o , multiple BCRs. This massive BCR cross-linking is thought to
provide a sufficient activation signal to over-ride the need for T cell help.

TD Antigens - Antigens that activate B cells with the help of T-cell are nown as T cell-
dependent (TD) antigens and include foreign proteins. B cells activated by TI antigens need
additional signals to complete activation, but instead of receiving them from T cells, they are
provided either by recognition and binding of a common microbial constituent or by extensive
crosslinking of BCRs to repeated epitopes on a bacterial cell possibly undergo immunoglobulin
class switching, and differentiate into short-lived plasmablasts that produce early, weak
antibodies mostly of class IgM, ut also some populations of long-lived plasma cells.

Activation of B cells by TD antigens-

Once a antibody binds a TD antigen, the antigen is taken up into the B cell through receptor-
mediated endocytosis, degrade , and presented.
1. The antigenic peptides are presented by class II MHC on their surface to CD4+ helper T cell
(i.e. TCR-Antigenic peptide- class II MHC trimolecular interaction). The t o cells interact,
forming a tight T-B cell conjugate.
2) Interaction between the B7- CD28 molecules. This is known as co-stimulatory signal. It
promote T cells to express CD40L.
3) CD40L – CD40 interaction = T cells express CD40L on its surface which can in eract with CD40,
which is expressed on the B cell to provide a signal that is essential for B c ll activation and
proliferation. CD40L promotes cell proliferation, immunoglobulin class switc ing, and somatic
hypermutation as well as sustai s T cell growth and differentiation
4) The B cell then expresses receptors for cytokines (IL-R) produced by the T cell, including IL-2,
IL-4 and IL-5. As a result of signals received from cytokines and from the CD40-CD40L
interaction, B cell proliferation occurs.
Activated B cells increase expression of surface MHC-II and also of another cell surface
molecule, B7.

Activation of B cells by TD antigen

B cell Differentiation
Activated B cells participate in a two-step differentiation process that yields both short-lived
plasmablasts for immediate protection and long-lived plasma cells and memory B cells for
persistent protection
• Affinity maturation- is the result of somatic hyper-mutation during subsequent exposure to
the antigen – This is an antigen driven process that generates antibodies with higher affinities
and this process and positive selection occurs in the germinal centers.
Positive selection- High affinity antibodies producing B cells are selected. No or Low affinity are
deleted. The variability in affinity occurs due to somatic hypermutation.
• Class-switching- similar recognition sites (specificities) but the effector role of the molecule
varies depending on the Ig class.
– Cytokines direct class switch from the original IgM. Class switching is process that leads to a
change in the isotype of Ig that the cell is producing. Class switching involves excision of DNA
which implies that the cell cannot revert back to producing the Ig once it used to. The sequence
of the gene segments on the heavy chain gene hence becomes important.

Cytokines mediated Class switching

 Differentiation into Plasma and Memory cells
Plasma cell – These are long-lived, non-proliferating antibody-secreting cell arising from B cell
differentiation. There is evidence that B cells first differentiate into a plasmablast-like cell, then
differentiate into a plasma cell. Plasma cells are generated later in an infection and, compared
to plasmablasts, have antibodies with a higher affinity towards their target antigen due to
affinity maturation in the germinal center (GC) and produce more antibodies. Plasma cells
typically result from the germinal center reaction from T cell-dependent activation of B cells,
however they can also result from T cell-independent activation of B cells.
Memory B cell - Dormant B cell arising from B cell differentiation. Their function is to circulate
through the body and initiate a stronger, more rapid antibody response (known as the
anamnestic secondary antibody response) if they detect the antigen that had activated their
parent B cell (memory B cells and their parent B cells share the same BCR, thus they detect the
same antigen). Memory B cells can be generated from T cell-dependent activation through both
the extrafollicular response and the germinal center reaction as well as from T cell-independent
activation of B1 cells.

B cell Differentiation
 T cell Development – Maturation, Activation & Differentiation
T cell maturation-
T-cells development initiates with the arrival of small numbers of lymphoid precursors migrating from
the bone marrow (through blood) into the thymus where they develop into mature T cells.
T-cell maturation involves the re-arrangement of the germ-line TCR genes and the expression of
various membrane markers.
In thymus, the developing T cells are termed as thymocytes (Pro T cells).
Pro T cells- These thymocytes proliferate and differentiate along developmental pathways that
produce functionally distinct sub-population of mature T-cells. The differentiating T-cells pass
through a series of stages that are marked by characteristic changes in their cell surface markers.
Initially, they are termed as double negative (DN) cells, as these cells are CD 4-, CD8-, CD3- & TCR-.
Then they differentiate to DN cells expressing CD3 & TCR but CD4-, CD8-. (Figure 1)

Pre T cell → Pro T cell → DN → DN (CD3 & TCR) → DP (CD4+ & CD8+) → SP (CD4+ or CD8+)

→
Figure 1 : T cell Maturation process

The most characteristic property of mature T-cells is that they identify only foreign antigen combined
with self MHC molecules. For this purpose, the thymocytes undergo two selection processes in
thymus.
Positive selection: Only those cells whose TCR recognizes a self-MHC molecule (bearing antigen)
are selected for survival. Cells that do not interact with MHC molecules undergo death by apoptosis.
Also, CD8+ binds with MHC I while CD4+ binds with antigen presented by MHC II molecules. This
results in MHC restriction.
This results into the generation of single positive (SP) cells which are CD4+ or CD8+ T cells. These
cells undergo another selection process to become mature T cells.
Negative selection: eliminates T cells that react too strongly with self-MHC or with self-MHC plus
selfpeptides. It is an extremely important factor in generating a primary T-cell repertoire that is self-
tolerant.Cells interacting with self antigens with high affinity undergo death by apoptosis. This
process is also known as Clonal deletion.
T-cell development is an expensive process for the host because about 98% of all thymocytes do not
mature.
T-cell Activation:
T-cells activation is initiated by interaction of the TCR-CD3 complex with a processed antigenic
peptide bound to either class-I (CD8+ cell) or class II (CD4+ cell) MHC molecules on the surface of an
antigen presenting cells.
Th cells Activation-
The interaction of mature Th cells (CD4+ cells) with Ag-MHC II complex generates activating signals
(a stimulatory signal induced by the CD28-B7 interaction) on the Th-cell and the antigen presenting
cell. (Figure 2)
Interaction of T cell with antigen initiates cascade of biochemical event that induces proliferation and
differentiation into memory cells or effector cells.

Figure 2 : Th cell activation and proliferation

Th cells differentiation -
The Th cells differentiate into
(a) Effector T cells - which are of two types: Th 1 subset and Th 2 subset
(b) Memory cells - long-lived, quiescent cells that respond with heightened reactivity to a subsequent
challenge with the same antigen, generating a secondary response.

Function of Effector cells - TH1 subset, secretes IL-2, IFN-γ, and TNF-α. The TH1 subset is
responsible for classic cell-mediated functions, such as delayed-type hypersensitivity and the
activation of cytotoxic T lymphocytes.
TH2 subset, secretes IL-4, IL-5, IL-6, and IL-10. This subset functions more effectively as a helper
for B-cell activation. (Figure 3)
 Figure 3 : Th cell differentiation

Tc cells Activation –
Activation of naive Tc cells and their subsequent proliferation and differentiation into effector T cells
require both a primary signal, delivered when the TCR complex and CD8 coreceptor interact with a
foreign peptide–MHC I molecule complex, and a co-stimulatory signal, delivered by interaction
between particular membrane molecules on the T cell and the target cell.
Additionally, IL 2 secreted by TH1interacts with IL 2-R {receptor} which initiates proliferation and
differentiation.

Tc cells differentiation –
The effector Tc cells differentiate into CTLs (Cytotoxic T Lymphocytes). These CTLs have lytic
capability and are critical in the recognition and elimination of altered self-cells (e.g., virus-infected
cells and tumor cells) and in graft-rejection reactions. (Figure 4)
 Figure 4 : CTL mediated killing of the target cell by Perforin pathway

CTLs Kill Cells in Two Ways (Figure 5)

(a) CTLs can initiates Fas/FasL apoptotic pathway.
(b) CTLs acquire the machinery to perform target/infected cell killing. The most striking feature is
the development of cytoplasmic granules that contain Perforins and Granzymes. The perforins
create a pore/ channel in the target cell membrane which facilitates the entry of granzyme inside
the cell. This enzyme initiates the caspase cascade reaction resulting into apoptosis of the target
(infected cell).

Figure 5 : CTL mediated Cytotoxicity by Fas & Perforin pathway

 Characteristics and Types of Antigen

ANTIGEN is a substances usually protein in nature and sometimes polysaccharide, that generates a specific immune
response and induces the formation of a specific antibody or specially sensitized T cells or both.
Antigen is defined as any substance which when introduced in the body, stimulates the production of an antibody
with which it reacts specifically.
Its ability to bind with antibodies or T-cell is referred to as antigenicity.
The word originated from the notion that they can stimulate antibody generation (However it is known that
the immune system does not only consist of antibodies)
Ex. Microbial Antigen- Bacteria (whole cell or coats, capsules, cell walls, flagella, fimbrae, and toxins, viruses, and
other microorganisms.
Non-microbial antigen-pollen, egg white, and proteins. These include:
a. allergens, including dust, pollen, hair, foods, dander, bee venom, drugs, and other agents causing allergic
reactions
b. foreign tissues and cells from transplants and transfusions
c. the body's own cells that the body fails to recognize as "normal self," such as cancer cells, infected cells, cells
involved in autoimmune diseases.

Although all antigens are recognized by specific lymphocytes or by antibodies, only some antigens are capable of
activating lymphocytes. Molecules that stimulate immune responses are called Immunogens.
IMMUNOGEN is a substance which produces an immune response as well as binds to its products i.e., antibodies or
sensitized T-cells, when injected into the host.
Immunogenicity is the ability to induce an Antibody- or Cell-mediated immune response. The
immunogenicity of the molecule depends upon its degree of foreignness. The greater the difference
between antigen and self components, the greater will be the intensity of immune response.
 Immunogenic molecule is Antigenic, but the reverse is not true.

Chemical Nature of Antigens (Immunogens)

A. Proteins The vast majority of immunogens are proteins. These may be pure proteins or they may be
glycoproteins or lipoproteins. In general, proteins are usually very good immunogens.
B. Polysaccharides Pure polysaccharides and lipopolysaccharides are good immunogens.
C. Nucleic Acids Nucleic acids are usually poorly immunogenic. However, they may become immunogenic when
single stranded or when complexed with proteins.
D. Lipids In general lipids are non-immunogenic, although they may be haptens.

Property of antigens/ Factors Influencing Immunogenicity

1. Foreignness
The prerequisite for immunogenicity is that the substance should be foreign to the body of recipient. As a result the
immune system must be able to distinguish between normal body components and foreign substances. Normally
the body has no tendency to recognize its own components as antigens, therefore does not initiate an immune
response against these.
For example, if serum albumin from a mouse is injected back into the same mouse or into another mouse, no
antibody production will occur. On the other hand, if the albumin is infected into an animal of another species,
antibody may be produced.
2. Molecular Size
The most active immunogens tend to have a molecular mass of 14,000 to 6,00,000 Da.
Examples: tetanus toxoid, egg albumin, thyroglobulin are highly antigenic. Insulin (5700 Da) is either non-antigenic
or weakly antigenic.
The molecule must have a minimum size (generally > 5000 Da) before it can be considered as antigen.
Hence small molecules like amino acids or monosaccharides are usually not antigenic. However, low molecular
substance can demonstrate immunogenicity, if coupled to a suitable carrier molecule like protein. These low
molecular substances are called haptens which are considered as ‘partial antigens’ that contain at least one
Antigenic determinant group.
3. Complexity
The rigidity and complexity of the molecule are the important factors for immunogenicity.
A rigid molecule is a good antigen, probably because it is easier to raise antibodies. It is presumed that presence of
an aromatic radical is essential for rigidity and antigenicity of a substance.
The immunogenicity is very much dependent on the complexity of the antigens. The more complex the substance is
chemically, the more immunogenic it will be. For example, a molecule containing a repeating unit of only one
amino acid is generally poor immunogen, even if the molecule is large; while a molecule with two or three
repeating amino acids can function as an immunogen.
Antigens are mainly proteins and some are polysaccharides.
4. Physical Form and Structural Stability
In general particulate antigens are more immunogenic than soluble ones.
Denatured antigens are more immunogenic than the native form.
Flexible molecule or shapes are poor antigens. Ex. Flagellin, starch. The molecule or the part of the molecule with
fixed shape is recognized easily by the cells of immune system and are considered good antigen.
5. Degradability
Antigens that are easily phagocytosed are generally more immunogenic.
This is because for most antigens (T-dependant antigens) the development of an immune response requires that
the antigen be phagocytosed, processed and presented to helper T cells by an antigen presenting cell (APC).
6. Dose and Route of Administration of the antigen
The dose of administration of an immunogen can influence its immunogenicity.
There is a dose of antigen above or below which the immune response will not be optimal.
Generally the subcutaneous route is better than the intravenous or intragastric routes.
The route of antigen administration can also alter the nature of the response. Antigen administered intravenously
is carried first to the spleen, whereas antigen administered subcutaneously moves first to local lymph nodes.
Route of administration of the antigen
- intravenous (spleen)
- subcutaneous (lymph nodes)
- intraperitoneal (lymph nodes)
- oral (mucosal - GALT)
- inhaled (mucosal - BALT))
7. Genetic Factors
Some substances are immunogenic in one species but not in another. Similarly, some substances are immunogenic
in one individual but not in others (i.e. responders and non-responders).
The species or individuals may lack or have altered genes that code for the receptors for antigen on B cells and T
cells.
They may not have the appropriate genes needed for the APC to present antigen to the helper T cells.
8. Specificity
Antigen Specificity- It depends on the specific active sites (Antigenic determinants) on the antigenic molecules.
Species Specificity- Tissues of all individuals in a particular species possess, species specific antigen.
Organ Specificity- Organ specific antigens are confined to particular organ or tissue. Certain proteins of brain,
kidney, thyroglobulin and lens protein of one species share specificity with that of another species.
Auto-specificity- The autologous or self antigens are ordinarily not immunogenic, but under certain circumstances
lens protein, thyroglobulin and others may act as autoantigens.
9. Age
Age can also influence immunogenicity.
Usually the very young and the very old have a diminished ability to elicit and immune response in response to an
immunogen.
10. Use of Adjuvants
Adjuvants enhance the antigenicity of the antigen.
 TYPES OF ANTIGEN
On the basis of order of their class (Origin)
1. Exogenous antigens
These antigens enter the body or system and start circulating in the body fluids and trapped by the APCs
(Antigen processing cells such as macrophages, dendritic cells, etc.)
The uptakes of these exogenous antigens by APCs are mainly mediated by the phagocytosis
Examples: bacteria, viruses, fungi etc
Some antigens start out as exogenous antigens, and later become endogenous (for example, intracellular
viruses)
2. Endogenous antigens
These are body’s own cells or sub fragments or compounds or the antigenic products.
The endogenous antigens are processed by the macrophages which are later accepted by the cytotoxic T –
cells.
Endogenous antigens include xenogenic (heterologous), autologous and idiotypic or allogenic (homologous)
antigens.
Examples: Blood group antigens, HLA (Histocompatibility Leukocyte antigens), etc.

On the Basis of genetic consideration

1. Autoantigens, for example, are a person’s own self antigens. Examples: Thyroglobulin, DNA, Corneal tissue, etc.
An autoantigen is usually a normal protein or complex of proteins (and sometimes DNA or RNA) that is
recognized by the immune system of patients suffering from a specific autoimmune disease.
These antigens are not the target of the immune system under normal conditions, but, due to genetic and
environmental factors, the normal immunological tolerance for such an antigen has been lost in these patients.
Examples: Nucleoproteins, Nucleic acids, etc.
2. Alloantigens are antigens found in different members of the same species (the red blood cell antigens A and B
are examples).
3. Heterophile antigens are identical antigens found in the cells of different species. Examples: Forrssman
antigen, Cross-reacting microbial antigens, etc.

On the basis of immune response

1. Complete Antigen or Immunogen
Possess antigenic properties, i.e. they are able to generate an immune response by themselves.
High molecular weight (more than 10,000 Da).
May be proteins or polysaccharides.
2. Incomplete Antigen or HAPTEN
Hapten refers to a group of substances, usually very
small in size, which do not induce an immune
response by themselves alone. It can induce an
immune response, if combined with another molecule
called carriers by forming hapten-carrier complex.
(figure)
[Karl Landsteiner gave the concept of Hapten
molecule].

 Haptens are unable to induce an immune response by itself, they require carrier molecule to act as a complete
antigen. The carrier molecule is a non-antigenic component and helps in provoking the immune response.
Example: Serum Protein such as Albumin or Globulin.
 Haptens can react specifically with its corresponding antibody, when conjugated with some carrier. Thus
antigenic hapten becomes immunogenic.
 Examples: DNP (dinitro phenol), Penicillin, sugars, amino acids, peptide hormone, Capsular polysaccharide of
pneumococcus, polysaccharide “C” of beta haemolytic streptococci, cardiolipin antigens, etc.
Conjugates of the haptens with large protein carriers are used to produce hapten-specific antibodies. These
antibodies are useful for measuring the presence of various substances in the body. For example, the original home
pregnancy test kit employed anti-hapten antibodies to determine whether a woman’s urine contained human
chorionic gonadotropin (HCG), which is a sign of pregnancy. However, as shown in the Clinical Focus, the formation
of drug-protein conjugates in the body can produce drug allergies that may be life-threatening.

ADJUVANT: a substance that, when mixed with an antigen, enhance the immune response (immunogenicity) to the
antigen. Adjuvants are often used to boost the immune response when an antigen has low immunogenicity or
when only small amounts of an antigen are available.
Mechanisms of action of adjuvants:
- Prolong the persistence of the antigen, thus giving the immune system more time to respond
- Increase the “size” of the antigen by causing aggregation,
- Stimulate lymphocyte proliferation and/or activation
- Stimulate a local inflammatory response, thus recruiting cells to the site of the antigen (granuloma formation)
- Enhance co-stimulatory signals
Commonly used adjuvants:
Alum (aluminum potassium sulfate) - precipitates the antigen, resulting in increased persistence of the antigen. It
increases
“size” of antigen thus facilitates its phagocytosis.
Incomplete Freund’s adjuvant (mineral oil-based) - increases persistence of the antigen.
Complete Freund’s adjuvant (mineral oil-based adjuvant containing dead Mycobacterium) - increases persistence
of the antigen, stimulates a chronic inflammatory response (granuloma), and co-stimulatory signals. Muramyl
dipeptide, a component of the mycobacterial cell wall, activates macrophages and DCs. It makes Freund’s complete
adjuvant far more potent than the incomplete form.
Alum and Freund’s adjuvants also stimulate a local, chronic inflammatory response that attracts both
phagocytes and lymphocytes. This infiltration of cells at the site of the adjuvant injection often results in
formation of a dense, macrophage-rich mass of cells called a granuloma.
Bacterial Lipopolysaccharides - stimulate nonspecific lymphocyte activation and proliferation, and costimulatory
signals.

SUPERANTIGENS
 Superantigens are the class of microbial antigens (viral, bacterial or mycoplasma proteins).
 They non-specifically activate large number of T cells. When the immune system encounters a conventional T-
dependent antigen, only a small fraction (1 in 104 -105) of the T cell population is able to recognize the antigen
and become activated (monoclonal/oligoclonal response). But, some antigens polyclonally activate a large
fraction of the T cells (up to 25%). These antigens are called superantigens.
 Superantigens simultaneously bind to MHC class II molecules on the APCs and to the Vβ-region of the TCR.
 A typical antigen must be processed by an APC, after which it binds to both α and β chain of the TCR.
Superantigens do not require processing. It bind to the β chain of the TCR directly and to the class II MHC
 molecule on the APC, an interaction that is sufficient to activate the T cell in the absence of any other co-
stimulatory signals.
 These are the most powerful cell mitogen discovered which can lead to massive T cell activation and excessive
cytokine release leading to systemic toxicity and are responsible for diseases like Staphylococcal food
poisoning, Staphylococcal Toxic shock syndrome, Streptococcal toxic shock like syndrome etc. Superantigen
also induces apoptosis in the superantigen-binding CD4 T cells, so T cells that can respond to the pathogen are
deleted.
 Exogenous SAGs (are soluble proteins secreted by bacteria)- Examples: Staphylococcal enterotoxins (food
poisoning), Staphylococcal toxic shock toxin (toxic shock syndrome), Staphylococcal exfoliating toxins (scalded
skin syndrome) and Streptococcal pyrogenic exotoxins (shock).
Endogenous SAGs (are cell-membrane proteins encoded by certain viruses that infect mammalian cells)- Ex.
Mouse mammary tumor virus (MTV)
 Although the bacterial superantigens are the best studied there are superantigens associated with viruses and
other microorganisms as well.
 The diseases associated with exposure to superantigens are, in part, due to hyper activation of the immune
system and subsequent release of biologically active cytokines by activated T cells.

Figure – Binding of Superantigens with TCR-MHC complex

EPITOPE-
Determinants of Antigenicity or Antigenic Determinants
 Epitope is immunologically active regions of an immunogen (or antigen) that binds to antigen-specific
membrane receptors on lymphocytes or to secreted antibodies. It is also called antigenic determinants.
 It is the smallest unit of antigenicity.
 The whole antigen does not evoke immune response and only a small part of it induces B and T cell response.
The small area of chemical grouping on the antigen molecule that determines specific immune response and
reacts specifically with antibody is called an antigenic determinant.
 The number of antigenic determinants on a small molecule varies with molecular size. For example, human
serum albumin (Mol. wt.70 KDa) has at least six different antigenic determinants, that means at least six
antibodies of different specificities can be produced after immunization.
 Epitopes may be overlapping or non-overlapping. Epitopes that are separated in location so that two different
antibodies can bind to same antigen at different epitopes, such epitopes/ determinants are called non-
overlapping epitopes. Sometimes two epitopes are so closely located that binding of antibody to one epitope
interfere with the binding of antibody to another epitope; such epitopes/ determinants are called overlapping
epitopes.
 It is established that antibodies recognize the three dimensional structure of the antigen rather than their
primary amino acid sequence.
 Epitopes are of three types (figure 1)
 1. Linear or Sequential epitopes are continuous sequence and found in polysaccharides as well as in both native
(nondenatured) and denatured proteins. Its specificity depends upon primary sequence. The typical size is 5-6
subunits in length
2. Conformational or Non-Sequential epitopes are discontinuous sequence (involve multiple subunits, often
located far apart in the primary sequence of the antigen molecule) and are thus found only in native (globular)
proteins.
3. Neoantigenic determinant are the epitopes that are introduced into the antigen by proteolysis or covalent
modifications. Such modifications alter the covalent structure of the antigen and produce the determinant on the
antigen.

Figure 1 - Types of Epitopes

Properties of B & T cell epitopes-

B cell epitope is a portion of antigen molecule that is recognized by B cell receptors.
 Antibody binds to an epitope by weak noncovalent interactions, which operate only over short distances.
 The antigen binding site of antibody and the epitope must have complementary shapes that place the
interacting groups near each other.
 A B-cell epitope must be accessible in order to be able to bind to an antibody; in general, protruding regions on
the surface of the protein are the most likely to be recognized as epitopes, and these regions are usually
composed of predominantly hydrophilic amino acids.
 B-cell epitopes can contain sequential or nonsequential amino acids. Sequential and nonsequential epitopes
generally behave differently when a protein is denatured, fragmented, or reduced. It is because sequential
epitope depend on folded of tertiary conformation of antigen protein.
 Some epitopes, called immunodominant, induce a more pronounced immune response than other epitopes in a
particular animal.
 Multiple and overlapping B-cell epitopes exist in complex or large proteins, some of which are
immunodominant. Ex. 64 different antibodies recognized a minimum of 25 different epitopes on BSA. Within an
animal, certain epitopes of an antigen are recognized as immunogenic, but others are not.
 Multiple epitopes on an antigen Multiple overlapping epitopes on an antigen

T cell epitope is the region of antigen molecules that are recognized by T cell receptors.
 Epitopes recognized by T cells are often internal, linear and hydrophobic sequences which are
exposed by antigen processing within APCs or altered self cells.
 T cells do not recognize soluble native antigen but rather recognize antigen that has been processed into
antigenic peptides, which are presented in combination with MHC molecules.
 Antigenic peptides recognized by T cells form trimolecular complex with a T-cell receptor and an MHC molecule.
 Endogenous antigens are processed into peptides within the cytoplasm, while exogenous antigens are
processed by endocytic pathway.
 Composed of mostly proteins but some lipids and glycolipids.

ABO Blood Group Antigens

At the beginning of the 20th century an Austrian scientist, Karl Landsteiner, noted that the RBCs of some individuals
were agglutinated by the serum from other individuals. He made a note of the patterns of agglutination and
showed that blood could be divided into groups. This marked the discovery of the first blood group system, ABO,
and earned Landsteiner a Nobel Prize.
Landsteiner explained that the markers (antigens) on the RBCs reacted with antibodies in the serum. He called the
antigens - A and B, and depending upon which antigen the RBC expressed, blood either belonged to blood group A
or blood group B. A third blood group contained RBCs that reacted as if they lacked the properties of A and B, and
this group was later called "O". Then the fourth blood group, AB, was added to the ABO blood group system. These
RBCs expressed both A and B antigens.
Therefore there are four basic ABO blood group phenotypes - O, A, B, and AB.(Table 1)

Table 1

The immune system forms antibodies against whichever ABO blood group antigens are not found on the
individual's RBCs (Table 1). ABO antibodies in the serum are formed naturally.
The ABO blood group antigens are of prime importance in blood transfusion. They are the most immunogenic of all
the blood group antigens. If incompatible blood is given in a transfusion, the donor cells are treated as foreign
invaders, and the patient's immune system attacks them accordingly. Not only is the blood transfusion rendered
useless, but a potentially massive activation of the immune system and clotting system can cause shock, kidney
failure, circulatory collapse, and death. To avoid a transfusion reaction, donated blood must be compatible with the
blood of the patient (recipient). Hence, before a blood transfusion, two blood tests known as a "type and cross
match" are done. First, the recipient's blood type is determined i.e., their ABO type and Rh D status. Then a "cross
match" is done to ensure that the donor RBCs actually do match against the recipient's serum.

The ABO blood group antigens are sugars (Figure 1). These antigens have a common oligosaccharide foundation
called the O antigen. The A and B antigens differ from the O antigen by the addition of one extra monosaccharide,
either N-acetylgalactosamine (for A) or galactose (for B) through an a -1,3 linkage to a galactose moiety of the O
antigen. The presence or difference of one monosaccharide is recognized by the immune system and elicit the
production of antibodies.

Figure 1 - Structures of A, B, and O Oligosaccharide Antigens

Abbreviations: Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine.
 Structural features of Immunoglobulins-
The structural details of Y-shaped immunoglobulins are:
 Antibody (or immunoglobulin) molecules are glycoproteins.
 They are composed of four polypeptide chains: two identical heavy chains (H) and two identical light chains (L).
These H & L chains are folded into different regions called domains.
 Light Chain Domains - VL (110 amino acids) and CL (110 amino acids)
 Heavy Chain Domains - VH (110 amino acids) and CH (three constant domains CH1, CH2 and CH3) (330-440
amino acids)
 The amino terminal ends of the polypeptide chains show considerable variation in amino acid composition and
are referred to as the variable (V) re gions to distinguish them from the relatively constant (C) regions.
 Mol. Wt.: Each heavy chain has abo ut twice the number of amino acids and molecular weight (~50,000) as each
light chain (~25,000), resulting in a total immunoglobulin monomer molecular weight of approximately 150,000.
 Heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain
disulfide bonds, forming a bilaterally symmetric structure.
 The V regions of H and L chains comprise the antigen-binding sites of the immunoglobulin (Ig) molecules. Each
Ig monomer contains two antigen-binding sites and is said to be bivalent.

 Hinge Region-This is the region at which the arms of the antibody molecule forms a Y. It is called the hinge
region because there is some flexibility in the molecule at this point. The hinge region is the area of the H chains
between the first and second C re gion domains and is held together by disulfide b onds. This flexible hinge
(found in IgG, IgA and IgD, but not IgM or IgE) region allows the distance between the two antigen-binding sites
to vary.
 Types of L chains: kappa and lambda are two types of L chains. Each Ig mole cule consist of any one type of L
chain.example µ2κ2 or µ2λ2
 Types of H chains: There are five types of H chains- γ, µ, α, δ, ε; present which give rise to five classes of Ig. The
five primary classes of immunoglobulins are IgG, IgM, IgA, IgD and IgE. These are distinguished by the type of
 heavy chain- γ, µ, α, δ, ε respectively found in the molecule. All these five types of antibodies have different
biological roles.

Functions of different types of antibodies

Ig G
 It provides immunity to the developing fetus.
 Activates the complement pathway of immune response.
 Mediates a process known as opsonization which refers to antibodies coating a pathogenic cell to attract the
phagocyte towards the antigenic surface of the pathogen.
 Facilitates the process of phagocytosis.
 Neutralization of toxins and pathogens.
 Since it is widely found circulating in the blood, it offers protection against pathogens in the blood and tissues.
Ig A
 It is found in the secretions and provides the first line of defense against the uptake of microbes and antigens
into the body.
 It limits inflammation.
 Participates in the immune response through the activation of the complement pathway.
 Provides immunity to the fetus and the newborn infant.
IgM
 It is the antibody that is found on the surface of the B-cell that helps in antigenic recognition.
 Activation of the complement pathway of the immune response.
 It is involved in opsonization and agglutination.
 Facilitates efficient activation of the immune system due to the more significant number of antigenic sites on its
surface.
 The ABO system of blood grouping consists of IgM antibodies that are specific to the ABO antigens expressed on
the surface of the RBCs.
 The known function of the IgD antibody is to act as a receptor on the surface of the B cell and participate in B
cell activation and differentiation.
IgE
 Immune protection against pathogens is invading through the gastrointestinal or respiratory barriers.
 It has a vital role in Type 1 hypersensitivity reactions or allergic response.
 MONOCLONAL ANTIBODIES

HYBRIDOMA TECHNOLOGY-
It is the technique of producing hybridoma for the production of Monoclonal antibodies.
Hybridomas are cells that have been engineered to produce a desired antibody in large amounts, to
producem onoclonal antibodies. Hybridoma technology was discovered in 1975 by two scientists,
Georges Kohler and Cesar Milstein. They jointly with Niels Jerne of Denmark were awarded the 1984
Noble prize for physiology and medicine.
Monoclonal antibody (MAb) is a single type of antibody that is directed against a specific antigenic
determinant (epitope). Monoclonal antibodies are valuable for the analysis of parasites antigen
monoclonal antibodies, as they are known, have opened remarkable new approaches to preventing,
diagnosing, and treating disease. Ex.- monoclonal antibody known as OKT3 is saving organ transplants
threatened with rejection, and preventing bone marrow transplants from setting off graft-versus-host
disease.
Production of Monoclonal Antibodies:
The establishment of hybridomas and production of MAbs involves the following steps:
1. Immunization: The very first step in hybridoma technology is to immunize an animal (usually a
mouse), with appropriate antigen. This enables increased stimulation of B-lymphocytes producing
desired antibody specificity. When the serum concentration of the antibodies is optimal, the animal is
sacrificed. The spleen is aseptically removed to obtain/isolate the B cells.
2. Cell Fusion: The thoroughly washed lymphocytes are mixed with HGPRT defective myeloma cells
using polyethylene glycol (PEG) and are allowed to grow. Fused cells are incubated in the HAT
(Hypoxanthine Aminopetrin Thymidine) medium. HAT medium (Hypoxanthine Aminopetrin Thymidine)
is used for preparation of monoclonal antibodies (Fig. 17.1). In this medium, the myeloma cells die, as
they cannot produce nucleotides by the de novo pathway and salvage medium is blocked by
Aminopterin. Unfused B cells die as they have a short life span. Only the B cell-myeloma hybrids survive,
since the HGPRT gene coming from the B cells is functional. These cells produce antibodies (a property
of B cells) and are immortal (a property of myeloma cells). (Fig. A)
3. Selection of Hybridomas: When the cells are cultured in HAT medium, only the hybridoma cells grow.
Selection of a single antibody producing hybrid cells is done. The suspension of hybridoma cells is so
diluted that the individual aliquots contain on an average one cell each. These cells, when grown in a
regular culture medium, produce the desired antibody.
4. Screening the Products: The hybridomas must be screened for the secretion of the antibody of
desired specificity which is done by two techniques namely ELISA and RIA. Thus, the screened
hybridoma cells are producing the desired antibody which is referred to as monoclonal antibody. After
selection, these cells are changed to larger tissue culture flasks for appreciable recovery of monoclonal
antibody. Fig. B
5. Cloning and Propagation: The single hybrid cells producing the desired antibody are isolated and
cloned.
6. Characterization and Storage: The monoclonal antibody has to be subjected to biochemical and
biophysical characterization for the desired specificity. The stability of the cell lines and the MAbs is also
characterized.
 Fig. A – Production of Hybridoma

Fig. B – Production of monoclonal antibodies

 Principle of HAT medium - The de novo synthesis of
nucleotides requires tetrahydrofolate which is formed
from dihydrofolate. The formation of tetrahydrofolate
(and therefore nucleotides) can be blocked by the
inhibitor Aminopterin. The salvage pathway involves the
direct conversion of purines and pyrimidine’s into the
corresponding nucleotides. Hypoxanthine guanine
phosphoribosyl transferase (HGPRT) is a key enzyme in
the salvage pathway of purines.
It converts hypoxanthine and guanine respectively to
inosin monophosphate and guanosine monophosphate.
Thymidine kinase (TK), involved in the salvage pathway
of pyrimidine’s converts thymidine to thymidine
monophosphate (TMP). Any mutation in either one of
the enzymes (HGPRT or TK) blocks the salvage pathway.
When cells deficient (mutated cells) in HGPRT are grown
in a medium containing hypoxanthine aminopterin and
Thymidine (HAT medium), they cannot survive due to
inhibition of de novo synthesis of purine nucleotides
(Note : Salvage pathway is not operative due to lack of
HGPRT). Thus, cells lacking HGPRT, die in HAT medium.

Human Monoclonal Antibodies:

The monoclonal antibodies produced by using
mice are quite suitable for in vitro use. However,
their administration to humans is associated with
immunological complications, since they are
foreign to human body. Therefore production of
human monoclonal antibodies is preferred.
However, it is difficult to produce human MAbs by
conventional hybridoma technology.
The following are the major limitations:
i. For ethical reasons, humans cannot be
immunized against antigens.
ii. The fused human lymphocyte-mouse myeloma
cells are very unstable.
iii. There are no suitable myeloma cells in humans
that can replace mouse myeloma cells.
Genetic Engineering Strategies for the Production
of Human- Mouse MAbs:
With the advances in genetic engineering, it is
possible to add certain human segments to a
mouse antibody. This is truly a hybridized
antibody and is referred to as humanized antibody
or chimeric antibody.
Substitution of Fv region of human Ig by mouse
Fv:
The DNA coding sequences for Fv regions of both L
and H chains of human immunoglobulin are
replaced by Fv DNA sequence (for L and H chains)
from a mouse monoclonal antibody (Fig. 17.4A).
The newly developed humanized MAb has Fc
region of Ig being human. This stimulates proper
immunological response. The chimeric antibodies
produced in this manner were found to be
effective for the destruction of tumor cells in vitro.
 Unit 3
Short & Long Important Questions

Question (Statement) - appeared in Year Marks

Q. No.
AKTU End Sem Exams
Properties and Functions of Haptens,
1 2022 2
Adjuvants and Immunogen.
Structure and Functions of Cells and
2 2020, 2022 2
Organs of immune system.
Structure, functions and characteristics
3 2020, 2022 10
of different classes of antibodies.
T cell & B cell maturation and its
4 2019, 2020 10
activation.
Innate immunity (First line of defence). 2022
5 10
Production and applications of 2021 10
6
Monoclonal antibodies
Compare between Humoral and Cell- 2021 10
7
mediated immunity
2019 2
8 Describe the process of inflammation.
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